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Characterization of Mycoplasma Alligatoris Immunodominant Antigens

Permanent Link: http://ufdc.ufl.edu/UFE0042224/00001

Material Information

Title: Characterization of Mycoplasma Alligatoris Immunodominant Antigens
Physical Description: 1 online resource (90 p.)
Language: english
Creator: Fabian, Niora
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: alligator, alligatoris, antigen, immunodominant, mycoplasmosis
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mycoplasma alligatoris is a bacterial pathogen that causes acute lethal multisystemic inflammatory disease in alligators. Previous Western blotting experiments revealed antibody response patterns of experimentally inoculated and naturally infected American alligators (Alligator mississippiensis). In all individuals surviving the acute infection, a significant immune response was observed for an antigen approximately 64 kDa in size as early as 6 wk post-inoculation (PI), increasing in intensity until euthanasia 16 wk PI. For some individuals, a second antigen approximately 50 kDa in size was immunoreactive. The importance of this distinct initial humoral response is unknown, as alligators surviving the natural infection developed immune responses to many other antigens several years later. The goal of this study was to identify and characterize the 64 kDa immunodominant antigen to better understand its potential role in host-pathogen interactions in M. alligatoris mycoplasmosis. Two-dimensional gel electrophoresis (2DGE) and affinity chromatography-based antigen isolation experiments were performed using M. alligatoris membrane proteins and total proteins. Following Western blot confirmation, LC/MS/MS peptide sequencing, and de novo peptide sequencing, protein matches were evaluated with statistical algorithm-based proteome analysis software. Top immunodominant antigen candidates were obtained from 2DGE samples. These candidates could be evaluated for immunodominant antigen expression in subsequent studies. Though the 64 kDa M. alligatoris antigen is still unknown, future work involving improved antigen enrichment and resolution as well as testing candidates for immunodominant antigen expression may confirm its identity. This work may explain relative virulence of M. alligatoris and provide insight on pathogenesis of other Mycoplasma species and related organisms.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Niora Fabian.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Brown, Daniel R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042224:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042224/00001

Material Information

Title: Characterization of Mycoplasma Alligatoris Immunodominant Antigens
Physical Description: 1 online resource (90 p.)
Language: english
Creator: Fabian, Niora
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: alligator, alligatoris, antigen, immunodominant, mycoplasmosis
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Mycoplasma alligatoris is a bacterial pathogen that causes acute lethal multisystemic inflammatory disease in alligators. Previous Western blotting experiments revealed antibody response patterns of experimentally inoculated and naturally infected American alligators (Alligator mississippiensis). In all individuals surviving the acute infection, a significant immune response was observed for an antigen approximately 64 kDa in size as early as 6 wk post-inoculation (PI), increasing in intensity until euthanasia 16 wk PI. For some individuals, a second antigen approximately 50 kDa in size was immunoreactive. The importance of this distinct initial humoral response is unknown, as alligators surviving the natural infection developed immune responses to many other antigens several years later. The goal of this study was to identify and characterize the 64 kDa immunodominant antigen to better understand its potential role in host-pathogen interactions in M. alligatoris mycoplasmosis. Two-dimensional gel electrophoresis (2DGE) and affinity chromatography-based antigen isolation experiments were performed using M. alligatoris membrane proteins and total proteins. Following Western blot confirmation, LC/MS/MS peptide sequencing, and de novo peptide sequencing, protein matches were evaluated with statistical algorithm-based proteome analysis software. Top immunodominant antigen candidates were obtained from 2DGE samples. These candidates could be evaluated for immunodominant antigen expression in subsequent studies. Though the 64 kDa M. alligatoris antigen is still unknown, future work involving improved antigen enrichment and resolution as well as testing candidates for immunodominant antigen expression may confirm its identity. This work may explain relative virulence of M. alligatoris and provide insight on pathogenesis of other Mycoplasma species and related organisms.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Niora Fabian.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Brown, Daniel R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042224:00001


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CHARACTERIZATION OF Mycoplasma alligatoris IMMUNODOMINANT ANTIGENS


By

NIORA J. FABIAN

















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2010































S2010 Niora J. Fabian





















To N.N., E.N., S.N., A.N., and my entire family









ACKNOWLEDGMENTS

I thank my main advisor, Dr. Daniel Brown, and my committee members for their

support and education. I would also like to thank Kevin Kroll for his kind advice with

Western blotting optimization and Mengmeng Zhu for her recommendations with IEF

and 2DGE equipment. Finally, I would like to thank Diane Duke and Linda Green of the

ICBR Hybridoma Lab, and Carolyn Diaz and Dr. Sixue Chen of the ICBR Proteomics

Core.









TABLE OF CONTENTS

page

ACKNOW LEDGMENTS............ ... .. .............. ... ..................... ..........

LIST O F TA B LES ................ ....... .................................................................. 7

LIST OF FIGURES ............. ......... ................................

ABSTRACT .......... .......... .... ..................... ................ 9

CHAPTER

1 INTRODUCTION ......................................................................... .. ......... ................... 11

Discovery and Characterization of M. alligatoris ...................... ................. 11
M ycoplasm osis ............... .............. ............... ........ ..................... 12
R eptile Im m unology ............. ......... ............... ......... ................................ 14
Immunodominance ...................... ....................... 17
Cellular Host Responses Affecting Immunodominance ............................... 18
Immunogenic Proteins and Epitopes ................................... ........ 19
Antigenic Variation and Im m unodom finance ...................... ................... .............. 22
Previous Studies on the Temporal Antibody Response of Crocodilians to M.
alligators ......... .... ................................................................. .. ...........................23
Objectives and Experimental Approaches ................................... 25
Long Term Goals of Research on M. alligators ..................................25
O bje c tiv e s ........................ .. ............................................. 2 5
Hypotheses ........... .... ..................................... ....... ...... ......... 26
Specific A im s .............. .......... ...................................... ............. .................. 26

2 MATERIALS AND M ETHO DS ............. ........... ... .......................... ................ 29

Affinity Chromatography Antigen Purification ................................... ................. 30
First Colum n Configuration .............................................. 31
Second Column Configuration ........................................._.. ........... .......... 32
Third Colum n Configuration .................................. .......2............. .......
SDS-PAGE Separation of Affinity Chromatography Elutions.............................. 33
Second Affinity Column Monoclonal Antibody Coupling Validation .....................33
2-Dimensional PAGE Antigen Separation ............................... .....................34
Active Rehydration ................. ......... ................................36
Isoelectric Focusing (IE F) ......... ................ .............................. ................ 37
Second Dimensional SDS-PAGE ......... ................................ ........ ......... 37
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) ............ ................38
W western Blotting ...................................................................................... 38
Phosphopentomutase Knockout Mutant Evaluation ........... ..... .............. ...........40
Peptide Sequencing and Protein Identification ............................ ..... 40
Protein Reduction, Alkylation and Enzymatic Digestion.................................. 40









L C -M S /M S ..................................................................................................... 4 2
D database S searching ............. ....... ...... .......... .. ...... .............. ...............42
De Novo Sequencing Protein Identifications ................................... ............... 45
P EA K S C riteria S ettings ................................................. ............ ............... ....46

3 R ESU LTS A N D D ISC U SS IO N ............................................. .............. ................47

Affinity C olum ns ............................. ..... ...........................47
Knockout Mutant Immunodominant Antigen Expression Evaluation....................53
2DG E Protein Separation .............. ........... .................................... .. ...... 54
2DGE Membrane Protein Separation .............................. .. .............. 54
2DGE Total Protein Separation ................... .............................55
Blue Native PAG E ............... ............... ............. ...... ........................ 57

4 CONCLUSIONS AND FUTURE DIRECTIONS....... ........................................ 68

C o n c lu s io n s ...................... .. .. ......... .. .. ......... ...................................... 6 8
Future Directions ............... ............. ............... ................... 68
Technical Im prove ents .................... ......... .. .......... ......... ...... ................70
Alternative Immunodominant Antigen Candidate Screening Techniques ...............72
Vaccine Development.... ....... .......... ............. ................ 73
Algorithm-Based Evaluations of Diagnostic Value............................................... 74
Alternatives to Hypotheses .............. .. ....................................... .... ...... 75
Responses to Future Potential M. alligatoris Outbreaks...................... ................ 75

APPENDIX: DE NOVO SEQUENCES AND FASTA ZP_06610777 FRAGMENT
PEPTIDE SEQUENCE ........... .... ........ .... ............................. 78

LIST OF REFERENCES .......................... ................ .................. 81

B IO G R A P H IC A L S K E T C H ......... .. .............................................................................. 90




















6









LIST OF TABLES


Table page

3-1 M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold
(peptide sources: in-gel trypsin or AspN digestion of third column
configuration LAMP antigen elutions separated by SDS-PAGE).........................65

3-2 M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold
(peptide sources: in-gel trypsin digestion of M. alligatoris membrane protein
separated by 2D G E). ................................................................ ........ ........ 65

3-3 M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold
(peptide sources: in-gel trypsin digestion of M. alligatoris total protein
separated by 2DGE). ............................................... .. .. .. ................. 66

3-4 M. alligatoris proteins identified by protein-protein BLAST searches of de
novo sequence data reported in Appendix Tables A-1, A-2 and A-3 ................. 67

A-1 De novo sequence data obtained from LC-MS/MS spectra using PEAKS
Studio software (peptide source: in-gel trypsin digestion of third column
configuration LAMP antigen elutions separated by SDS-PAGE)......................78

A-2 De novo sequence data obtained from LC-MS/MS spectra using PEAKS
Studio software (peptide source: in-gel Asp-N digestion of third column
configuration LAMP antigen elutions separated by SDS-PAGE) ....................79

A-3 De novo sequencing data obtained from LC-MS/MS spectra using PEAKS
Studio software (peptide source: in-gel trypsin digestion of total protein
separated by 2DG E). ............. ..... ....... ................................. .............. ....... 80









LIST OF FIGURES


Figure page

1-1 Representative Western blots of M. alligatoris antigens probed with plasma
from crocodilians exposed to M. alligatoris................ ................ ................. 28

3-1 Coupling validation of affinity column (second configuration). .............................58

3-2 M. alligatoris LAMP antigen eluted from affinity chromatography column (third
configuration) ...................... ............................................................. ........ 60

3-3 Secondary antibody-only control ............................................. .... ............... 61

3-4 Western blot of M. alligatoris total protein extracted from a
phosphopentomutase knockout mutant (342), and from an irrelevant
knockout mutant, NanA (209) as a control .............. .... ..................62

3-5 M. alligatoris membrane proteins separated by 2DGE..................... ........ 63

3-6 M. alligatoris total proteins separated by 2DGE............ ............................... 64

A-4 Peptide fragment sequence of immunodominant antigen candidate
ZP_06610777 as seen in the FASTA database used for Scaffold 2
evaluations. ........... ..... ................................................. ........... 80









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

CHARACTERIZATION OF Mycoplasma alligatoris IMMUNODOMINANT ANTIGENS

By

Niora J. Fabian

August 2010

Chair: Daniel R. Brown
Major: Veterinary Medical Sciences

Mycoplasma alligatoris is a bacterial pathogen that causes acute lethal

multisystemic inflammatory disease in alligators. Previous Western blotting

experiments revealed antibody response patterns of experimentally inoculated and

naturally infected American alligators (Alligator mississippiensis). In all individuals

surviving the acute infection, a significant immune response was observed for an

antigen approximately 64 kDa in size as early as 6 wk post-inoculation (PI), increasing

in intensity until euthanasia 16 wk PI. For some individuals, a second antigen

approximately 50 kDa in size was immunoreactive. The importance of this distinct initial

humoral response is unknown, as alligators surviving the natural infection developed

immune responses to many other antigens several years later. The goal of this study

was to identify and characterize the 64 kDa immunodominant antigen to better

understand its potential role in host-pathogen interactions in M. alligatoris

mycoplasmosis. Two-dimensional gel electrophoresis (2DGE) and affinity

chromatography-based antigen isolation experiments were performed using M.

alligatoris membrane proteins and total proteins. Following Western blot confirmation,

LC/MS/MS peptide sequencing, and de novo peptide sequencing, protein matches were









evaluated with statistical algorithm-based proteome analysis software. Top

immunodominant antigen candidates were obtained from 2DGE samples. These

candidates could be evaluated for immunodominant antigen expression in subsequent

studies. Though the 64 kDa M. alligatoris antigen is still unknown, future work involving

improved antigen enrichment and resolution as well as testing candidates for

immunodominant antigen expression may confirm its identity. This work may explain

relative virulence of M. alligatoris and provide insight on pathogenesis of other

Mycoplasma species and related organisms.









CHAPTER 1
INTRODUCTION

Discovery and Characterization of M. alligatoris

Mycoplasma alligatoris is a lethal bacterial pathogen of American alligators

(Alligator mississippiensis) and caimans, while crocodiles are comparatively resistant.

In 1995, an outbreak of mycoplasmosis resulted in the death or euthanasia of 60

captive bull alligators in St. Johns County, Florida, USA (Clippinger et al., 2000). Cause

of the outbreak remains unknown. Clinical symptoms included rapidly progressive

lethargy, anorexia, muscle weakness, paraparesis, bilateral ocular discharge, edema of

the limbs, periocular, facial and cervical areas, and death (Brown et al., 2001a).

Pathology findings included necrotizing pneumonia, polyserositis, pericarditis,

necrotizing myocarditis, lymphocytic interstitial nephritis, lymphocytic periportal

hepatitis, splenic hyperplasia, pyogranulomatous meningitis, and synovitis (Clippinger et

al., 2000; Brown et al., 2001a; Brown et al., 2001b; Brown et al., 2001c; Richey, 2001).

There were no significant abnormalities in blood cell counts or plasma biochemistries of

the affected animals. Results for toxicant, heavy metal, mineral, and vitamin analyses

of tissues and serum were unremarkable. Routine aerobic and anaerobic bacterial

culture of blood and lung tissue samples did not confirm etiology of the disease. No

viruses were isolated from pneumonic lung tissue samples. However, a previously

uncharacterized Mycoplasma species, Mycoplasma alligatoris, was cultured from

multiple tissues, peripheral blood, synovial fluid, and cerebrospinal fluid of the affected

alligators. The type strain A21JP2T was characterized using standard taxonomic

methods and was utilized in additional studies.









Disease following experimental M. alligatoris inoculation via the glottis of adult and

juvenile alligators and via the trachea of hatchlings was clinically similar to that

observed in the 1995 outbreak (Clippinger et al., 2000; Brown et al. 2001b, Pye et al.,

2001). Shortly after the natural outbreak, a pilot experimental inoculation study was

performed to fulfill Henle-Koch-Evans postulates. Healthy, seronegative juvenile

(approximately 1 m long) alligators were inoculated by intracoelomic injection or by

instillation through the glottis with 106 CFU of M. alligatoris strain A21JP2T. Three of

four inoculated animals died within 3 wk PI, demonstrating significant virulence of the

pathogen. Next, in a dose-response study, healthy subadult (approximately 1.5 to 2 m

long) female alligators were inoculated by instillation through the glottis with 102, 104, or

106 CFU of M. alligatoris strain A21JP2T in 1 mL of SP4 broth. The Henle-Koch-Evans

postulates were fulfilled by results of these studies for M. alligatoris as the etiological

agent of primary fatal mycoplasmosis of alligators (Evans, 1976). Additional research

has included genome sequencing of M. alligatoris and its close relative M. crocodyli,

characterization of putative M. alligatoris virulence mechanisms, and an immunological

survey of M. alligatoris of captive and natural populations of alligators.

Mycoplasmosis

Mycoplasma-host relationships can range from innocuous commensalism to acute

fatal disease (Brown, 2002). While differences in pathogenicity may be explained by

the presence of certain virulence factors, the detailed pathogenic mechanisms of

mycoplasmas are still generally poorly understood (Matsushita and Okabe, 2001; Brown

et al., 2004). Like other members of its genus, M. alligatoris has several characteristic

features, including a small genome size in comparison to most other prokaryotes. This

reduction in genome size is likely an evolutionary result of adopting a parasitic lifestyle,









in which most nutrients are directly acquired from the host (Fraser et al., 1995;

Himmelreich et al., 1997).

Many Mycoplasma species are able to change their surface antigen profile at high

frequency, including M. pulmonis (Watson et al., 1988), M. hyorhinis (Rosengarten and

Wise, 1990; Rosengarten and Wise, 1991; Yogev et al., 1991), M. hominis (Olson et al.,

1991), M. fermentans (Theiss et al., 1993; Wise et al., 1993), M. gallisepticum

(Markham et al., 1993; Yogev et al., 1994) and M. bovis (Behrens et al., 1994). It

remains to be demonstrated whether the main function of this surface variation is

actually immune evasion and/or whether these antigenic or structural components are

involved in pathogenesis during Mycoplasma-host interactions. It is currently unknown

if M. alligatoris displays a high frequency of antigenic variation, but no homologs for

systems of antigenic variation known in other Mycoplasma species have been identified

in the M. alligatoris genome.

A notable physical characteristic of Mycoplasma is the complete lack of a cell wall.

Thus, the mycoplasmal cell surface lacks the highly conserved lipopolysaccharide and

peptidoglycan antigens that act as endotoxins, eliciting strong immune responses in

animals (Dziarski, 1982). Instead, membrane surface proteins are thought to play a

large role in the pathogenesis of many species. For example, studies have shown that

approximately 10 membrane proteins of M. galactiae are responsible for eliciting

immune responses in sheep (Tola et al., 1997). In some species, initial attachment to

host cells is mediated by adhesion proteins, which can create a host response that

exacerbates disease (Baseman et al., 1982; Razin et al., 1998). For example, M.

pneumoniae, M. bovis, and M. hyopneumoniae rely on cytadhesins to attach to host









cells, a critical step in their pathogenic invasion (Baseman et al., 1982; Zhang, et al.,

1995; Sachse et al. 2000).

Thus far, there is no evidence that M. alligatoris invades host cells, but rather

appear to aggregate on host cell peripheries (Hunt and Brown, 2005; Hunt and Brown,

2007). Lesion severity correlates with large numbers of M. alligatoris cells in diseased

tissues, suggesting the pathogen's virulence may be due to a spreading factor.

However, classic-tip mediated cytadherence is not involved in all adherence pathways.

For example, surface localized elongation factor, Tu (EF-Tu) and the pyruvate

dehydrogenase El3 subunit of M. pneumonia help mycoplasmal binding to fibronectin,

a glycoprotein found in the extracellular matrix primarily serving as a substrate for

mammalian cell adhesion (Balasubramanian et al., 2008). As infection progresses,

mycoplasmas can significantly affect the immune system by inducing either suppression

or polyclonal stimulation of B and T lymphocytes. This in turn induces expression of up-

and down-regulating cytokines, subsequently increasing the cytotoxicity of

macrophages, natural killer cells, and T cells. For example, M. hyorhinis was shown to

release a 200 kDa factor into the supernatants of infected cultured cells that suppresses

cytotoxic T-cell responses to alloantigens (Teh et al., 1988). There is also evidence that

M. pneumoniae, M. arthritidis, and M. fermentans stimulate IL-10 production, a down-

regulating cytokine that suppresses T-cell proliferation (Rawadi et al., 1996; Razin et al.,

1998).

Reptile Immunology

Reptiles and other modern poikilotherms have innate and adaptive immune

defenses similar to those of modern mammals and birds (Sunyer et al., 1998a). Innate

immunity of reptiles includes histaminergic cells, complement, phagocytes and natural









killer cells, peptide regulatory factors, and fever (Brown, 2002; Merchant et al., 2007).

Fever is important for stimulating inflammation, increasing antibody production, and

decreasing serum iron availability. Reptile tissues are rich in mast cells and antigen-

specific immunoglobulin-bearing basophils. Monocytes and macrophages, the latter

being phagocytic and capable of processing and presenting antigens and producing

cytokines, are also present. Heterophils are reptilian phagocytic granulocytes

functionally equivalent to mammalian neutrophils. In an acute response to bacterial

infection, heterophils accumulate locally and degranulate. This aggregation attracts

macrophages, resulting in granuloma formation. Cytokines characterized in reptiles

include interferon, interleukin-1, and transforming growth factor beta.

Another key component of reptilian innate immunity is the complement system

(Zimmerman et al., 2010). The complement system involves of a series of circulating

plasma proteins that kill invading bacteria either through opsonization or by lysis. The

reptilian complement system contains the same pathways of the mammalian system

and additionally contains multiple highly-polymorphic isoforms of C3 (Sunyer et al.,

1998b). The classical pathway, the last of the three pathways to evolve, is activated by

the immunoglobins IgG and IgM. Several Mycoplasma species can become lysed by

activated complement. Mycoplasmal activation of complement by both the classical and

alternative pathways has also been documented (Razin et al., 1998; Brown, 2002).

Adaptive immunity includes lymphoid tissues and lymphocytes, major

histocompatibility (MHC) receptors, lymphocyte receptors, and antibodies. Immune

memory is also evident in reptiles (Montali et al., 1988). Reptile spleen does not

possess germinal centers known for B-cell clonal selection and antibody affinity









maturation in mammals. Instead, lymphocytes originate from bone marrow, spleen and

thymus in adult reptiles (Zapata and Amemiya, 2000). However, splenectomy can

completely or partially suppress the humoral response, indicating importance of the

spleen in reptile immunity (Kanakambika and Muthukkaruppan, 1972; Hussein et al.,

1979). Reptiles lack true lymph nodes, but have mucosal-associated lymphoid

reticulum in addition to T- and B-lymphocytes.

Functions of reptile immunoglobulins include neutralization, agglutination

(antibody interaction with particulate antigen), precipitation (antibody interaction with

soluble antigen), opsonization (enhanced phagocytosis of antigen upon opsonin

binding), complement fixation, and blocking interaction of viruses or toxins with their

cognate receptor structures exposed on the host cell membrane (Origgi, 2007). The

type of antibody that offers protection depends on the infectious strategy and lifestyle of

the pathogen. For example, when opsonizing antibodies such as IgG are available,

opsonization of extracellular pathogens will be more efficient (Murphy et al., 2008).

The major antibody isotypes in reptiles are IgM (the ancestor of all

immunoglobulins) and IgY (the ancestor of IgG and IgE). In addition, a low molecular

weight form of IgY lacking the Fc region exists (Warr et al., 1995; Bengten et al., 2000).

The IgY heavy chain has a molecular mass of 65-70 kDa, and the light chain is 19-21

kDa (Hatta et al., 1993; Sun et al., 2001). The whole IgY molecule is approximately 180

kDa by SDS-PAGE and approximately 167 kDa By MALDI-TOF (Hatta et al., 1993, Sun

et al., 2001). A sequential IgM-lgY response occurs as well as affinity maturation.

Features such as specificity, diversity, and memory are comparatively less extensive in

reptiles than in mammals, not due to a lack in the number of any of the genetic









components of the immunoglobulin gene systems, but because of comparatively low

antibody heterogeneity and poor affinity maturation (Hsu, 1998). Specific antibody titers

after mucosal or systemic mycoplasmal infection are lower in reptiles compared to

mammals or birds (Hsu, 1998; Brown, 2002). In secondary responses, antibody titers

and affinities are rarely any higher than in primary responses. Thus, their differences

are not likely due to restrictions in primary antibody production.

In adult tortoises and crocodilians, mycoplasmosis elicits a humoral immune

response to immunodominant antigens approximately six weeks after initial exposure

(Schumacher et al., 1993; Brown et al., 2001c). Captive alligators naturally exposed to

a sub-lethal dose of M. alligatoris during the 1995 outbreak had detectable specific

antibody titers for at least six years PI (Brown et al., unpublished). However, it cannot

be ruled out that these findings were possibly due to the animals remaining chronically

infected or experiencing multiple exposures to M. alligatoris. If subjected to multiple

exposures, any antibodies remaining from the previous immune response would be

immediately available to bind newly invading pathogens. If sufficient antibody already

existed and cleared the pathogen completely, it is be possible that no secondary

immune response would follow (Murphy et al., 2008). However, if antigen persisted, a

secondary B-cell response would be initiated.

Immunodominance

Though a substantial amount of research is devoted to discovering and

characterizing immunodominant antigens of many medically significant pathogens, the

concept of immunodominance is often interpreted differently among investigators or not

clearly defined. In part, this confusion may be due to the broadness and complexity of

the topic. In an infection, the innate immune response provides an immediate but non-









specific response. However, if a pathogen successfully evades the host's innate

response, the innate immune system must activate the adaptive immune system. Long-

term active immunological memory is acquired after infection by activation of T and B

cells and subsequent production of antigen-specific antibodies. Immunological memory

and specific antibodies formed against certain antigens help the immune system more

effectively identify the pathogen and clear the infection. What is not clear is why the

humoral system mounts a significant response to certain epitopes and not others.

Like other immunogens, an immunodominant antigen contains at least one epitope

that adequately stimulates the immune system to produce specific antibodies.

However, the immunodominant antigen is unique because it is the first of all the

antigens from a given immunogenic source to elicit a detectable humoral response.

This antigen could truly elicit the first response, for example, because it is structurally or

functionally the first to make physical contact host cells. In contrast, it could simply elicit

the strongest response, either due to higher expression levels compared to other

antigens, or due to higher affinity to host immune cells and molecules. Important events

occur in both innate and adaptive immunity before antibodies are produced. Therefore,

factors responsible for immunodominance might take place within the innate system,

adaptive system or both systems.

Cellular Host Responses Affecting Immunodominance

Multiple variables involving the host response are thought to affect the ultimate

outcome of immunodominance of an antigen. These variables include MHC binding

affinity, the formation of stable MHC-peptide complexes on the surface of antigen

presenting cells, efficiency of cellular processing capable of generating the relevant

MHC binding peptides, availability of T-cell receptors capable of recognizing complexes









between the processed MHC binders and MHC and oxidative stress (Yewdell and Del

Val, 2004; Gaddis et al., 2006; Sette and Sundaran, 2006; Yewdell, 2006; Weiskopf et

al., 2010). However, the rules governing all of these processes are still mostly

unknown.

Adaptive host responses depend on major histocompatibility complex (MHC)

molecules. Pathogens express various amino acid sequences that may successfully

bind to MHC-I or MHC-II molecules and provide targets for specific T cells. For

example, the MHC class I binding groove appears to have evolved to bind a low fraction

(approximately 1%) of peptides without evolutionary pressure to avoid or enhance

vertebrate immune recognition (Istrail et al., 2004). Large numbers of host T cell

receptors are distributed clonally on the cell surface to recognize a variety of MHC-

antigen complexes. Despite the large number of potential combinations, pathogen-

specific T cells seem to recognize a relatively small number of epitopes. This apparent

preference, or immunodominance, reflects the final product of innumerable positive and

negative factors that direct antigen presentation and T cell activation (Yewdell and

Bennink, 1999). Furthermore, B cells and T cells must be specific for the same antigen

in order to interact and allow B cell activation, proliferation and differentiation into

antibody-secreting cells (Murphy et al., 2008).

Immunogenic Proteins and Epitopes

Surface proteins and adhesins are commonly regarded as potential

immunodominant antigens among bacterial pathogens. Lipoproteins are considered to

be highly immunogenic due to their surface exposure and amino-terminal lipoylated

structure (Chambaud et al., 1999). The plasma membrane of prokaryotic organisms is

protein-rich and contains many important enzymes, receptors and transporters (Sigler









and Hofer, 1997). The integral membrane proteins of various bacterial pathogens have

been found to be powerful inducers of the immune system, specifically T cells. It is

thought that the acyl chains of conjugated lipids have an adjuvant effect by binding to

the hydrophobic groove of major histocompatibility complex (MHC) molecules,

positioning an epitope for T cell interaction (Deres et al., 1989; Akins et al., 1993; Rees

et al., 1989; Frankenburg et al., 1996). Membrane proteins are also linked to

pathogenesis in other pathogens. For example, a Vibrio vulnificus membrane-bound

lipoprotein, IlpA, functions as an adhesion molecule. It is considered to be a virulence

factor because it can induce cytokine production in human immune cells. An IpA-

deleted V. vulnificus mutant was shown to significantly decreased adherence to human

intestinal epithelial cells, in turn reducing cytotoxicity (Lee et al., 2010).

There is also evidence of immunodominance among other types of bacterial

proteins. For example, many of the immunodominant antigens described in

Mycoplasma mycoides subsp. mycoides small colony type are intracellular enzymes of

intermediary metabolism, not phase-variable surface lipoproteins (Jores et al., 2009).

Some of these are surface-localized and able to bind mucin.

Given the diversity of primary, secondary and tertiary structures, proteins have a

potentially wide range of epitopes. However, the extent of immune recognition does not

reflect this vast pool of potential candidates (Laver et al., 1990). Instead, only certain

epitopes may dominate an infection. For example, immunogenicity of a particular

species within a multi-species infection in a host may be based more on the structural

and biochemical nature of their epitopes rather than how many individual organisms of a

given species exist in the infection. Epitopes can be mapped using protein microarrays,









and with the ELISPOT or ELISA techniques. Various algorithms can be used to predict

immunodominant sites of linear epitopes based on a putative antigen's protein

sequence. Predictions derived by each method should be compared, and only

sequences identified by all three methods should be selected for epitope synthesis for

experimental procedures. Genetic sequences coding for epitopes that are recognized

by common antibodies can be fused to genes, thus aiding further molecular

characterization of the gene product.

The estimated molecular masses of immunodominant M. alligatoris antigens

discovered in previous studies (Brown et al., unpublished) are not consistent with the

predicted sizes of any of the surface-associated glycosidases (165.4 kDa

hyaluronidase, 58.5 kDa sialidase, or 113.7 kDa beta-galactosidase) thought to be

important virulence factors of M. alligatoris. However, the clear immunodominance

especially of the 64 kDa antigen suggests a potential role in host-pathogen interaction in

M. alligatoris mycoplasmosis. Functional studies directed by genome annotation

revealed that M. alligatoris secretes glycosidases, including sialidase, 3-galactosidase

and hyaluronidase (Brown et al., 2004). A combination of virulence factors

unprecedented among mycoplasmas, it is thought that these enzymes liberate glycans

from host extracellular matrix (ECM) and glycosylated cell-surface molecules during

nutrient scavenging and allow invasiveness of M. alligatoris infection.

An antibody response may not always be protective, but it can potentially have

diagnostic value. Since immunodominant antigens elicit the first detectable response in

a host, they can serve as diagnostic markers of disease, especially when a rapid

diagnosis is desired. For example, immunodominant antigens have been identified and









used in diagnostic applications for acute giardiasis and early-onset periodontitis

(Califano et al., 1991; Weiland et al., 2003).

It is not clear if immunodominant antigens have a particularly important role in

protective immunity. In any case, immunization and challenge studies would be

required to validate the protective function of an immunodominant antigen. The answer

may be revealed in the biological function (if any) of the antigen that is to be neutralized

or opsonized by its specific antibodies. For example, antibodies against proteins with

functions in pathogenicity exposed to host cells later in infection may more likely be

protective than those produced against those simply seen by the host immune system

in the initial infection process. Antibodies against proteins that aid in adhesion to host

cells may also be protective since they would theoretically help prevent pathogen

invasion. Though surviving alligators in the M. alligatoris outbreak produced antibodies

against particular proteins, it is currently unknown if they contained any protective

function to the animals.

Antigenic Variation and Immunodominance

Many pathogens, including Mycoplasma spp., Campylobacter fetus,

Pneumocystis carinii, and Giardia lamblia, as well as vector-borne parasites such as

Borrelia spp., Anaplasma and related genera, African trypanosomes, Plasmodium spp.,

and Babesia spp. utilize antigenic variation to evade the immune system. Surface

lipoproteins play an important role in the pathogenesis of some Mycoplasma species by

allowing escape from immune response (Citti and Rosengarten, 1997). It has been

shown that M. arthritidis has an expanded repertoire of variant membrane proteins with

expression subject to independent, reversible phase variation (Droesse et al., 1995).

The complete genomes of other pathogenic bacteria, such as Helicobacter pylori,









Treponema pallidum and Mycobacterium tuberculosis, contain large families of

repeated genes with polymorphic sequences that are possibly involved in antigenic

variation (Vinall et al., 2002; Rindi et al., 2007; Radolf and Desrosiers, 2009).

While immunodominant antigens can be products of antigenic variation, they can

also be highly conserved proteins. For example, the majority of previously defined

immunogenic vaccinia virus epitopes are conserved in variola virus and demonstrate

the efficacy of using vaccinia virus in the smallpox vaccine (Sette et al., 2009). If M.

alligatoris immunodominant antigens are highly conserved, they would serve as

potential vaccine components as well as M. alligatoris mycoplasmosis diagnostic tools.

Previous Studies on the Temporal Antibody Response of Crocodilians to M.
alligatoris

To study the relationship between humoral immunity and progression of disease,

the temporal patterns of antibody responses of American alligators experimentally

inoculated and naturally infected with M. alligatoris were examined. In previous studies,

6 adult alligators were inoculated by instillation into the glottis of 1 mL of broth

containing 106 CFU of M. alligatoris. Four negative control alligators received either

inoculation with sterile broth or no treatment. For comparison, caimans and crocodiles

(n = 6 infected and 2 negative controls of each species) were also inoculated similarly.

Blood samples were obtained at weekly intervals for 4 wk PI, then biweekly, until death

or euthanasia 16 wk PI. M. alligatoris was cultured as early as 1 wk PI from peripheral

blood of 3 inoculated alligators. Plasma from alligators naturally exposed during the

1995 epizootic was collected opportunistically. Western blots of M. alligatoris whole cell

lysate, as well as a fraction enriched for lipid-associated membrane protein antigens,









were probed with plasma from inoculated caimans and crocodiles (Brown et al.,

unpublished).

A positive immune response in individuals of all species surviving the experimental

infection against an immunodominant 64 kDa antigen was detectable as early as 6 wk

PI and increased in intensity until euthanasia 16 wk PI (Figure 1-1A). For some

individuals, a second, approximately 50 kDa antigen evoked a humoral immune

response. No difference in the patterns of humoral responses of caimans, crocodiles,

and alligators could be discerned. Plasma samples were also obtained opportunistically

in 1996, 1997, and 2000 from alligators exposed during the natural epizootic that

occurred in 1995. When M. alligatoris antigens were probed with plasma from these

individuals, the same immunodominant bands as well as many others were revealed

(Figure 1-1B). This result indicated that after the initial acute infection, antibodies were

eventually made against substantially less immunostimulatory antigens. These more

complex humoral responses could be due to these individuals having chronic infections

or subsequent exposures to sublethal doses of M. alligatoris.

There is evidence from previous experiments that the immunodominant 64 kDa

antigen exists in the LAMP-enriched fraction (Brown et al., unpublished). However, as

techniques that enrich for LAMP fractions can still contain soluble protein contamination,

more work would need to be done to establish the protein's cellular location. First, its

protein identity must be determined by peptide sequencing with a subsequent

evaluation for immunodominance of its knockout mutant. Next information on the

identified protein's classification, function and cellular location can be collected.









Objectives and Experimental Approaches

Long Term Goals of Research on M. alligatoris

The long term goal of M. alligatoris research is to improve our understanding of

certain host-pathogen interactions by using the alligator-M. alligatoris model. This goal

involves work to (1) explain relative virulence of M. alligatoris, (2) develop predictive

models of pathogen emergence and evolution, and (3) to gain insight on virulence of

other Mycoplasma species and related organisms.

Infectious diseases represent a substantial global threat to human and animal

health. Infectious diseases of wildlife should not be overlooked, as many newly

recognized infectious diseases stem from zoonotic agents with wildlife reservoirs. In

order to develop new countermeasures such as vaccines and therapeutics, we must

first have a better understanding of the mechanisms of pathogenesis, virulence factors,

and host susceptibility. Genomics and proteomics provide tools to help us better

understand these specialized fields. In turn, we are able to identify new drug targets

and vaccine component candidates and improve diagnostics tools.

Objectives

Some species of Mycoplasma are pathogenic bacteria affecting diverse hosts,

ranging from humans to desert tortoises. Since very little is known regarding M.

alligatoris (e.g. the natural reservoir, routes of transmission and host range of the

pathogen), outbreaks are potentially very difficult to treat or control. Research to date

has shown that crocodilians surviving mycoplasmosis seroconvert around 6 to 8 wk PI,

forming specific antibodies against at least two bacterial components of the LAMP

fraction. The identities of these antigens are currently unknown.









The objectives of this work were to obtain protein identity candidates for the M.

alligatoris 64 kDa immunodominant antigen and to test their respective knockouts for

immunodominant antigen expression. These results may provide a basis for future

work evaluating candidate knockout virulence in infection experiments. Further

characterization such as antigen epitope mapping may facilitate diagnostic and

therapeutic efforts. For example, antigen cloning and purification would be performed to

test potential vaccine or diagnostic test components. Results could serve as a model

for other pathogenic species with similar characteristics.

This work may provide more evidence for specific types of proteins that cause

significant host immunostimulation during the acute infection phase. This information

may help better predict bacterial cell targets of the vertebrate humoral immune system.

Specifically, this could increase our knowledge of the host response of a non-

mammalian system.

Hypotheses

1. Based on previous observations and the general importance of membrane surface
proteins in Mycoplasma pathogenesis, it is hypothesized that the approximately
64kDa-sized immunodominant antigen is associated with the M. alligatoris cell
membrane.

2. The approximately 64 kDa-sized immunodominant antigen is a highly conserved
protein.

Specific Aims

The specific aim of the study was to identify the 64 kDa immunodominant antigen

of M. alligatoris. This could be accomplished through several main approaches:

1. Antigen Isolation. Affinity column chromatography will be used to capture the
antigen that binds to specific antibodies in plasma samples collected 8-16 wk PI
from exposed alligators. Two methods will be utilized. One column will be
covalently bound with monoclonal antibodies against alligator light chain
immunoglobulin, which will capture antibodies from alligator plasma. A small









fraction of the captured antibodies should be selective for the immunodominant
antigen from M. alligatoris cell lysate. In the second method, a column will be
covalently bound with whole seropositive alligator plasma, which will be used to
capture the antigen.

The alternative approach was isolation by 2DGE or BN-PAGE. Western blotting
with plasma collected from infected alligators will be used to confirm isolation of
antigen.

2. Proteomic Analysis. Peptide sequencing was performed on the purified putative
antigen. Tandem mass spectrometry allowed protein identification with further
validation by de novo sequencing. Complete amino acid sequences for the
antigen could be determined from the annotated M. alligatoris genome (GenBank
NZ_ADNC00000000).

3. Confirmation with Knockout Mutants. Mutants (in an existing M. alligatoris
knockout library) corresponding to protein identifications with high confidence
scores would be tested for immunodominant antigen expression by Western blot
probed with infected alligator plasma. A lack of a reactive protein, or a peptide
differing in size from the wild-type M. alligatoris 64 kDa antigen (due to the
presence of the inserted transposon) would confirm its identity.











Caiman Crocodi






1 2 3 4 6 8 10 1216 1 2 3 4 6
M 1 2 3 4 6 8 10 12 16 1 2 3 4 6


C203



-



ohm


Alligator
64 kDa
.- k < 50 kDa


8 10 12 16 1 2 3 4 6 8 10 12 16


B271




I I 64 kDa
S 50 kDa


M 96 97 90 96 00


Figure 1-1. Representative Western blots of M. alligatoris antigens probed with plasma
from crocodilians exposed to M. alligatoris. A) Caimans, crocodiles and
alligators were experimentally exposed to M. alligatoris. Samples were
obtained serially from weeks 1 through 16 post-inoculation (M = molecular
mass standards). B) Samples obtained opportunistically in 1996, 1997, and
2000 from two alligators, "C203" and "B271," that were exposed during the
1995 natural epizootic in St. Augustine, FL (M = molecular mass standards).









CHAPTER 2
MATERIALS AND METHODS

Plasma samples. Alligator plasma samples were obtained during previous M.

alligatoris infection studies and stored at -80 C (Brown et al., 2001b). For all affinity

chromatography columns and Western blots in this current study, plasma from the

following animals were used: accession number AA165099 collected 8 wks and 16 wk

post-inoculation (PI), AA165089 collected 10 wk PI and AA165094 collected 10 wk PI.

These individuals received 106 CFU of M. alligatoris (ATTC 700619, specimen

A21JP2T, first passage isolate of pure colony) by instillation through the glottis and were

euthanized at the end of the study 16 wk PI. Plasma samples used in this study were

ELISA positive against M. alligatoris whole cell lysate and shown to detect the 64 kDa

band in preliminary experiments. Plasma samples were diluted 1:100 in 1x phosphate

buffered saline (PBS) for all experiments.

Mycoplasma culture. Wild-type M. alligatoris previously frozen at -20 C was

cultured at 30 C in ATCC medium 988 SP4 containing 0.5% (wt/vol) glucose and 20%

(vol/vol) fetal bovine serum to the early log phase, as detected by acidification of the

medium. To verify a MS protein identification, phosphopentomutase knockout and Nan

A (irrelevant knockout) mutant cultures were grown in SP4 medium was cultured to the

early log phase, as detected by acidification of the medium, at 30 C in SP4 broth

containing 0.5% (wt/vol) glucose and 20% (vol/vol) fetal bovine serum, supplemented

with 10 mg/mL tetracycline.

Knockout mutant library construction. A large-scale transposon mutagenesis

library of tetR-selected M. alligatoris clones was previously generated by methods

identical to Dybvig et al. (2008). After transformation by electroporation, mutagenization









by random transposon insertion was performed using plasmid pTF20 carrying the

minitransposon Tn4001TF2. Approximately 2,000 individual colonies were expanded

and filter-cloned to assure homogeneity. Unique single-insertion knockout mutations

have been obtained for approximately 650 of 950 clones precisely mapped by direct

genomic DNA sequencing in both directions from the point of insertion, via extension

from primers complementary to each IS256 arm of the transposon.

Affinity Chromatography Antigen Purification

Affinity columns. HiTrap NHS-activated HP columns (GE Healthcare Life

Sciences, Piscataway, NJ) were prepared according to manufacturer's instructions.

The columns allow covalent coupling of ligands containing primary amino groups. The

medium is based on SepharoseTM (highly cross-linked agarose) beads with 6 atoms

spacer arms attached to the matrix by epichlorohydrine and activated by N-

hydroxysuccinimide. The substitution level is = 10 pmol NHS groups/mL medium.

Nonspecific adsorption of proteins to HiTrap columns is negligible due to the hydrophilic

properties of the base matrix. Three different affinity column configurations as described

below were tested. After each elution, the column was washed 6 times with 1 mL of 1x

PBS to clean and neutralize the column, sealed and stored at 4 OC.

Whole cell lysate preparation. Whole cell lysate used in the affinity

chromatography column (first configuration) was previously prepared. Washed wild-

type M. alligatoris pellets were disrupted with lysis buffer containing NaHCO3 and

Na2CO3 (pH 10.0), and samples were diluted to 100 mg protein/mL with 2.2% boric acid

(Horowitz and Cassell, 1978). Aliquots were stored at -80 C until use in an affinity

column.









Lipid associated membrane protein (LAMP) antigen preparation. LAMP

antigen used in the affinity chromatography columns (second and third configurations)

was previously prepared (Wang and Lo, 1996). Cells were lysed in ice-cold 50 mM

Tris-HCI buffer (pH 8.0), 0.15 M NaCI, 1 mM EDTA containing Triton X-114 with

vortexing and sonication. The NaCI concentration was adjusted to 0.5 M, followed by

centrifugation at 20,840 g for 20 min at 5 oC to remove insoluble materials. Triton X-114

phase fractionation was performed by incubating lysates at 37 OC, inducing

condensation of the detergent. Cloudy and partially biphasic appearing lysates were

then centrifuged at 5,200 g for 10 min at 30 OC. After removing aqueous supernatants,

the detergent-enriched phase was mixed and stored in aliquots at -80 OC until use in

affinity columns.

First Column Configuration

In the first configuration, a 1 mL column was used to couple 8 wk PI plasma from

alligator AA165099 for 6 hours at 4 OC. After the coupling procedure, the remaining

coupling sites of the column were deactivated and washed. The column was then

incubated with 10 mg wild-type M. alligatoris whole cell lysate for 12 hr. After washing

with 1x PBS, 15 consecutive 200 uL-fractions were eluted with 100 mM glycine HCI (pH

3.0). The fractions were immediately neutralized with 100 mM TRIS buffer (pH 9.0)

during the collection process (final pH approximately 7.0). Fractions were then pooled

into 3 groups based on the order of their elution and microconcentrated with 30 kDa

size exclusion centrifugal filters (Millipore, Billerica, MA) in 50 mM TRIS buffer (pH 6.8).









Second Column Configuration

Protein G-purified monoclonal mouse (subclass IgG2b kappa) anti-alligator IgY

light chain (HL1740 5H1-3B6) was previously generated at the ICBR Hybridoma Core

Lab, Cancer Genetics Research Complex, University of Florida, Gainesville, FL. In the

second configuration, a 1 mL column was used to couple 1.25 mg mouse anti-alligator

light chain for 16 hr. Coupling was validated as described below. After the coupling

procedure, the column was deactivated and washed, and 8 wk PI plasma from alligator

AA165099 was injected into the column and incubated for 12 hr at 4 OC. The column

was washed with 1x PBS and then incubated with 10 mg wild-type M. alligatoris whole

cell lysate for 12 hr. After additional washing with 1x PBS, 15 consecutive 200-uL

fractions were collected. The fractions were immediately neutralized, pooled into 3

groups and microconcentrated as previously described. After being coupled with the

monoclonal antibody, the affinity column was used for several replicate binding and

elution experiments to see if the column could be reused.

Third Column Configuration

In the third configuration, a 5 mL column was used. To optimize contact with

binding sites, samples were recirculated through the column with a variable flow

peristaltic pump (Fisher Scientific, Pittsburgh, PA) and silicone capillary tubing. Eight

wk PI plasma from alligator AA165099 was coupled during 12 hr of circulation at 4 OC.

The column was washed with 1x PBS and 3.8 mg LAMP antigen was recirculated for 12

hr. After washing with 1x PBS, 15 consecutive 1 mL-fractions were collected. The

fractions were immediately neutralized, pooled into 3 groups and microconcentrated as

previously described.









SDS-PAGE Separation of Affinity Chromatography Elutions

After microconcentration, pooled elution samples were prepared in 2x Laemmli

sample buffer with 350 mM DTT added fresh (Laemmli, 1970) or 4x NuPage LDS

sample buffer (Invitrogen Life Technologies, Carlsbad, CA) and separated by SDS-

PAGE on 7.5% Tris-HCI Ready gels at 70 V along with pre-stained protein standards.

Second Affinity Column Monoclonal Antibody Coupling Validation

The following validation was performed to confirm that mouse monoclonal

antibodies were covalently bound to the stationary-phase beads in the second affinity

column configuration. After two 1 mL-1x PBS washes, the column was injected with 1

mL of alkaline phosphatase conjugate goat anti-mouse IgG (H+L) (Promega

Corporation, Madison, WI) diluted 1:100 in 1x PBS, sealed and incubated for about 20

hr at 4 OC. After incubation, the column was washed 6 times with 1 mL 1x PBS. Twenty

consecutive 2-drop (approximately 80 uL) fractions were collected starting with the first

wash, and another series of 20 consecutive 2-drop fractions were collected starting with

the last wash. Next, forty consecutive 2-drop fractions were collected as soon as the

first 400 uL of the elution buffer was injected into the column. Next, 50 uL-aliquots of

each fraction (first washes, final washes, elutions, positive controls, and negative

control) were transferred to wells of a 96-well microtiter plate in the same order of their

collection from the column. The anti-mouse IgG (H+L) alkaline phosphatase conjugate

was used in the plate at four dilutions (1:100, 1:500, 1:1000, and 1:5000) for positive

controls. The negative control was 1x PBS. The plate was sealed and stored at 4 C

overnight. After rinsing the plate with 1x PBS three times, 0.1 mg substrate (4-

nitrophenyl phosphate disodium salt hexahydrate) was added to each well. Presence of









antibody was determined by color reaction and absorbance readings of the plate wells

at 405 nm.

The remaining fraction volumes were then pooled intro groups (first washes, final

washes, first elutions and final elutions), prepared in 4x NuPage LDS sample buffer plus

350 mM DTT and separated by SDS-PAGE along with a positive control on a precast

7.5% polyacrylamide Tris-HCI Ready gel (Bio-Rad Laboratories, Hercules, CA) at 70 V

along with pre-stained protein standards (Bio-Rad Laboratories). The gel was prepared

and subjected to silver staining with the SilverSNAP Stain Kit II (ThermoScientific

Pierce, Rockford, IL). If the coupling procedure was successful, the expected outcome

was to see at least one band at a molecular weight corresponding to structural

components of the goat anti-mouse IgG molecule.

2-Dimensional PAGE Antigen Separation

Membrane Protein Preparation. Wild-type M. alligatoris native membrane

protein was extracted from approximately 100 mg of 1x PBS-washed pelleted cells (wet

weight) with the proprietary components of the ProteoExtract Native Membrane Protein

Extraction Kit (Calbiochem, EMD Chemicals Inc., La Jolla, CA) according to

manufacturer instructions. The purpose of using this kit was to extract integral

membrane and membrane-associated proteins based on association of proteins with

cellular membranes rather than on their hydrophobicity. It was not used specifically to

preserve the native state of the extracted proteins, as they were later treated under

denaturing conditions in preparation for and during 2DGE. Approximately 50 U

Benzonase (Novagen, EMD Chemicals Inc.), a broad-spectrum endonuclease, was

added to each approximately 100 mg cell pellet (wet weight) during the protein

extraction protocol, directly after addition of the protease inhibitor cocktail provided in









the kit. The purpose of adding an endonuclease was to degrade and remove nucleic

acids, thus reducing sample viscosity. Samples were incubated at room temperature

for an additional 30 minutes with gentle shaking. Protein concentrations were quantified

with Bradford or DC assays (Bio-Rad Laboratories). After overnight precipitation with

100% ice-cold acetone, samples were centrifuged at 14,000 g for 15 min at 4 OC,

supernatant was removed and pellets were dried by vacuum centrifugation for 2-3 min

immediately before rehydration and isoelectric focusing.

Total Protein Preparation. Total protein was extracted by two different methods.

In the first method, wild-type M. alligatoris total protein was extracted by adding 1 mL

radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCI, 1.0% IGEPAL CA-630,

0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) (Sigma-Aldrich Co., St.

Louis, MO) and 10 uL Halt Protease Inhibitor Single-Use Cocktail (ThermoScientific

Pierce) to each approximately 100 mg (wet weight) 1x PBS-washed cell pellet.

Samples were incubated at 4 OC for 30 minutes with gentle shaking and then

centrifuged at 14,000 g for 20 min at 4 OC. Protein concentrations were quantified by

Bradford or DC assays, precipitated with ice-cold 100% acetone and resuspended to

0.5-1 ug/uL in 2-D Rehydration/Sample Buffer (Bio-Rad Laboratories) containing 7 M

urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer (pH 3-10), 0.04 M DTT, and 0.002%

Bromophenol Blue) immediately before rehydration and isoelectric focusing.

The ReadyPrepTM total protein extraction kit (Bio-Rad Laboratories) was used

according to manufacturer instructions for the second extraction method. First, wild-

type M. alligatoris cell pellets of approximately 100 mg (wet weight) were washed with

ice cold 50 mM Tris buffer (pH 6.8) and centrifuged at 14,000 g for 30 min at 4 OC. After









removal of supernatant, the cell pellets were incubated in 50 mM Tris buffer (pH 6.8), 1x

Halt protease inhibitor cocktail and 50 U Benzonase at 4 C for 1 hr with gentle shaking.

After incubation, samples were centrifuged again and the supernatant was removed.

To each pellet, 1 mL of complete 2-D Rehydration/Sample Buffer with 0.2% (w/v) IPG

Buffer pH 3-10 NL ampholyte (GE Healthcare Life Sciences) was added as well as an

additional 1x Halt protease inhibitor cocktail and 50 U Benzonase. The complete 2-D

Rehydration/Sample Buffer contains 7M urea, 2M thiourea, 1% (w/v) ASB-14 detergent,

40 mM Tris base, 0.001% Bromophenol Blue, and TBP reducing agent. The samples

were chilled on ice and sonicated with an ultrasonic probe with 30 sec bursts 3 times,

remaining on ice for 30 sec between each sonication treatment. Samples were then

centrifuged at 14,000 g for 30 min at 18 C to pellet cellular debris. Supernatant was

transferred to a clean tube and the insoluble pellet was discarded. Protein samples

were quantified with CB-X Protein assays (G-Biosciences, Maryland Heights, MO) and

diluted in more complete 2-D Rehydration/Sample Buffer 1 and 0.2% (w/v) IPG Buffer

pH 3-10 NL ampholyte to obtain appropriate concentrations for isoelectric focusing (IEF)

as described below.

Active Rehydration

Protein samples (200, 300 or 500 ug) were loaded onto 11 cm, pH 4-7 linear

gradient immobilized pH gradient (IPG) strips (Bio-Rad Laboratories). The proteins

were subjected to active rehydration and resolved in the first dimension in a PROTEAN

IEF apparatus (Bio-Rad Laboratories). Active rehydration of the IPG gel strip was first

carried out at 50 V for 8 hr. Active rehydration allows a more complete uptake of the

protein in the sample into the IPG (Kinter and Sherman, 2000). The process of active









rehydration occurs most effectively with rehydration buffer containing the sample to be

separated, allowing the proteins to enter the gel along its entire length. As a result,

more proteins are detected than in experiments with passive rehydration.

Isoelectric Focusing (IEF)

Active rehydration was immediately followed by IEF in the PROTEAN IEF

apparatus. This procedure separates proteins according to their electric charge

differences along a pH gradient. Each protein migrates until it reaches a point in the pH

gradient corresponding to its isoelectric point (pl), the pH at which a molecule has a

zero net charge. An anode "positive" end and cathode "negative" end are created by

passing an electric current through the ampholyte medium (sample buffer), causing

proteins above their pl, which have a negative net charge, to migrate toward the anode.

Likewise, proteins below their pl, which have a positive net charge, migrate toward the

cathode. IEF was performed under the following conditions: a linear increase of voltage

to 250 V for 30 min, a linear increase to 500 for 1 hr, a linear increase 8000 V for 2.5 hr,

a rapid increase to 8000 V for 40,000 Vhr, followed by a decrease to 50 V for 20 hr.

After IEF, the strips were equilibrated in a solution of 50 mM Tris pH 8.8, 6 M urea, 30%

glycerol, 2% SDS, 0.002% bromophenol blue, and 1% DTT, followed by a second

equilibration solution of 2.5% iodoacetamide, each for 15 min.

Second Dimensional SDS-PAGE

Electrophoresis in the second dimension was performed on 8-16% Tris-Bis

Criterion gels (Bio-Rad Laboratories) in 1x SDS running buffer at 60 V for 30 min, then

120 V until the loading dye front reached the gel bottom. For gels of membrane

proteins, a small gel plug containing approximately 20 ug wild-type M. alligatoris total

protein was inserted in the gel just prior to electrophoresis to be used as a reference









along with the molecular weight marker. The total protein for the gel plug was extracted

with RIPA Buffer plus Halt protease inhibitor as described above, then mixed with an

equal volume of 2% agarose and 0.001% Coomassie Brilliant Blue R-250 powder (Bio-

Rad Laboratories).

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE)

BN-PAGE. BN-PAGE allows isolation of protein complexes from biological

membranes and total cell and tissue homogenates without disrupting their native state.

The anionic dye Coomassie blue G-250 has hydrophobic properties, allowing it to

interact with the surface of membrane proteins. Importantly, interacting with the dye

makes membrane proteins water-soluble while preserving their native structure. Thus,

no detergent is necessary to solubilize samples. In addition, binding the dye molecules

imparts a negative charge on all proteins, making membrane proteins less likely to

aggregate (Wittig et al., 2006).

Wild-type M. alligatoris native membrane proteins were extracted with

ProteoExtract Native Membrane Protein Extraction Kit as described above, or with the

NativePAGETM Sample Prep Kit (Invitrogen Life Technologies) according to

manufacturer instructions. Protein concentrations were quantified with DC assays.

Samples extracted by both methods were separated by BN-PAGE on a 4-16% gradient

NativePAGE Novex Bis Tris gel using amperage as low as 1 mA. Duplicate gels were

stained and subjected to Western blotting as described below.

Western Blotting

For each set of samples analyzed by Western blotting, one gel was stained after

PAGE with standard Coomassie Blue stain (Merril, 1990), Bio-SafeTM Coomassie Stain

(Bio-Rad Laboratories), or Colloidal Blue Staining Kit (Invitrogen Life Technologies), and









a duplicate gel was electroblotted onto a 0.2 um nitrocellulose or polyvinylidene fluoride

(PVDF) membrane. Transfers were performed at 15 V for 30 min on a Semi-Dry

Transfer Cell Transblot SD (Bio-Rad Laboratories) with 10% methanol 0.01% SDS

transfer buffer, or at 100 V for 3 hr by wet transfer with 2-[N-morpholino]ethanesulfonic

acid (Sigma-Aldrich Co.) plus 0.01% SDS transfer buffer. The membranes were

blocked with 5% nonfat dried milk in PBS-0.2% sodium azide (PBS/A)-0.5% or

SuperBlock (ThermoScientific Pierce) for 1 hr at 25 C or overnight at 4 C, then

incubated with either AA165099 8 wk PI or pooled alligator plasma 25 C for 1 hr.

Previously prepared (Brown et al., 2001) biotinylated polyclonal rabbit anti-alligator

immunoglobulins (Ig) light chain and heavy chain antibodies were used for detecting

bound alligator in Western blots. Bound specific antibodies were detected by incubation

with biotinylated polyclonal rabbit anti-alligator Ig. Membranes were incubated in

alkaline phosphatase-conjugated streptavidin diluted 1:1,000 in 1x PBS. The blots were

developed in 1 Step NBT/BCIP (ThermoScientific Pierce) until bands became visible.

To evaluate the validity of the elution samples from the third affinity column, a

secondary antibody-only control was performed. Affinity column elution samples were

prepared in 5x lane marker sample buffer (ThermoScientific Pierce) and separated by

SDS-PAGE on a 7.5% Tris-HCI Ready gel. After electroblotting and blocking, the PVDF

membrane was incubated with biotinylated secondary antibodies for 1 hr at 25 C, and

then developed as described above. Membranes were washed with 1x PBS-0.05%

Tween 20 three times for 5 min in between all steps.

In some transfer experiments, a second PVDF membrane was included in the gel-

membrane-blot paper sandwich, where it was placed directly on top of the gel. This top









membrane was subjected to Colloidal Gold Total Protein Stain (Bio-Rad Laboratories)

after electroblotting according to manufacturer's instructions to obtain the protein pattern

of the gel. The gold staining was discontinued after appearing unsuccessful.

Phosphopentomutase Knockout Mutant Evaluation

To demonstrate the approach to confirming immunodominant antigen candidates

identified by other methods, total protein was extracted from a M. alligatoris

phosphopentomutase knockout mutant. The control was an irrelevant M. alligatoris

sialic acid lyase knockout mutant (NanA). Approximately 100 mg (wet weight) PBS-

washed cell pellets were briefly boiled in 50 mM TRIS buffer, 2x Laemmli sample buffer

and 0.1 % Triton-X 100. Samples were kept on ice until separation by SDS-PAGE on a

7.5% Tris HCI Ready gel. Approximately 10 ug of protein was loaded per lane. One gel

half was electroblotted to a PVDF membrane and subjected to Western blotting with

high infection alligator plasma as previously described. The other gel half, containing

sample duplicates, was stained with Coomassie blue.

Peptide Sequencing and Protein Identification

Protein Reduction, Alkylation and Enzymatic Digestion

Proteins of interest were excised from gels based on corresponding Western

blotting results and submitted for tandem mass spectrometry. Proteins selected for

analysis were from eluted LAMP antigen (third affinity column configuration) separated

by SDS-PAGE as well as total protein and membrane protein 2DGE separation. Tryptic

peptides were prepared by ICBR Proteomics Core, University of Florida, Gainesville,

FL. for LC-MS/MS analysis by the following method. To each excised gel spot, 200 mL

of 50 mM NH4HCO3 in 50% v/v acetonitrile was added and the sample was vortexed.

The gel particles were brought to complete dryness with a vacuum centrifuge. Freshly









prepared 45 mM DTT solution was added to the gel pieces for 45 min at 55 C. The

DTT solution was removed and an equal volume of freshly prepared 100 mM

iodoacetamide was added, followed by 45 min incubation at room temperature. The

iodoacetamide solution was removed and the gel pieces were washed with 200 mL of

50 mM NH4HCO3 in 50% v/v acetonitrile (pH 8.4) for 30 min while vortexing. The wash

procedure was repeated three times. The gel particles were again brought to complete

dryness with a vacuum centrifuge.

Approximately 5-10 uL of 12.5 ng/uL trypsin (Promega Corporation) in 25 mM

NH4HCO3 (pH 8.4) was added to each gel piece and kept at 4 C for 30 min. The

enzyme solution as replaced with enough 25 mM NH4HCO3 (pH 8.4) to cover the gel

particles and incubated for 12-16 hr at 37 C. The reaction was stopped by adding 5%

aqueous acetic acid to a final concentration of 0.5% acetic acid. After shaking and

centrifugation, supernatant containing tryptic peptides was transferred to clean 0.5 mL

tubes. The original gel pieces were treated with an additional 50 pL of acetonitrile,

agitated by sonication or gentle vortexing for 10 min, and centrifuged to obtain

additional supernatant containing tryptic peptides. The pooled extracted peptides were

completely dried with a vacuum centrifuge. To each sample, 15 pL of loading buffer

(3% acetonitrile [ACN], 1% acetic acid, and 0.1 % trifluoroacetic acid [TFA]) was added,

followed by agitation by brief sonication or vortexing. For comparison, a sample was

also separately digested with endoproteinase AspN (ThermoScientific Pierce) according

to manufacturer's instructions to produce different peptide fragments due to cleavage at

N-terminally at aspartic acid and cysteic acid.









LC-MS/MS

Tandem mass spectrometry was performed with a nanoelectrospray ionization

hybrid quadrupole time-of-flight mass spectrometer at the ICBR Proteomics Core,

University of Florida, Gainesville, FL.

The enzymatically-digested samples were concentrated and desalted onto a

capillary trap, an Acclaim a PepMap [L-PrecolumnTM cartridge (Dionex, Sunnyvale, CA),

with 0.1% v/v acetic acid for 5 min with a flow rate of 10 pL/min. Samples were next

loaded onto an Acclaim PepMapl00 Nano-HPLC column (Dionex) for reversed-phase

chromatography. Peptides were eluted over a linear gradient from the column starting

at 3% solvent A, 97% solvent B and finished at 60% solvent A, 40% solvent B for 60.

Solvent A consisted of 0.1% v/v acetic acid, 3% v/v ACN, and 96.9% v/v H20. Solvent

B consisted of 0.1% v/v acetic acid, 96.9% v/v ACN, and 3% v/v H20. LC-MS/MS

analysis was carried out on a QSTAR XL or QSTAR Elite (Applied Biosystems,

Framingham, MA) mass spectrometer. The focusing potential and ion spray voltage

was set to 275 V and 2600 V, respectively. The information-dependent acquisition

(IDA) mode of operation was employed, in which a survey scan from m/z 400-1200 was

acquired followed by collision-induced dissociation of the three most intense ions.

Survey and MS/MS spectra for each IDA cycle were accumulated for 1 and 3 s,

respectively.

Database Searching

LC-MS/MS spectra were extracted, deisotoping was performed, and charge states

were deconvoluted by ABI Analyst version 1.1 or 2.0 software. All LC-MS/MS data

were submitted for database searching using Mascot (Matrix Science, London, UK;

version 2.0.01). A second, more stringent analysis was performed with Scaffold 2









(version Scaffold-02-03-01, Proteome Software Inc., Portland, OR) software, which

uses the two algorithms Mascot and X! Tandem. Only results in Scaffold 2 were

examined due to higher stringency. However, all data had to be first submitted to

Mascot before they could be run in Scaffold 2.

Scaffold 2. Scaffold 2 identifies proteins from MS/MS data by comparing it to a

database of known proteins and validating with Mascot*/Sequest*/X! Tandem*/Phenyx*

searches. Sequence coverage is automatically calculated within the software. Protein

identification results are displayed as probability scores, allowing you to balance the

number of proteins and the confidence level of each identification. The algorithm for

calculating the peptide probabilities from the search engine scores is described by

Keller et al. (2002). The algorithm for calculating the protein probabilities from the

peptide probabilities is described in Nesvizhskii et al. (2003). The algorithm for

combining results from multiple searches is described by Searle et al. (2008). The

algorithm for grouping proteins across samples is described by Searle (2010).

Mascot. Mascot is a software package that converts mass spectral data into

protein identities. Mascot compares the observed spectra to a database of known

proteins and determines the most likely matches. Each match is then assigned a P-

value equaling the likelihood that the match is incorrect (E~P-N). Mascot is also one of

the algorithms used in the software package Scaffold 2.

X! Tandem. X! Tandem, the second algorithm in Scaffold 2, is by itself an open-

source software package that matches the acquired MS/MS spectra to a model

spectrum based on peptides in a specified protein database. Each match is assigned

an E-value equaling the likelihood that the match is incorrect (E~P-N). The model









spectrum is based on the presence or absence of y and b ions. The program considers

semi-tryptic peptides, sequence polymorphisms and uses probability-based scoring. It

automatically searches for missed cleavages, semi-tryptic peptides, post-translational

modifications, and point mutations. The algorithm for X! Tandem is described in Craig

and Beavis (2003).

An analysis was performed on eluted antigen samples with the anole (Anolis

carolinensis) protein database available in GenBank (NC_010972) in Mascot and

Scaffold 2. To date, no other reptilian genome was more complete. The purpose of this

analysis was to evaluate the unintended possibility of the eluted antigen being part of

the alligator immunoglobulin molecule instead of the desired M. alligatoris antigen. This

database, containing only 328 proteins, was chosen because of the 276 American

alligator (A. mississippiensis) protein sequences currently available through GenBank,

none were immunoglobulin-associated.

Trypsin or AspN enzymes were specified in Mascot MS/MS ion searches and

Scaffold 2 searches. Mascot settings included a fragment ion mass tolerance of 0.30

Da and a parent ion tolerance of 0.30 Da. lodoacetamide derivative of Cys,

deamidation of Asn and Gin, oxidation of Met, were specified in Mascot as variable

modifications.

In Scaffold 2, Mascot and X! Tandem were searched with a fragment ion mass

tolerance of 0.50 Da and a parent ion tolerance of 0.50 Da. lodoacetamide derivative of

cysteine was specified in Mascot and X! Tandem as a fixed modification. S-

carbamoylmethylcysteine cyclization (N-terminus) of the n-terminus, deamidation of

asparagine and oxidation of methionine were specified in Mascot as variable









modifications. S-carbamoylmethylcysteine cyclization (N-terminus) of the n-terminus,

deamidation of glutamine, oxidation of methionine and iodoacetamide derivative of

cysteine were specified in X! Tandem as variable modifications. Peptide identifications

were accepted if they could be established at greater than 95.0% probability as

specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications

were accepted if they could be established at greater than 99.0% probability and

contained at least 2 identified unique peptides. Protein probabilities were assigned by

the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar

peptides and could not be differentiated based on MS/MS analysis alone were grouped

to satisfy the principles of parsimony.

De Novo Sequencing Protein Identifications

LC-MS/MS spectra were extracted, deisotoping was performed, and charge

states were deconvoluted by ABI Analyst version 1.1 or 2.0 software. Data were

converted to XML format and submitted for de novo sequencing using PEAKS Studio

5.1 (Bioinformatics Software, Inc.)

PEAKS Studio 5.1. De novo sequencing is a process in which peptide

sequences are derived from the masses of their fragments as shown on a tandem mass

spectrum without the use of a protein sequence database for reference. This process

minimizes false positives, and when combined with database protein identification,

offers optimal result validation. PEAKS Studio is a tandem MS program with

applications to determine protein/peptide sequences and respective quantitative

properties. One major beneficial feature is that confidence is not only on the peptide

sequence, but also on each amino acid assignment. Fragmentation flexibility includes

y/b ions or c/z ions. The program also has capabilities to perform an internal database









search engine, meta protein identification (inChorus) tool, peptide sequence tag

homology search program/sequence reconstruction method (SPIDER), and post

translational modification identification and quantification. According to the PEAKS

manufacturer, the algorithm first computes a y-ion matching score and a b-ion matching

score at each mass value according to the peaks around it. If there are no peaks

around a mass value, a penalty value is assigned. The algorithm then efficiently

computes many amino acid sequences that maximize the total scores at the mass

values of b-ions and y-ions. These candidate sequences are further evaluated by a

more accurate scoring function, which also considers other ion types such as immonium

ions and internal-cleavage ions. The problem of ion absence is addressed because the

PEAKS model assigns a score (or penalty) for each mass value. The software also

computes a "positional confidence" for each amino acid in the final result by examining

the consensus of the top-scoring peptides.

PEAKS Criteria Settings

PEAKS data refinement included deisotoping, deconvolution and centroiding.

Error tolerance parameters included 0.5 Da for parent ion and 0.5 Da for fragment ion.

Deamidation, oxidation, pyro-glu from Q, and carbamidomethylation post translational

modification tolerances were selected. Only results with an ALC (%) score of at least

80 were reported or further evaluated.









CHAPTER 3
RESULTS AND DISCUSSION

Affinity Columns

The first affinity column configuration had alligator plasma directly coupled to its

SepharoseTM beads and was then incubated with wild-type M. alligatoris whole cell

lysate antigen. After multiple attempts to optimize elution, sample solubilization, and

SDS-PAGE resolution, protein was visualized on a 7.5% Tris-HCI gel by Coomassie

blue staining. Very faint bands on the gel corresponding to approximately 64 kDa, 250

kDa, and >250 kDa were seen (not shown). However, Western blots were not

performed on these elution samples, and thus immunodominance of the 64 kDa protein

could not be determined. The very faint stain intensity of the 64 kDa band approached

the minimum protein concentration detectable by basic Coomassie blue staining, or 50-

100 ng/mm3 gel volume (Weiss et al., 2009). Nanoelectrospray ionization experiments

on a QSTAR XL MS/MS system have shown that 1-2 fmol of sample digest already in

HPLC solvent is sufficient for protein identification concentration, but LC-MS/MS results

are improved with higher concentrations (Chen, 2006). With in-gel digestions, there is

significant peptide loss from the gel matrix during sample preparation (Chen, 2006).

Though sequencing may have still worked with the low protein concentration of the

given sample, efforts were instead made to increase affinity column elution

concentrations before attempting LC-MS/MS.

It is not known why multiple bands were seen in the separated elution samples.

After alligator plasma coupling in the first column, PBS washes were collected and

separated by SDS-PAGE. The last PBS washes, right before elution, were confirmed to

be protein-free by SDS-PAGE separation and Coomassie blue staining. Therefore, it









was thought that all non-specific binding of M. alligatoris proteins were removed prior to

elution. However, it is possible that non-specific material coincidentally containing a 64

kDa protein was retained in the column until the elution process. For example, the IgY

heavy chain has a molecular mass of 65-70 kDa (Hatta et al., 1993; Sun et al., 2001).

The 64 kDa protein may have been part of the alligator immunoglobulin molecule or any

other plasma protein of a similar molecular weight that leached from the stationary

phase. Molecular weight alone would not strongly indicate that the larger (250 kDa and

>250 kDa) bands from the first affinity column elutions were from residual

immunoglobulins. The whole IgY molecule has been estimated to be approximately 180

kDa by SDS-PAGE and approximately 167 kDa by MALDI-TOF (Sun et al., 2001; Hatta

et al., 1993). Formations of protein dimers, protein modifications resulting in increased

molecular weight, or problems such as poor sample solubility and resolution may have

also affected the protein separation patterns on the gels.

The second affinity column configuration had mouse monoclonal anti-alligator IgY

light chain coupled directly to its beads. The column was then incubated with alligator

plasma, followed by incubation with wild-type M. alligatoris LAMP antigen. The coupling

validation study demonstrated that the monoclonal mouse anti-alligator IgY light chain

did indeed covalently bind to the column's beads (Figure 3-1). However, a lack of

protein staining on SDS-PAGE-separated elutions indicated unsuccessful antigen

capture. It is possible that the antibodies in the column were unable to bind antigen.

For example, once alligator Igs were bound to the monoclonal antibodies by their light

chains, there may have been a physical barrier preventing specific antigens access to

their respective antibody binding sites. Another likely issue was simply poor antigen









enrichment. For example, perhaps too few M. alligatoris antigen-specific IgY molecules

were able to bind to the monoclonal antibodies simply due to naturally low

concentrations relative to other plasma proteins.

Maximum binding efficiency would be necessary for all of the antibodies to be

captured by the coupled mouse monoclonal antibodies. All of these factors would result

in a relatively low number of antigen-specific immunoglobulins in the second column

configuration. Assuming that their orientations in the column would allow successful

antigen capture, this configuration would still only capture a very small total amount of

antigen. Without any previous knowledge of the immunodominant antigen identity, there

was no accurate way to predict its relative abundance within a certain volume of LAMP

or whole cell lysate. Lacking this knowledge, it is uncertain that the column was

incubated with the ideal amount of antigen material.

IgY concentrations, including those specific for M. alligatoris antigens, were not

known for the alligator plasma samples used in the affinity columns. Though plasma

concentrations of immunoglobulin isotypes are not well documented in reptiles,

predictions regarding alligator antigen-specific IgY plasma concentrations can be made

from other animal models. Total protein values, including globulin values, were

measured for the alligator plasma samples as part of a comprehensive biochemistry

profile. Most of the alligators, including both normal and infected animals, had total

protein and globulin values within the normal ranges for A. mississippiensis: 5.31.3

g/dL and 3.51.0 g/dL, respectively. These A. mississippiensis reference values are

also relatively similar to normal total protein and globulin values observed in mammals

(Diethelm, 2005; Diethelm and Stein, 2006). Total IgG levels can be measured by rate









nephelometry. For example, serum levels of IgG, comprising part of the globulin

fraction, have been determined in healthy humans (600-1,400 mg/dL), mares

(1,352.91 mg/dL), and dogs (1,560 116 mg/dL) (O'Reilly, 1993; Ginel et al., 1997;

Murphy et al., 2008). IgG subclass concentrations can be determined by

immunoradiometric assay (IRMA) using monoclonal antibodies specific to individual

subclasses of IgG conjugated to polyglutamated-carboxymethyl sephacryl beads. For

example, IgG1, IgG2, IgG3, and IgG4 subclasses range at about 300-7,000 mg/L in

healthy humans, and all subclasses can increase to varying degrees during processes

such as chronic schistosomiasis (Boctor and Peter, 1990). In diseased humans, the

same IgG subclasses ranged from 1,400-15,000 mg/L (Boctor and Peter, 1990). These

values might be used to predict upper limits for IgY subclass concentrations in healthy

or infected alligators. Specific antibody titers after infection are generally lower in

reptiles compared to mammals or birds, and reptilian secondary responses rarely have

antibody titers and affinities higher than in primary responses (Hsu, 1998; Brown, 2002).

In addition, only a small percentage of these circulating antibodies might be specific for

a single antigen, for example, if multiple antigens are stimulating the host immune

system at the same time.

The third affinity column configuration was similar to the first configuration in that

alligator plasma was directly coupled to its beads. However, this column was five times

greater in volume, and like the second configuration, wild-type M. alligatoris LAMP was

used as a source of antigen. Both the alligator plasma and antigen were recirculated

with a peristaltic pump to increase overall binding. A protein band from this source

(pooled elution aliquots) was submitted for LC-MS/MS three separate times, each with









slight differences in its treatment. The first submission was a 64 kDa band previously

subjected to SDS-PAGE separation on a 7.5% Tris-HCI gel and detected by Coomassie

blue staining (Figure 3-2A), subjected to in-gel trypsin digestion prior to LC-MS/MS.

The protein band was immunoreactive with alligator plasma by Western blot (Figure 3-

2B). The second submission was similarly treated and separated by SDS-PAGE, but

subjected to in-gel AspN (flavastacin) digestion and LC-MS/MS. This restriction

enzyme, a zinc metalloendopeptidase, selectively cleaves peptide bonds N-terminal to

aspartic acid residues (Tarentino et al., 1995). The purpose of using a different

restriction enzyme was to increase the number and types of peptide fragments available

for LC-MS/MS analysis. Because fixation with methanol and acetic acid can sometimes

interfere with enzymatic digestion of the protein, Bio-SafeTM Coomassie stain was used

as a fixative-free stain alternative for other sample aliquots. Therefore, the third

submission for LC-MS/MS was a 64 kDa band visualized with Bio-SafeTM Coomassie

stain and subjected to in-gel trypsin digestion.

All affinity column elution samples submitted for LC-MS/MS were confirmed by

Western blot to have an approximately 64 kDa immunoreactive band, as represented in

Figure 3-2B. To ensure that these Western blot results were accurately confirming the

presence of M. alligatoris antigen, a secondary antibody-only (anti-alligator Ig) control

Western blot of the same column elution sample was performed. A valid control result

would have no band develop. However, a positive signal on the control was seen at

approximately 64 kDa (Figure 3-3). Such results indicated possible non-specific binding

or alligator antibodies leached off from the column, which were then recognized by the

secondary anti-alligator Ig antibody.









Protein matches from the three elution samples were evaluated in Scaffold 2

under specific stringency settings (Table 3-1). Top matches included Lmp3 protein,

putative (ORF01306), viral A-type inclusion protein, putative (ORF00114), DNA

polymerase III subunit, putative (ORF01370) and lipoprotein, putative (ORF00858).

DNA polymerase III subunit, putative, from the trypsin-digested Bio-SafeTM Coomassie-

stained sample, had the highest amino acid coverage (14%). However, the overall

quality of spectra from these biological samples was low. Quality of spectra was

evaluated in Scaffold 2 by X! Tandem and Mascot probability score scatterplots and

distributions describing the number of correct and incorrect assigned spectra for the

entire biological sample. Matches were evaluated for extent of amino acid coverage,

match probability scores, the m/z versus relative intensity spectrum, spectrum/model

error data and fragmentation tables. Based on these factors, none of the eluted antigen

protein matches were of strong confidence. LC-MS/MS data from elution samples were

evaluated for protein matches in the M. crocodyli genome to see if proteins homologous

to M. alligatoris would also show up. However, no M. crocodyli matches homologous to

any of the M alligatoris matches were found.

If the rabbit polyclonal anti-alligator Ig detected alligator IgY heavy chain, a

positive signal for the secondary antibody only-control Western would appear about 67-

70 kDa. To investigate the possibility IgY heavy chain leaching off the column and

being present in elution samples, the LC-MS/MS data was also compared to the

genome of the green anole, Anolis carolinensis, in Scaffold. This database, containing

only 328 proteins at the time of analysis, allowed only a limited evaluation. The only









immunoglobulin heavy chain associated amino acid sequences in the Anolis database

were the following:

gi|1576950831gbIABV66132.1 immunoglobulin Y heavy chain constant region
gil 57695081 gblABV66131.1 immunoglobulin Y heavy chain constant region
gi|1576950791gbIABV66130.1 immunoglobulin D heavy chain constant region
gill 576950771gblABV66129.1 immunoglobulin Y delta Fc
gi|1576950751gbIABV66128.1 immunoglobulin M heavy chain constant region
gill 573809241gblABV46492.1 immunoglobulin heavy chain constant region

The most relevant match was immunoglobulin light chain kappa variable region

(gi|171464617). However, Scaffold 2 algorithms indicated that confidence for this

match was overall low. In addition, its predicted molecular weight (14 kDa) did not

explain control Western blot observations at approximately 64 kDa.

Standard protein-protein BLAST (BLASTP) searches were also performed using

de novo sequences obtained from two affinity column elution sample submissions: the

in-gel AspN digestion and in-gel trypsin digestion, both stained with regular Coomassie.

The actual peptide sequences obtained through PEAKS Studio are reported in Tables

3-3 and 3-4. The best matches found by performing BLASTP searches on the de novo

sequences (M. alligatoris glycosyl hydrolase family 2, ABC transporter permease

protein, and M. crocodyli Holliday junction DNA helicase, etc.) were not significant

(Table 3-6).

Knockout Mutant Immunodominant Antigen Expression Evaluation

Phosphopentomutase (deoB) was identified with an initial Mascot search from the

in-gel trypsin digested Coomassie stained elution sample against the M. alligatoris

genome database. This identification was not significant (Mascot score = 43).

However, deoB was previously identified as a putative immunogenic S. aureus protein

by MALDI-TOF by other researchers (Glowalla et al., 2009). Since a deoB mutant was









available in the M. alligatoris mutant library, it was evaluated for immunodominant

antigen expression. Total protein was extracted from the deoB mutant as well from as

an irrelevant M. alligatoris knockout, sialic acid lyase NanA. Samples were prepared in

2x Laemmli sample buffer and separated by SDS-PAGE on a 7.5% Tris HCI gel. A

Western blot probed with infected alligator plasma revealed identical immunoreactivity

patterns. A band for both mutants developed at approximately 60 kDa (Figure 3-4).

This result indicated that phosphopentomutase was not the correct immunodominant

antigen.

2DGE Protein Separation

Both membrane proteins and total protein of M. alligatoris, separated by 2DGE

and blotted onto PVDF membranes, were further analyzed by immunoblotting. The

Western blots were used to select protein spots from their corresponding 2-D gels.

2DGE Membrane Protein Separation

Two immunoreactive areas containing membrane proteins were excised (indicated

by circles) from a 2-D gel (Figure 3-5A). These areas on the gel best aligned with two

positive signals on its corresponding Western blot (Figure 3-5B), which appeared at

approximately 50 kDa and 30 kDa. Each protein spot was separately subjected to in-

gel trypsin digestion and LC-MS/MS. Best matches in Scaffold 2 were reported (Table

3-2). The quality of the MS/MS data was high, and protein matches had high

confidence as indicated by various Scaffold parameters. However, the likelihood that

any of these matches were the actual immunodominant antigen seemed low due to

several issues. An unstained molecular marker was used for these gels. The marker

was visualized in the gel used for selecting the protein spot once it was stained with Bio-

SafeTM Coomassie. However, the gel used for the Western transfer could not be









stained prior to electroblotting, and thus the membrane itself did not have a visible

molecular marker. The gel used for electroblotting was stained afterwards to visualize

any remaining proteins. This post-transfer gel was aligned with the Western blot to

estimate where the molecular marker bands should have belonged on the membrane.

Other physical references were aligned between the membrane and the gels to excise

the two spots. Despite best efforts, accuracy could not be guaranteed. None of the

other 2DGE membrane protein Western blots developed a positive signal. It could not

be explained why there were two positive signals instead of one, or why they appeared

at approximately 50 kDa and 30 kDa.

2DGE Total Protein Separation

One immunoreactive protein area could be detected by alligator plasma (Figure 3-

6B) on a 2DGE total protein Western blot. Replicate gels and Westerns were run to see

if the same protein identifications could be repeated independently. This band

corresponded to a protein approximately 100 kDa in molecular weight (Figure 3-6A).

The results of the immunoreactive protein area that was investigated (enciricled) by in-

gel trypsin digestion and LC-MS/MS are elaborated in Table 3-3. Five protein matches

were found using highest stringency settings: Min. Protein = 99.9%, Min # Peptides = 5,

Min. Peptide = 95%.

One of the top matches from the total protein sample had 33% amino acid

sequence coverage with a sequence fragment named "lipoprotein" from the original

FASTA database (Figure A-1). The match had relatively good amino acid coverage in

Scaffold 2 compared to other matches. Overall confidence in this match was increased

since it was identified in two independent 2DGE gels and LC-MS/MS sequencing

experiments, as well as from de novo analysis. When the complete sequences were









made available in GenBank (after database searches were performed in this study), the

protein name was changed to "conserved hypothetical protein" (accession no.

ZP_06610777.1). A BLASTP search of the lipoprotein sequence had 100% query

coverage (E value e-130) with ZP_06610777.1. This full sequence had a theoretical

molecular weight of 89.5 kDa. A lipid addition covalently bound to the protein could

result in a molecule with a greater molecular weight. This possibility could explain the

immunoreactive protein visualized at 100 kDa.

The next best BLASTP matches to the lipoprotein sequence were all lipoproteins

or membrane-associated in Mycoplasma, including: M. crocodyli lipoprotein

(YP_003560325.1), M. fermentans (BAH70134.1), M. pulmonis lipoprotein

(NP_326468.1), and M. gallisepticum str. F conserved hypothetical protein

(ADC31412.1). Thought some streptococcal proteins were also in the BLASTP match

results, their E-values were too high to be considered significant.

Horizontal streaks were a prevalent problem for the 2DGE experiments, especially

noticeable in the gels (Figures 3-5 and 3-6). Several modifications were made to

address horizontal streaks seen on the 2-D gels and Western blots. Common

contaminants include salt, detergents, peptides, nucleic acids, lipids, and phenolic

compounds (Chang et al., 2003). Sample preparation modifications included using

different protein extraction methods, ensuring that salt concentration was adequately

low, and treating protein samples with endonuclease to reduce nucleic acid

contamination. Different agarose and polyacrylamide overlays were tested to see if a

better seal could be made between the IPG strip and the gel before second dimensional

electrophoresis. Since no major reductions in horizontal streaks could be seen with









these adjustments, it is thought that the major problem was inadequate isoelectric

focusing.

Blue Native PAGE

Wild-type M. alligatoris native membrane protein was extracted and separated BN-

PAGE on a 4-16% gradient NativePAGE Novex Bis Tris gel. Gels were stained and

duplicate gels were subjected to Western blotting. However, Westerns were never

successful. The inability to remove an adequate amount of Coomassie stain from the

gels prior to electroblotting resulted in extremely high background during

immunoblotting.










M W E

250
150
100

50
37

25 ,
15 + Control


A

Figure 3-1. Coupling validation of affinity column (second configuration). Monoclonal
mouse (subclass IgG2b kappa) anti-alligator IgY light chain (HL1740 5H1-
3B6) was coupled directly to the column's SepharoseTM beads. The column
was then incubated with goat anti-mouse IgG (H+L) alkaline phosphatase
conjugate. A) Samples were prepared in 4x NuPage sample buffer,
separated by SDS-PAGE on a 7.5% Tris-HCI Ready gel and visualized by
silver stain. W = first 12 pooled 80-ul PBS washes collected from the column
before elution, containing excess goat anti-mouse IgG (H+L) Alkaline
Phosphatase Conjugate that did not couple to beads. E = first 12 pooled 80-
ul fractions eluted from the column with glycine HCI elution buffer. The anti-
mouse IgG (H+L) alkaline phosphatase conjugate was used as a positive
control. Note: the last PBS wash samples, which did not contain any protein
as indicated by absorbance readings at 405 nm, were not run on this gel. B)
PBS wash fractions prior to elution as well as elution fractions were
consecutively collected from the column. Absorbance of each fraction after
treatment was read at 405 nm in a plate reader. PBS washes = 40
consecutive 50-ul PBS wash fractions collected. Elutions = 40 consecutive
50-ul glycine HCI elution fractions collected. Positive (+) Controls = anti-
mouse IgG (H+L) alkaline phosphatase conjugate was used at four dilutions,
which were measured in the following order: 1:100, 1:500, 1:1000, and
1:5000. The negative control was 1x PBS.













51300 t -


**,





Neg. Cor rol

PBS Washes Elutions +Controls


Fractions collected from affinity column (second configuration)


B



Figure 3-1. Continued


3.438



1.875



0313
0-00


-1 250


5.000











E M
ME
250 2

150 150


75
75
^ -- .- ..... .- .











Figure 3-2. M. alligators LAMP antigen eluted from affinity chromatography column
(third configuration). A) Samples were prepared in 2x Laemmli sample buffer
with DTT, separated by SDS-PAGE on a 7.5% Tris-HCI Ready gel, and
visualized by Coomassie blue staining. E = pooled elution fractions #4-9. M
= pre-stained molecular mass standards. Bands indicated by the arrow were
excised. Peptides produced by both in-gel trypsin and Asp-N digestion were
subjected to LC-MS/MS. B) Western blot of LAMP antigens eluted from
affinity chromatography column (third configuration) probed with infected
alligator plasma (accession number AAA165099). E = pooled elution
fractions #4-9 from first antigen incubation of third column configuration. M =
pre-stained molecular mass standards.





























Figure 3-3. Secondary antibody-only control. Affinity column elution samples from the
third column configuration were separated by SDS-PAGE on a 7.5% Tris-HCI
Ready gel and electroblotted onto a PVDF membrane. After incubation with
biotinylated polyclonal rabbit anti-alligator Ig antibodies, the membrane was
developed to reveal a band at the same molecular weight as that from the
third column configuration elutions (approximately 64 kDa). The
corresponding band on a Bio-SafeTM Coomassie-stained duplicate gel (not
shown) was excised. Tryptic peptides were subjected to LC-MS/MS, the
results of which are elaborated in Table 3-1.





























Figure 3-4. Western blot of M. alligatoris total protein extracted from a
phosphopentomutase knockout mutant (342), and from an irrelevant knockout
mutant, NanA (209) as a control. The blot was probed with infected alligator
plasma (accession number AA165099) and developed, revealing identical
band patterns.












































Figure 3-5. M. alligators membrane proteins separated by 2DGE. A) Membrane
proteins (300 ug) were resolved in a 4-7 linear pH gradient in the first
dimension, a 8-16% gradient SDS-PAGE in the second dimension and
visualized by Bio-SafeTM Coomassie staining. The results of the proteins that
were identified (encircled) by in-gel trypsin digestion and LC-MS/MS are
elaborated in Table 3-2. Gel image colors were reversed to allow better
visualization of the proteins and molecular marker. B) A duplicate gel was
electroblotted onto a PVDF membrane and probed with a mix of plasma from
three infected alligators: AA1 650998 wk PI, AA165094 10 wk PI, and
AA165089 10 wk Pl. Two immunoreactive areas were observed, as indicated
by arrows. The molecular marker in this particular Western blot could not be
visualized properly. Note, images in A and B are not to scale.























25 "

15Q


15. ... .. .j j 0ir ..

150
I fl- ________
4w~


37 -











B


Figure 3-6. M. alligatoris total proteins separated by 2DGE. A) Total proteins (300 ug)
were resolved in a 4-7 linear pH gradient in the first dimension, and an 8-
16% gradient SDS-PAGE in the second dimension. The immunoreactive
protein area (encircled) was selected for in-gel trypsin digestion and LC-
MS/MS. De novo sequencing was also performed. Best protein matches are
elaborated in Table 3-3. B) A duplicate gel was electroblotted onto a PVDF
membrane and probed with plasma from infected alligator AA165099 16 wk
PI. An immunoreactive area was observed, indicated by the arrow.


V

....-..
-~L~C

..-,- -- -
--~311~1~511--

f ~-


t
~-~


-NOW










Table 3-1. M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold (peptide sources: in-gel trypsin orAspN digestion of third
column configuration LAMP antigen elutions separated by SDS-PAGE).
Theoretical Column Sequence Top
GenBank Accession Mol Wt elution sample coverage Mascot sequence
Putative identifications Number (kDa) submitted (%) Ion Score
M protein repeat protein ZP_06610681.1 110 3rd 5 n/a
Hypothetical protein MALL_0758 ZP_06610553.1 50.4 3rd 13 1.5
Hypothetical protein MALL_0396 ZP_06610600.1 35.8 3rd 14 24.2
Hypothetical protein MALL_0656 ZP_06610235.1 58.9 3rd 7 14.8
Translation initiation factor IF-2 ZP_06610653.1 66.4 1st 4 26.1
Protein identifications and related information according to their reference sequences GenBank. Theoretical molecular weights are based on
complete peptide sequences. LC-MS/MS data were searched against both M. alligatoris and M. crocodyli, though no M. crocodyli matches were
reported due to extremely low confidence scores.
Sequence coverage % and top Mascot sequence ion scores of protein matches as seen in Scaffold based on LC-MS/MS data searched against
incomplete FASTA peptide sequences. Criteria settings in Scaffold: Min. Protein = 99.9%, Min # Peptides = 2, Min. Peptide = 95%. Column
elution sample submitted: (1st) in-gel trypsin digestion, Coomassie blue stain; (2nd) in-gel AspN digestion, Coomassie blue stain, (3rd) in-gel
trypsin digestion, Bio-SafeTM Coomassie stain.

Table 3-2. M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold (peptide sources: in-gel trypsin
digestion of M. alligatoris membrane protein separated by 2DGE).


Putative identifications
Conserved hypothetical protein
Translation elongation factor G
Hypothetical protein MALL_0411
Phenylalanyl-tRNA synthetase, beta subunit


GenBank
Accession
Number*
ZP_06610710.1
ZP_06610590.1
ZP_06610774.1
ZP_06610855.1


Theoretical
Mol Wt (kDa)
79.9
76.8
81.7
79.0


Sequence
coverage
(%)
27
23
25
10


Top
Mascot sequence
Ion Score
123.3
86.3
76.9
38.5


ABC transporter, ATP-binding protein ZP_06610702.1 79.4 31 71.0
Chaperone protein DnaK ZP_06610514.1 67.1 15 52.9
S1 RNA binding domain protein ZP_06610410.1 79.9 15 63.8
Acyltransferase ZP_06610154.1 27.8 26 87.7
Ribosomal protein L ZP_06610409.1 24.9 26 83.2
Ribosomal protein S5 ZP_06610363.1 24.0 35 64.2
Protein identifications and related information according to their reference sequences GenBank. Theoretical molecular weights are based on
complete peptide sequences. LC-MS/MS data were searched against M. alligatoris only.
Sequence coverage % and top Mascot sequence ion scores of protein matches as seen in Scaffold based on LC-MS/MS data searched against
incomplete FASTA peptide sequences. Criteria settings in Scaffold: Min. Protein = 99.9%, Min # Peptides = 4, Min. Peptide = 95%.










Table 3-3. M. alligatoris proteins identified by LC-MS/MS and evaluated in Scaffold (peptide sources: in-gel trypsin
digestion of M. alligatoris total protein separated by 2DGE).
GenBank Top
Accession Theoretical Sequence Mascot sequence
Putative identifications Number Mol Wt (kDa) coverage % Ion Score
D-xylulose 5-phosphate/D-fructose 6-
phosphate phosphoketolase (M. crocodyli) gi1291600221 89 12 68.7**
Conserved hypothetical protein ZP_06610777.1 89.5 33 69.8**
Dihydrolipoyl dehydrogenase ZP_06610150 78.3 17 59.6**
Translation elongation factor Tu ZP_06610740.1 43.2 18 94.3
Hypothetical protein MALL_0531 ZP_06610333.1 98.5 15 85.7
*Protein identifications and related information according to their reference sequences GenBank. LC-MS/MS data were searched against M.
alligatoris and M. crocodyli. Theoretical molecular weights are based on complete peptide sequences.
**Sequence coverage % and top Mascot sequence ion scores of protein matches as seen in Scaffold based on LC-MS/MS data searched against
incomplete FASTA peptide sequences. All identifications were derived from samples subjected to Bio-SafeTM Coomassie staining and in-gel
trypsin digestion. Criteria settings in Scaffold: Min. Protein = 99.9%, Min # Peptides = 5, Min. Peptide = 95%.
***Protein identified twice independently (separate 2DGE and LC-MS/MS experiments).










Table 3-4. M. alligatoris proteins identified by protein-protein BLAST searches of de novo sequence data reported in
Appendix Tables A-1, A-2 and A-3 (peptide source: in-gel trypsin digestion of third column configuration LAMP
antigen elutions separated by SDS-PAGE and in-gel trypsin digestion of total proteins separated by 2DGE).


Genome
Database
Searched
M. alligatoris
M. alligatoris
M. crocodyli
M. crocodyli
M. crocodyli
M. crocodyli
M. crocodyli
M. alligatoris

M. alligatoris
M. alligatoris

M. alligatoris
M. alligatoris

M. alligatoris
M. crocodyli
M. crocodyli
M. crocodyli


GenBank
Accession No.
ZP_06610777.1
ZP_06610753.1
YP_003559943.1
YP_003559946.1
YP_003560083.1
YP_003560325.1
YP_003559885.1
ZP_06610216.1


Source
2DGE
total
protein





Affinity
column
(LAMP)


ZP_06610729.1
YP_003559917.1
YP_003560256.1
YP 003559949.1


Protein ID
Conserved hypothetical protein
RDD family protein
Putative lipoprotein
Putative lipoprotein
Phosphoglycerate kinase
Lipoprotein
Hypothetical protein MCRO_0232
Glycosyl hydrolase family 2, sugar
binding domain protein
ABC transporter, permease protein
Glucosamine-6-phosphate
deaminase
Putative metallophosphoesterase
Type I restriction modification DNA
specificity domain protein
Hypothetical protein MALL_0336
Holliday junction DNA helicase
Thioredoxin-disulfide reductase
Cation transporting P-type ATPase


Theoretical
Mol Wt (kDa)
89.5
25
137.5
41.3
42.7
89.1
26

95.4
37.7

49.8
30.2

46.7
143.5
35.9
33.5
101


BLASTP
Score
(bits)
21.6
21.2
24.3
22.7
20.8
20.8
19.2

23.9
23.5

23.1
23.9

23.9
23.1
22.3
21.9
21.6


BLASTP
Expected
Value
4.8
6.5
0.32
1
3.2
3.9
10


Trypsin ZP_06610133.1
Trypsin ZP_06610871.1

AspN ZP_06610601.1
AspN ZP_06610215.1


Digest
Trypsin
Trypsin
Trypsin
Trypsin
Trypsin
Trypsin
Trypsin
Trypsin


AspN
AspN
AspN
AspN









CHAPTER 4
CONCLUSIONS AND FUTURE DIRECTIONS

Conclusions

It was initially hypothesized that the approximately 64 kDa immunodominant

antigen was (1) associated with the mycoplasma cell membrane and (2) a highly

conserved protein. Objectives to test these hypotheses included obtaining candidates

for the M. alligatoris 64 kDa immunodominant antigen and testing their respective

knockout mutants for immunodominant antigen expression. Despite multiple

approaches and persistent efforts to characterize this antigen, empirical evidence did

not yield definitive conclusions about its identity. The main accomplishment of the

project was establishing several putative proteins that could be considered for

subsequent analyses. Preliminary testing of candidates for immunodominant antigen

expression was also demonstrated for one (albeit low-confidence) match.

A putative lipoprotein, "conserved hypothetical protein" was considered to be the

most promising result. This identity was obtained from two separate 2DGE gels and

LC-MS/MS sequencing experiments as well as from de novo sequencing. This was

also the only match found by LC-MS/MS that was also obtained by independent de

novo sequencing analysis. BLASTP analysis of its full sequence offered evidence of a

conserved protein amongst Mycoplasma species, as functional importance of

orthologous genes did not change. However, based on E-values, it could not be

concluded that this protein was conserved outside of the Mycoplasma genus.

Future Directions

Results in this study helped characterize a potential immunodominant antigen.

However, future work must be done to fully prove that the purified antigen is indeed









immunodominant. Antigen separation and sequencing experiments must be optimized

and repeated to make sure results are reproducible. Full M. alligatoris protein

sequences now available in GenBank would be utilized to have a stronger database.

Next, immunodominance of the candidates) must be evaluated in vitro. An immediate

follow-up to obtaining confident matches, possibly including other candidates from these

experiments (Tables 3-2 and 3-3) would include testing their respective M. alligatoris

knockout clones for immunodominant antigen expression by Western blot. There are at

least half a dozen knockout mutants for the conserved hypothetical protein

(ZP_06610777.1) available in our M. alligatoris knockout mutant library. Alternatively, a

systematic screening could be performed on all knockout mutants in the library by

Western blot, regardless of preliminary candidate protein identifications.

As was done with preliminary results, comparing highly confident MS results to

homologous genes in closely related species could reveal biological importance of the

immunodominant antigen. Further characterization such as epitope mapping may also

enhance future diagnostic and therapeutic efforts such as vaccine candidate screenings

and design. Analysis of immunogenic epitopes could be valuable for the design of a

diagnostic test. If these experiments prove to be meaningful, other immunodominant

antigens such as the 50 kDa antigen should be examined with similar approaches.

Biological importance may also be demonstrated in an infection study using the

immunodominant antigen knockout clone to inoculate naive alligators and perform a

comprehensive evaluation of its resulting pathogenesis. For example, if the antigen

were an important virulence factor, its respective knockout clone might cause a

reduction in disease. If the antigen were vital for the generation of protective antibodies,









infection with its knockout might create a more severe disease. However, if the antigen

were vital to initial colonization of the host, its respective knockout clone might not be

able to cause any disease. Western blots probed with plasma from naturally-infected

alligators 1-5 years PI displayed 50 and 64 kDa immunodominant bands in addition to

many others. Future studies might include identifying the other antigens to see if there

is a profound difference between "early-response" and "later-response" antigens.

As previously discussed, the full ZP_06610777.1 protein sequence had a

theoretical molecular weight of 89.5 kDa. A lipid addition covalently bound to the

protein could result in a molecule with a greater molecular weight. This possibility could

explain the immunoreactive protein visualized at 100 kDa. Other future work might

include determining changes in molecular weight due to lipid additions on mycoplasma

membrane-associated proteins or lipoproteins.

Technical Improvements

2DGE protein samples were generally of high quality, reflected in good MS/MS

data and subsequently reflected in more confident protein matches. Identifications with

the highest confidence came from membrane proteins separated by 2DGE, followed by

total protein separated by 2DGE. However, it could not be ruled out that these

preliminary results were exclusively correlated to the 64 kDa antigen as 2DGE

immunoreactive band results were obtained without excluding other potential

immunodominant antigens of other molecular weights (e.g. at 50 kDa). There was

always possibility of mechanical or human error in selecting bands from gels,

highlighting the importance of repeating experiments. Therefore, the likelihood of

selecting the correct immunodominant antigen, at least initially, is suspected to be low.

One reason for this is that there was no prior enrichment of the immunodominant









antigen when separating total protein by 2DGE. If the immunodominant antigen is

indeed a membrane protein, then there was only slight enrichment when separating

membrane proteins. Another reason was that horizontal streaking patterns indicated

imperfect resolution of individual proteins. Therefore, one protein spot could potentially

contain multiple or aggregated proteins. This possibility was also suggested by the

observation that multiple, dissimilar protein matches were found in Scaffold 2, even with

high stringency settings.

It is known that 2DGE of total protein might not be sufficient to separate the

complete proteome at one time, even for bacteria with a limited number of ORFs

(Regula et al., 2000). Limiting factors include the dynamic range of 2DGE, which

prevents detection of the low abundance proteins, solubilization of membranes, and the

poor resolution of basic proteins, proteins that are too large (>150 kDa) or too small

(<10kDa) (Regula et al. 2000; Chang et al., 2003). Even if proteins appear to be

adequately resolved, it is still possible to pick up more than one protein in an area that

appears to correspond to Western blot patterns. 2DGE does not always have high

reproducibility, which also decreases the chances of selecting the right gel spots for

analysis.

Developing an optimal IEF method for mycoplasmas would take time but would be

extremely useful for future research. This technique is capable of extremely high

resolution with proteins differing by a single charge being fractionated into separate

spots. The standard IEF program steps used in this experiment were originally

developed with E. coli. Thus, optimal settings for mycoplasmas could be quite different.

In a study of the immunoproteome of Mycoplasma mycoides subsp. mycoides small









colony type, horizontal streaking problems were not apparent (Jores et al., 2009). It

might be worthwhile following this study's methods as closely as possible for sample

preparation and 2DGE with special regard to IEF steps.

The number of 2-D gel protein spots identified in this study could not be accurately

measured due to significant horizontal streaking issues seen in the gels. Therefore, it

could not be confirmed that the protein patterns seen for the total protein gels were

within the expected range. Genome sequencing and annotation of the type strain

A21JP2T has revealed 803 open reading frames (ORFs). Therefore, with ideal 2DGE of

total proteins, it would have been expected to see a similar number of protein spots.

Even a higher number of total protein spots could be expected if, by chance, there are

any proteins remaining to be annotated.

A strong point of this study was that two approaches were used to identify protein

matches: LC-MS/MS and de novo sequencing. Ideally, de novo sequencing should

always be included to have higher confidence in protein matches in Scaffold or similar

software, as well as the in the quality of the MS/MS spectra themselves. De novo

sequencing is useful because it can help validate matches obtained by LC/MS/MS. The

method is a good control because amino acid sequences are obtained without a protein

database, helping eliminate false positives. However, once the sequences obtained de

novo are compared to a protein database, the quality of the protein match results will

depend on the quality of the given database.

Alternative Immunodominant Antigen Candidate Screening Techniques

One way to identify immunodominant proteins for potential development of

improved control measures is 2DGE combined with tandem MS analyses. In

mycoplasmas, this method is especially appealing since high A+T content and using









TGA as a tryptophan codon instead of a stop codon can interfere with traditional genetic

approaches such as using E. coli expression libraries. With the genome sequence of

M. alligatoris available, the essential prerequisite for this approach already exists.

Phage display-based identification has also been used for finding novel antigens with

potential diagnostic applications. Mycoplasma mycoides subsp. mycoides small colony

type, which causes contagious bovine pleuropneumonia, has been evaluated by this

method (Naseem et al., 2010). Furthermore, researchers found that best performing

antigens for diagnostic applications included a conserved hypothetical protein (Naseem

et al., 2010). Candidates were expressed as GST-fusion proteins in E. coli, purified and

used as solid phase antigens in ELISAs. Theoretically, the same method could be

applied to M. alligatoris, and the diagnostic potential of recombinant antigens could be

tested using the sera of experimentally infected animals and control animals. A rigorous

evaluation of candidate diagnostic sensitivity and specificity would be necessary. A

complement fixation test could also be used to screen candidates for diagnostic tests.

Non-laboratory based methods to identify possible immunodominant antigens

include sorting all known M. alligatoris proteins by theoretical molecular weight and

focus on candidates that are approximately 64 kDa in molecular weight. An alternative,

possibly more practical approach would be to use Gene Ontology, a collaborative

bioinformatics project, to sort proteins by cellular location and/or function. However,

Gene Ontology is not currently available for M. alligatoris.

Vaccine Development

Live attenuated vaccines are already available against various pathogenic

Mycoplasma species (May et al., 2009). Similarly designed vaccines could potentially

be effective in alligators against M. alligatoris. However, reptile vaccines have not been









developed and will not likely be developed due to lack of a significant economic

incentive. However, candidates could still theoretically be tested as targets of

opsonizing antibodies in vitro and as vaccine candidates in a crocodilian model of

sepsis.

Antigen over-expression and purification would need to be performed to test

potential vaccine or diagnostic test components. A PCR strategy could be developed to

generate a synthetic construct, which could be cloned into a prokaryotic expression

vector. A new strategy for rapidly selecting and testing genetic vaccines has been

developed, in which a whole genome library is cloned into a bacteriophage express

vector containing prokaryotic promoters upstream of the insertion site (March et al.,

2006). The phage library is plated on E. col cells, immunoblotted, and probed with

hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine

candidates.

Algorithm-Based Evaluations of Diagnostic Value

To find potential protein epitopes of diagnostic value, various algorithms can first

be employed. Prediction of antigenic epitopes can be performed using the algorithm of

Hopp and Woods (1981), Zimmerman et al. (1968), and Levitt (1978) (from

www.expasy.ch/cgi-bin/protscale.pl). The secondary structure of polypeptides can be

determined by the methods of Garnier-Robson (Garnier and Robson, 1989) and Chou-

Fasman (Chou and Fasman, 1978). Surface properties of the structural proteins, such

as hydrophilicity,flexibility, accessibility and antigenicity, can be analyzed by the

methods of Kyte-Doolittle (Kyte and Doolittle, 1982), Karplus-Schulz (Karplus and

Schulz, 1985), Emini (Emini et al., 1985) and Jameson-Wolf (Jameson and Wolf, 1988),

respectively. Peptides resulting from these methods with good hydrophilicity, high









accessibility and strong antigenicity could be chosen for further investigation such as

testing purified antigens for immunoreactivity with infected animal sera.

Alternatives to Hypotheses

The immunodominant antigen protein has been proposed to be a membrane

associated protein or lipoprotein. However, the alternative to this hypothesis (that the

antigen is cytosolic) is still possible. In another study, staphylococcal proteins like

enolase and EF-Tu, normally characterized as predominantly cytosolic proteins, were

shown to be located on the surface of S. aureus and other bacterial pathogens

(Glowalla et al., 2009). A certain number of the proteins identified are enzymes that

play a crucial role in the biogenesis of the bacterial cell wall, supporting their surface

location. The exact mechanism of the extracellular translocation of these proteins is

unknown. However, secretion followed by noncovalent, receptor-mediated

reassociation with the cell wall was suggested for pneumococcal a-enolase. Therefore,

it is not surprising that some of the proteins identified in this screen were also found in

secreted or cytosolic protein fractions. The possibility that the 64 kDa immunodominant

antigen is in fact a cytosolic protein could be tested with the same methods already

discussed.

Responses to Future Potential M. alligatoris Outbreaks

Ideal antibiotic targets of M. alligatoris have not yet been identified. Work to

characterize such targets at the molecular level could be done. Treatment of

mycoplasmoses usually involves antibiotics that inhibit DNA replication or protein

synthesis. Drugs targeting spreading factors such as sialidases might be effective. In

vitro drug susceptibility for M. alligatoris has been previously studied by determining the

minimum inhibitory concentration (MIC) for multiple antibacterial agents. The MIC for









doxycycline, enrofloxacin, sarafloxacin, oxytetracycline, tilmicosin, and tylosin were < 1

pg/mL (Helmick et al., 2002). High efficacy in vivo has not yet been demonstrated. In

response to the 1995 outbreak, oxytetracycline was administered to all alligators

intramuscularly at approximately 10 mg/kg once per week for five weeks, then once

every two weeks for 3 months (Clipppinger et al., 2000). However, control of the

disease was not ideal, as many animals still died. Autologous vaccinations might be

explored as a mode of therapy. In an effort to treat farmed crocodiles in Zimbabwe

afflicted with M. crocodyli infection, it was found that use of an autogenous vaccine was

more effective in treating symptoms than antibiotic therapy (Mohan et al., 2001).

Because M. alligatoris pathogenesis is so rapid, survival of an alligator would

likely depend mostly on innate immunity and the present health status of the affected

individual. Processes would likely include phagocytosis of M. alligatoris cells, which

relies on opsonization of bacteria by antibodies and complement. Recognition of

opsonizing antibodies bound to the surface of the bacterial cell via Fcy receptors of

neutrophils is necessary for the induction of the oxidative burst and thus for killing of the

phagocytosed bacteria and induction of a long-term immune response. Based on

ELISA seroprevalence of M. alligatoris and lack of reoccurring outbreaks, it is assumed

that formation of anti-mycoplasma antibodies offers adequate long-term protection

against reinfection. However, experimental studies involving the reinoculation of

previously infected crocodilians have not been done.

The original cause of the 1995 M. alligatoris outbreak is still unknown.

Understanding the pathology of and prevention of the pathogen is important for

protecting the health of both captive and non-captive crocodilians. Further work is









necessary to better understand virulence of M. alligatoris and to gain insight on

virulence of other Mycoplasmas and related microorganisms.










APPENDIX
DENOVO SEQUENCES AND FASTA ZP_06610777 FRAGMENT PEPTIDE
SEQUENCE

Table A-1. De novo sequence data obtained from LC-MS/MS spectra using PEAKS
Studio software (peptide source: in-gel trypsin digestion of third column
configuration LAMP antigen elutions separated by SDS-PAGE).


Sequence
TTLPQ[2]VPGK
TTLPEVPGK
MLSGM[4]APLK
MLSGFAPLK
SSGMLLNNALK
LSSPATLNSR
SSGMLLNNALK
LSSPATLDSR
LSSPATLN[2]SR
SSGMLLDNALK
SSGMLLN[2]NALK
SSLSNALPALK
VATVSPLR
AVGYLVSGYKR
AVGYLVSGYQR
SSMALVRPSK
SKAVGYLVSGYKR
SQAVGYLVSGYKR
TTLPEVPGK
TTLPQ[2]VPGK


TLC
7.697482109
7.697482109
7.667612553
7.667612553
9.341050148
8.448849678
9.178469658
8.305230141
8.305230141
9.036511421
9.036511421
9.031148911
6.554719925
9.01044178
9.01044178
8.176656723
10.51771927
10.51771927
7.233291149
7.233291149


ALC(%)
85.52757899
85.52757899
85.19569503
85.19569503
84.91863771
84.48849678
83.44063325
83.05230141
83.05230141
82.15010383
82.15010383
82.10135373
81.93399906
81.91310709
81.91310709
81.76656723
80.90553284
80.90553284
80.36990166
80.36990166


Rank
1
2
2
1
1
1
1
2
1
2
1
1
1
1
5
1
1
3
2
1


m/z
471.2416687
471.2416687
482.2719727
482.2719727
574.2905884
523.3428345
574.3457031
523.826416
523.826416
574.8052368
574.8052368
550.8555908
421.7575684
606.8172607
606.8172607
538.2861938
714.3775635
714.3775635
471.2472839
471.2472839


Mass
940.52295
940.52295
962.49286
962.5259
1146.6067
1044.5564
1146.6067
1045.5403
1045.5403
1147.5906
1147.5906
1099.6238
841.50214
1211.6663
1211.6299
1074.5854
1426.7932
1426.7568
940.52295
940.52295


Note: Only results with an ALC (%) score of 80 or higher was allowed. Sequence = the sequence of the
peptide (including modifications if present) as determined by de novo sequencing. TLC = Total local
confidence (the confidence that we have in the peptide sequence), calculated by adding the positional
confidence for each amino acid in the peptide sequence. ALC = Average local confidence (the
confidence that we have in the peptide sequence), calculated by adding the positional confidence for
each amino acid in the peptide sequence and dividing by the total number of amino acids. Rank = the
sequences for a particular spectrum (ID) as sorted by score (TLC). m/z = Measured mass/charge value,
in Daltons, for the peptide. Z = Calculated charge value for the peptide. Mass is calculated using the
measured m/z and calculated z, used as the experimental mass of the peptide.










Table A-2. De novo sequence data obtained from LC-MS/MS spectra using PEAKS
Studio software (peptide source: in-gel Asp-N digestion of third column
configuration LAMP antigen elutions separated by SDS-PAGE).


Sequence
DAASAPSPVSL
SVVP
DVVNGAPSH
DPLLL
DGGLGPGGGGN[2]
GGLH
DGGLGPGGGGDG
GLH
Q[2]AVP
EAVP
DVGAY
DLTLGAFPAGSLSY
LPPV
DLTLGAM[3]PAGSL
SYLPV
DVHGTTAGMT
DLLGTAM[3]HGGL
H
DLLGTAFHGGLH
DGGMHNLH
DVKMLSGM[3]APL
AK
DVKMLSGM[3]APL
AQ
DVQMLSGM[3]APL
AK
DVKMLSGFAPLAK
DLLGTAMH


TLC
10.04759789
3.622637272
7.526432037
4.110112667

12.30580235

12.30580235
3.277987957
3.277987957
4.094697952

14.72895813

14.72895813
8.15039444

9.773213387
9.773213387
6.502185345

10.51620102

10.51620102

10.51620102
10.51620102
6.45478344


ALC(%)
91.34179896
90.5659318
83.62702264
82.20225334

82.0386823

82.0386823
81.94969893
81.94969893
81.89395905

81.82754517

81.82754517
81.5039444

81.44344489
81.44344489
81.27731681

80.89385399

80.89385399

80.89385399
80.89385399
80.684793


Rank
1
1
1
1


m/z
1014.758728
401.2255859
448.2688599
570.4898071


1 611.6010132


611.6010132
415.2221375
415.2221375
524.3009033


1 606.6923828

5 606.6923828
1 495.3058167

1 619.3223877
2 619.3223877
1 440.7293701

1 688.9056396

4 688.9056396


688.9056396
688.9056396
429.2340698


Mass
1013.5028
400.23218
894.4195
569.3424


2 1221.5376


1221.5376
414.21143
414.21143
523.2278


3 1816.9609

3 1816.928
2 988.42834

2 1236.5923
2 1236.6252
2 879.36566

2 1375.7205

2 1375.6841


1375.6841
1375.7534
856.41125


Note: Only results with an ALC (%) score of 80 or higher was allowed.










Table A-3. De novo sequencing data obtained from LC-MS/MS spectra using PEAKS
Studio software (peptide source: in-gel trypsin digestion of total protein
separated by 2DGE).
Sequence TLC ALC(%) Rank m/z Z Mass
VRHK 3.717977762 92.94944406 1 538.2775269 1 538.334
VLNWTSTK 7.769430637 86.32700708 1 480.7709045 2 959.5651
QATVSLPR 6.852394581 85.65493226 2 435.7689819 2 870.49225
KATVSLPR 6.852394581 85.65493226 1 435.7689819 2 870.5286
LSSPATLNSR 8.360575676 83.60575676 1 523.2537231 2 1044.5564
VATVSLPR 6.68298769 83.53734612 1 421.717926 2 841.50214
NTDLAKR 5.774003029 82.48575756 1 408.7219543 2 816.4454
NTDLAQR 5.774003029 82.48575756 2 408.7219543 2 816.409
NTN[2]LAKR 5.774003029 82.48575756 3 408.7219543 2 816.4454
Note: Only results with an ALC(%) score of 80 or higher was allowed.



ORF01382 lipoprotein {Mycoplasma alligatoris A21JP2T (AS)}:
MNKTKKLVITGLASASLLSALAVISCGSSTNGGSDNGSNTGTGSQSGQIDKNKEILLAVDGPQQGMYDHV
VAEFAKSESYKLGYRIRLIKKDVFGALDTFVGHTDRNVVPDLFYAPQDRITDLAQRNVVSDLDTFDKSLFD
DVLAVTGASSDEKTQARTFGTVVGANEGDKTFSPVRKLFGIRHNQEAIVLASTKELAGVKADMANDKTNT
LEDLVKSGEAFIRLQDF

Figure A-1. Peptide fragment sequence of immunodominant antigen candidate
ZP_06610777 as seen in the FASTA database used for Scaffold 2
evaluations. A protein-protein BLAST search of this sequence had 100%
query coverage (E-value 3e-130) with fully sequenced M. alligatoris
conserved hypothetical protein (ZP_06610777).









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BIOGRAPHICAL SKETCH

Niora J. Fabian was born in Boston, Massachusetts. After graduating from Boston

Latin School in 2002, she attended Boston University. In 2006, she received a B.A.

cum laude in biology with a specialization in marine biology. Throughout her

undergraduate career, she gained research experience in various fields of biology.

During the summer of 2005, Niora completed a research fellowship at the Conservation

and Research for Endangered Species (CRES), in San Diego, California. Working in a

molecular diagnostics and pathology laboratory at CRES, she was a contributing author

to her third publication. After college graduation, she worked as a veterinary technician

at a mixed species animal hospital. She also helped rehabilitate wildlife as a volunteer

with Project Wildlife in San Diego.

From 2008 to 2010, Niora worked as a graduate assistant at the University of

Florida's College of Veterinary Medicine in the Department of Infectious Diseases and

Pathology, earning a master's degree in veterinary medical sciences. At the same time,

she was employed as a technician at an animal hospital in Gainesville, FL, furthering

her experience in veterinary emergency medicine. Additionally, she has worked as a

research animal care technician from 2008 until the present. In this position, she was

responsible for intensive neonatal medical treatment and behavioral data collection for

dogs afflicted with metabolic diseases. A member of the class of 2014, Niora will attend

vet school at the University of Florida's College of Veterinary Medicine.





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1 CHARACTERIZATION OF Mycoplasma a lligatoris IMMUNODOMINANT ANTIGENS By NIORA J. FABIAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Niora J. Fabian

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3 To N.N E.N., S.N. A.N., and my entire family

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4 ACKNOWLEDGMENTS I thank my main advisor, Dr. Daniel Brown, and my committee members for their support and education. I would also like to thank Kevin Kroll for his kind advice with Western blotting optimization and Mengmeng Zhu for her recommendations with IEF and 2DGE equipment. Finally, I would like to thank Diane Duke and Linda Green of the ICBR Hybri doma Lab, and Carolyn Diaz and Dr. Sixue Chen of the ICBR Proteomics Core.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 7 LIST OF FIGURES .............................................................................................................. 8 ABSTRACT .......................................................................................................................... 9 CHAPTER 1 INTRODUCTION ........................................................................................................ 11 Discovery and Characterization of M. alligatoris ....................................................... 11 Mycoplasmosis ........................................................................................................... 12 Reptile Immunology .................................................................................................... 14 Immunodominance ..................................................................................................... 17 Cellular Host Re sponses Affecting Immunodominance ............................................ 18 Immunogenic Proteins and Epitopes ......................................................................... 19 Antigenic Variation and Immunodominance .............................................................. 22 Previous Studies on the Temporal Antibody Response of Crocodilians to M. alligatoris .................................................................................................................. 23 Objectives and Experimental Approaches ................................................................. 25 Long Term Goals of Research on M. alligatoris ................................................. 25 Objectives ............................................................................................................. 25 Hypotheses .......................................................................................................... 26 Specific Aims ........................................................................................................ 26 2 MATERIALS AND METHODS ................................................................................... 29 Affinity Chromatography Antigen Purification ............................................................ 30 First Column Configuration ......................................................................................... 31 Second Column Configuration ................................................................................... 32 Third Column Configuration ....................................................................................... 32 SDS-PAGE Separation of Affinity Chromatography Elutions .................................... 33 Second Affinity Column Monoclonal Antibody Coupling Validation .......................... 33 2 -Dimensional PAGE Antigen Separation ................................................................. 34 Active Rehydration ............................................................................................... 36 Isoelectric Focusing (IEF) .................................................................................... 37 Second Dimensional SDS -PAGE ........................................................................ 37 Blue Native Polyacrylamide Gel Electrophoresis (BN -PAGE) .................................. 38 Western Blotting ......................................................................................................... 38 Phosphopentomutase Knockout Mutant Evaluation ................................................. 40 Peptide Sequencing and Protein Identification .......................................................... 40 Protein Reduction, Alkylation and Enzymatic Digestion ..................................... 40

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6 LC -MS/MS ............................................................................................................ 42 Database Searching ............................................................................................ 42 De Novo Sequencing Protein Identifications ....................................................... 45 PEAKS Criteria Settings ...................................................................................... 46 3 RESULTS AND DISCUSSION ................................................................................... 47 Affinity Columns .......................................................................................................... 47 Knockout Mutant Immunodominant Antigen Expression E valuation ........................ 53 2DGE Protein Separation ........................................................................................... 54 2DGE Membrane Protein Separation ................................................................. 54 2DGE Total Protein Separation ........................................................................... 55 Blue Native PAGE ...................................................................................................... 57 4 CONCLUSIONS AND FUTURE DIRECTIONS ......................................................... 68 Conclusions ................................................................................................................ 68 Future Directions ........................................................................................................ 68 Technical Impr ovements ............................................................................................ 70 Alternative Immunodominant Antigen Candidate Screening Techniques ................ 72 Vaccine Development ................................................................................................. 73 Algorithm -Based Evaluations of Diagnostic Value .................................................... 74 Alternatives to Hypotheses ......................................................................................... 75 Responses to Future Potential M. alligatoris Outbreaks ........................................... 75 APPENDIX: DE NOVO SEQUENCES AND FASTA ZP_06610777 FRAGMENT PEPTIDE SEQUENCE ............................................................................................... 78 LIST OF REFERENCES ................................................................................................... 81 BIOGRAPHICAL SKETCH ................................................................................................ 90

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7 LIST OF TABLES Table page 3 -1 M. alligatoris proteins identified by LC MS/MS and evaluated in Scaffold (peptide sources: in -gel trypsin or AspN digestion of third column configuration LAMP antigen elutions separated by SDS PAGE). ........................ 65 3 -2 M. alligatoris proteins identified by LC MS/MS and evaluated in Scaffold (peptide sources: in -gel trypsin digestion of M. alligatoris membrane protein separated by 2DGE). ............................................................................................. 65 3 -3 M. alligatoris proteins identified by LC MS/MS and evaluated in Scaffold (peptide sources: in -gel trypsin digestion of M. al ligatoris total protein separated by 2DGE). ............................................................................................. 66 3 -4 M. alligatoris proteins identified by proteinprotein BLAST searches of de novo sequence data reported in Appendix Tables A -1, A 2 and A 3 ................... 67 A-1 De novo sequence data obtained from LC MS/MS spectra using PEAKS Studio software (peptide source: ingel trypsin digestion of third column configuration LAMP antigen elutions separated by SDS PAGE). ........................ 78 A-2 De novo sequence data obtained from LC MS/MS spectra using PEAKS Studio software (peptide source: ingel Asp -N digestion of third column configuration LAMP antigen elutions separated by SD SPAGE). ........................ 79 A-3 De novo sequencing data obtained from LC MS/MS spectra using PEAKS Studio software (peptide source: ingel trypsin digestion of total protein separated by 2DGE). ............................................................................................. 80

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8 LIST OF FIGURES Figure page 1 -1 Representative Western blots of M. alligatoris antigens probed with plasma from crocodilians exposed to M alligatoris .......................................................... 28 3 -1 Coupling validation of affinity column (second configuration). ............................. 58 3 -2 M. alligatoris LAMP antigen eluted from affinity chromatograph y column (third configuration).. ........................................................................................................ 60 3 -3 Se condary antibody only control. ......................................................................... 61 3 -4 Western blot of M. alligatoris total protein extracted from a phosphopentomutase knockout mutant (342), and from an irrelevant knockout mu tant, NanA (209) as a control. ........................................................... 62 3 -5 M. alligatoris membrane proteins separated by 2DGE. ........................................ 63 3 -6 M. alligatoris total proteins separated by 2DGE. ................................................... 64 A-4 Peptide fragment sequence of immunodominant antigen candidate ZP_06610777 as seen in the FASTA database us ed for Scaffold 2 evaluations. ............................................................................................................ 80

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CHARACTERIZATION OF Mycoplasma alligatoris IMMUNODOMINANT ANTIGENS By Niora J. Fabian August 2010 Chair: Daniel R. Brown Major: Veterinary Medical Sciences Mycoplasma alligatoris is a bacterial pathogen that causes acute lethal multisystemic inflammatory disease in alligators. Previous Western blotting experiments revealed antibody response patterns of experimentally inoculated and naturally infected American alligators ( Alligato r mississippiensis). In all individuals s urviving the acute infection, a significant imm une response was observed for an antigen approximately 64 kDa in size as early as 6 wk post inoculation (PI), increasing in intensity until euthanasia 16 wk PI. For s ome individuals, a second antigen approximately 50 kDa in size was immunoreactive. The importance of this distinct initial humoral response is unknown, as alligators surviving the natural infection develop ed immune responses to many other antigens several years later The goal of this study was to identify and characterize the 64 kDa immunodominant antigen to better understand its potential role in host pathogen interactions in M. alligatoris mycoplasmosis. Two -dimensional gel electrophoresis ( 2DGE ) and a ffinity chromatography -based antigen isolation experiments were performed using M. alligatoris membrane proteins and total proteins. Followin g Western blot confirmation, LC/MS/MS peptide sequencing, and de novo peptide sequencing, protein matches were

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10 evaluated with statistical algorithm -bas ed proteome analysis software. Top immunodominant antigen candidates were obtained from 2DGE samples. These candidates coul d be evaluated for immunodominant antigen expression in subsequent studies. Though th e 64 kDa M. alligatoris antigen is still unknown, future work involving improved antigen enrichment and resolution as well as testing candidates for immunodominant antigen expression may confirm its identity. This work may explain relative virulence of M. alligatori s and provide insight on pathogenesis of other Mycoplasma species and related organisms.

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11 CHAPTER 1 INTRODUCTION Discovery and Characterization of M. alligatoris Mycoplasma alligatoris is a lethal bacterial pathogen of American alligators (Alligator mississippiensis ) and caimans, while crocodiles are comparatively resistant. In 1995, an outbreak of mycoplasmosis resulted in the death or euthanasia of 60 captive bull alligators in St. Johns County, Florida, USA (Clippinger et al., 2000). Cause of the outbreak remains unknown. Clinical symptoms included rapidly progressive lethargy, anorexia, muscle weakness, paraparesis, bilateral ocular discharge, edema of the limbs, periocular, facial and cervical areas, and death (Brown et al., 2001a). Pathology findings included necrotizing pneumonia, polyserositis, pericarditis, necrotizing myocarditis, lymphocytic interstit ial nephritis, lymphocytic periportal hepatitis, splenic hyperplasia, pyogranulomatous meningitis, and synovitis (Clippinger et al., 2000; Brown et al., 2001a; Brown et al., 2001b; Brown et al., 2001c; Richey, 2001). There were no significant abnormalitie s in blood cell counts or plasma biochemistries of the affected animals. Results for toxicant, heavy metal, mineral, and vitamin analyses of tissues and serum were unremarkable. Routine aerobic and anaerobic bacterial culture of blood and lung tissue sam ples did not confirm etiology of the disease. No viruses were isolated from pneumonic lung tissue samples. However, a previously uncharacterized Mycoplasma species Mycoplasma alligatoris was cultured from multiple tissues, peripheral blood, synovial fl uid, and cerebrospinal fluid of the affected alligators. The type strain A21JP2T was characterized using standard taxonomic methods and was utilized in additional studies.

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12 Disease following experimental M. alligatoris inoculation via the glottis of adul t and juvenile alligators and via the trachea of hatchlings was clinically similar to that observed in the 1995 outbreak (Clippinger et al. 2000; Brown et al. 2001b, Pye et al., 2001). Shortly after the natural outbreak, a pilot experimental inoculation study was performed to fulfill Henle Koch Evans postulates. H ealthy, seronegative juvenile (approximately 1 m long) alligators were inoculated by intracoelomic injection or by instillation through the glottis with 106 CFU of M. alligatoris strain A21JP2T. Three of four inoculated animals died within 3 wk PI, demonstrating significant virulence of the pathogen. Next i n a dose -response study, healthy subadult (approximately 1.5 to 2 m long) female alligators were inoculated by instillation through the glottis with 102, 104, or 106 CFU of M. alligatoris strai n A21JP2TMycoplasmosis in 1 mL of SP4 broth. T he Henle-Koch-Evans postulates were fulfilled by results of these studies for M. alligatoris as the etiological agent of primary fatal mycoplasmosis of alligators (Evans, 1976). Additional research has included genome sequencing of M. alligatoris and its close relative M. crocodyli characterization of putative M. alligatoris virulence mechanism s, and an immunological survey of M. alligatoris of captive and natural populations of alligators. Mycoplasmahost relationships can range from innocuous commensalism to acute fatal disease (Brown, 2002). While differences in pathogenicity may be explained by the presence of certain virulence factors, the det ailed pathogenic mechanisms of m ycoplasmas are still generally poorly understood (Matsushita and Okabe, 2 001; Brown et al., 2004). Like other members of its genus, M. alligatoris has several characteristic features, including a small genome size in comparison to most other prokaryotes. This reduction in genome size is likely an evolutionary result of adopting a parasitic lifestyle,

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13 in which most nutrients are directly acquired from the host (Fraser et al. 19 95; Himmelreich et al., 1997). Many Mycoplasma species are able to change their surface antigen profile at high frequency, including M. pulmonis (Watson et al., 1988), M. hyorhinis (Rosen garten and Wise, 1990; Rosengarten and Wise 1 991; Yogev et al., 1991), M. hominis (Olson et al., 1991), M. fermentans (Theiss et al., 1993 ; Wise et al., 1993), M. gallisepticum (Markham et al., 1993; Yogev et al., 1994) and M. bovis (Behrens et al., 1994). It remains to be demonstrated whether the m ain function of this surface variation is actually immune evasion and/or whether these antigenic or structural components are i nvolved in pathogenesis during Mycoplasmahost interactions. It is currently unknown if M. alligatoris displays a high frequency of antigenic variation, but no homologs for systems of antigenic variation known in other Mycoplasma species have been identified in the M. alligatoris genome. A notable physical ch aracteristic of Mycoplasma is the complete lack of a cell wall. Thus, the mycoplasmal cell surface lacks the highly conserved lipopolysaccharide and peptidoglycan antigens that act as endotoxins, eliciting strong immune responses in animals (Dziarski, 1982). Instead, membrane surface proteins are thought to play a large ro le i n the pathogenesis of many species For example, studies have shown that approximately 10 membrane proteins of M. galactiae are responsible for eliciting immune responses in sheep ( Tola et al., 1997). In some species, initial attachment to host cells is mediated by adhesion proteins which can create a host response that exacerbates disease (Bas eman et al., 1982; Razin et al., 1 998). For example, M. pneumoniae, M. bovis and M. hyopneumoniae rely on cytadhesins to attach to host

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14 cells, a critical step in their pathogenic invasion (Baseman et al., 1982; Zhang, et al. 1995; Sachse et al. 2000). Thus far, there is no evidence that M. alligatoris invades host cells, but rather appear to aggregate on host cell peripheries (Hunt and Brown, 2005; Hunt and Brown, 2007). Lesion severity correlates with large numbers of M. alligatoris cells in diseased tissues, suggesting the pathogens virulence may be due to a spreading factor. However, classic -tip mediated cytadherence is not involved in all adherence pathways For example, surface localized elongation factor, Tu (EF -Tu) and the pyruvate dehydrogenase E1 subunit of M. pneumonia help mycoplasmal binding to fibronectin, a glycoprotein found in the extracellular matrix primarily serving as a substrate for mammal ian cell adhesion (Balasubramanian et al., 2008). As infection progresses, mycoplasmas can significantly affect the immune system by inducing either suppression or polyclonal stimulation of B and T lymphocytes. This in turn induces expression of up and down -regulating cytokines, subsequently increasing the cytotoxicity of macrophages, natural killer cells, and T cells. For example, M. hyorhinis was shown to release a 200 kDa factor into the supernatants of infected cultured cells that suppresses cytotox ic T -cell respon ses to alloantigens (Teh et al., 1988). There is also evidence that M. pneumoniae M. arthritidis and M. fermentans stimulate IL-10 production, a down regulating cytokine that suppresses T -cell proliferation (Rawadi et al., 1996; Razin et al., 1998). Reptile Immunology Reptiles and other modern poikilotherms have innate and adaptive immune defenses similar to those of modern mammals and birds (Sunyer et al., 1998 a ). Innate immunity of reptiles includes histaminergic cells, complement, phagocytes and natural

PAGE 15

15 killer cells, peptide regulatory factors, and fever (Brown, 2002 ; Merchant et al., 2007). Fever is important for stimulating inflammation, increasing antibody pro duction, and decreasing serum iron availability. Reptile tissues are rich in mast cells and antigenspecific immunoglobulin -bearing basophils. Monocytes and macrophages, the latter being phagocytic and capable of processing and presenting antigens and pr oducing cytokines, are also present. Heterophils are reptilian phagocytic granulocytes functionally equivalent to mammalian neutrophils. In an acute response to bacterial infection, heterophils accumulate locally and degranulate. This aggregation attrac ts macrophages, resulting in granuloma formation. Cytokines characterized in reptiles include interferon, interleukin1, and transforming growth factor beta. Another key component of reptilian innate immunity is the complement system (Zimmerman et al., 2010 ). The complement system involves of a series of circulating plasma proteins that kill invading bacteria either through opsonization or by lysis. The reptilian complement system contains the same pathways of the mammalian system and additionally contai ns multiple highly -polymorphic isoforms of C3 (Sunyer et al., 1998b). The classical pathway, the last of the three pathways to evolve, is activated by the immunoglobins IgG and IgM. Sever al Mycoplasma species can become lysed by ac tivated complement. My coplasmal activation of complement by both the classical and alternative pathways has also been documented (Razin et al., 1 998; Brown, 2002). Adaptive immunity includes lymphoid tissues and lymphocytes, major histocompatibility (MHC) receptors, lymphocyte receptors, and antibodies. Immune memory is also evident in reptiles ( Montali et al., 1988 ). Reptile spleen does not possess germinal centers known for B -cell clonal selection and antibody affinity

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16 maturation in mammals. Instead, lymphocytes originate f rom bone marrow, spleen and thymus in adult reptiles ( Zapata and Amemiya, 2000). However, splenectomy can completely or partially suppress the humoral response, indicating importance of the spleen in reptile immunity ( Kanakambika and Muthukkaruppan, 1972; Hussein et al., 1979). Reptiles lack true lymph nodes, but have mucosal associated lymphoid reticulum in addition to T and B -lymphocytes. Functions of reptile immunoglobulins include neutralization, agglutination (antibody interaction with particulate antigen), precipitation (antibody interaction with soluble antigen), opsonization (enhanced phagocytosis of antigen upon opsonin binding), complement fixation, and blocking interaction of viruses or toxins with their cognate receptor structures exposed on the host cell membrane ( Origgi 2007). The type of antibody that offers protection depends on the infectious strategy and lifestyle of the pathogen. For example, when opsonizing antibodies such as IgG are available, opsonization of extracellular pathogens will be more efficient (Murphy et al., 2008). The major antibody isotypes in reptiles are IgM (the ancestor of all immunoglobulins) and IgY (the ancestor of IgG and IgE). In addition, a low molecular weight form of IgY lacking the Fc region exists ( Warr et al., 1995; Bengtn et al., 2000). The IgY heavy chain has a molecular mass of 65-70 kDa and the light chain is 1921 kDa (Hatta et al., 1993 ; Sun et al., 2001 ). T he whole IgY molecule is approximately 180 kDa by SDS -PAGE and approximately 167 kDa By MALDI -TOF ( Hatta et al., 1993, Sun et al., 2001). A sequential IgM -IgY response occurs as well as affinity maturation. Features such as specificity, diversity, and memory are comparatively less extensive in reptiles than in mammals, not due to a lack in the number of any of the genetic

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17 components of the immunoglobulin gene systems, but because of comparatively low antibody heterogeneity and poor affinity maturation ( Hsu, 1998). Specific antibody titers after mucosal or systemic mycoplasmal infection are lower in reptiles compared to mammals or birds ( Hsu, 1998 ; Brown, 2002). In secondary responses, antibody titers and affinities are rarely any higher than in primary responses. Thus, their differences are not likely due to restrictions in primary antibody production. In adult tortoises and crocodilians, mycoplasmosis eli cits a humoral immune response to immunodominant antigens approximately six weeks after initial exposure (Schumacher et al., 1993; Brown et al., 2001c) C aptive alligators naturally exposed to a sub-lethal dose of M. alligatoris during the 1995 outbreak h ad detectable specific antibody titers for at least six years PI ( Brown et al., unpublished ). However, it cannot be ruled out that these findings were possibly due to the animals remaining chronically infected or experiencing multiple exposures to M. alligatoris If subjected to multiple exposures, any antibodies remaining from the previous immune response would be immediately available to bind newly invading pathogens. If sufficient antibody already existed and cleared the pathogen completely, it is be possible that no secondary immune response would follow (Murphy et al., 2008). However, if antigen persisted, a secondary B -cell response would be initiated. Immunodominance Though a substantial amount of research is devoted to discovering and characteriz ing immunodominant antigens of many medically significant pathogens, the concept of immunodominance is often interpreted differently among investigators or not clearly defined. In part, this confusion may be due to the broadness and complexity of the topi c. In an infection, the innate immune response provides an immediate but non-

PAGE 18

18 specific response. However, if a pathogen successfully evades the hosts innate response, the innate immune system must activate the adaptive immune system. Longterm active im munological memory is acquired after infection by activation of T and B cells and subsequent production of antigen-specific antibodies. Immunological memory and specific antibodies formed against certain antigens help the immune system more effectively id entify the pathogen and clear the infection. What is not clear is why the humoral system mounts a significant response to certain epitopes and not others. Like other immunogens, an immunodominant antigen contains at least one epitope that adequately stimu lates the immune system to produce specific antibodies. However, the immunodominant antigen is unique because it is the first of all the antigens from a given immunogenic source to elicit a detectable humoral response. This antigen could truly elicit the first response, for example, because it is structurally or functionally the first to make physical contact host cells. In contrast, it could simply elicit the strongest response, either due to higher expression levels compared to other antigens, or due t o higher affinity to host immune cells and molecules. Important events occur in both innate and adaptive immunity before antibodies are produced. Therefore, factors responsible for immunodominance might take place within the innate system, adaptive system or both systems. Cellular Host Responses Affecting Immunodominance Multiple variables involving the host response are thought to affect the ultimate outcome of immunodominance of an antigen. These variables include MHC binding affinity, the formation of stable MHC -peptide complexes on the surface of antigen presenting cells, efficiency of cellular processing capable of generating the relevant MHC binding peptides, availability of T -cell receptors capable of recognizing complexes

PAGE 19

19 between the processed MHC binders and MHC and oxidative stress ( Yewdell and Del Val, 2004; Gaddis et al., 2006; Sette and Sundaran, 2006; Yewdell, 2006; Weiskopf et al., 2010). However, the rules governing all of these processes are still mostly unknown. Adaptive host responses d epend on major histocompatibility complex (MHC) molecules. Pathogens express various amino acid sequences that may successfully bind to MHC I or MHC -II molecules and provide targets for specific T cells. For example, the MHC class I binding groove appear s to have evolved to bind a low fraction (approximately 1%) of peptides without evolutionary pressure to avoid or enhance vertebrate immune recognition (Istrail et al., 2004). Large numbers of host T cell receptors are distributed clonally on the cell sur face to recognize a variety of MHC antigen complexes. Despite the large number of potential combinations, pathogenspecific T cells seem to recognize a relatively small number of epitopes. This apparent preference, or immunodominance, reflects the final product of innumerable positive and negative factors that direct antigen presentation and T cell activation ( Yewdell and Bennink 1999). Furthermore, B cells and T cells must be specific for the same antigen in order to interact and allow B cell activation, proliferation and differentiation into antibody -secreting cells (Murphy et al., 2008). Immunogenic Proteins and Epitopes Surface proteins and adhesins are commonly regarded as potential immunodominant antigens among bacterial pathogens. Lipoproteins ar e considered to be highly immunogenic due to their surface exposure and amino-terminal lipoylated structure ( Chambaud et al., 1999). The plasma membrane of prokaryotic organisms is protein-rich and contains many important enzymes, receptors and transporte rs ( Sigler

PAGE 20

20 and Hofer, 1997). The integral membrane proteins of various bacterial pathogens have been found to be powerful inducers of the immune system, specifically T cells. It is thought that the acyl chains of conjugated lipids have an adjuvant effect by binding to the hydrophobic groove of major histocompatibility complex (MHC) molecules, positioning an epitope for T cell interaction ( Deres et al ., 1989; Akins et al., 1993; Rees et al ., 1989 ; Frankenburg et al ., 1996 ). Membrane proteins are also link ed to pathogenesis in other pathogens. For example, a Vibrio vulnificus membrane-bound lipoprotein, IlpA, functions as an adhesion molecule. It is considered to be a virulence factor because it can induce cytokine production in human immune cells. An IpAdeleted V. vulnificus mutant was shown to significantly decreased adherence to human intestinal epithelial cells, in turn reducing cytotoxicity (Lee et al., 2010). There is also evidence of immunodominance among other types of bacterial proteins. For example, many of the immunodominant antigens described in Mycoplasma mycoides subsp. mycoides small colony type are intracellular enzymes of intermediary metabolism, not phasevariable surface lipoproteins ( Jores et al., 2009). Some of these are surface -loc alized and able to bind mucin. Given the diversity of primary, secondary and tertiary structures, proteins have a potentially wide range of epitopes. However, the extent of immune recognition does not reflect this vast pool of potential candidates ( Laver et al 1990). Instead, only certain epitopes may dominate an infection. For example, immunogenicity of a particular species within a multi -species infection in a host may be based more on the structural and biochemical nature of their epitopes rather th an how many individual organisms of a given species exist in the infection. Epitopes can be mapped using protein microarrays,

PAGE 21

21 and with the ELISPOT or ELISA techniques. Various algorithms can be used to predict immunodominant sites of linear epitopes based on a putative antigens protein sequence. Predictions derived by each method should be compared, and only sequences identified by all three methods should be selected for epitope synthesis for experimental procedures. Genetic sequences coding for epitopes that are recognized by common antibodies can be fused to genes, thus aiding further molecular characterization of the gene product. The estimated molecular masses of immunodominant M. alligatoris antigens discovered in previous studies ( Brown et al., unpu blished) are not consistent with the predicted sizes of any of the surfaceassociated glycosidases (165.4 kDa hyaluronidase, 58.5 kDa sialidase, or 113.7 kDa beta-galactosidase) thought to be important virulence factors of M. alligatoris. However, the clear immunodominance especially of the 64 kDa antigen suggests a potential role in host -pathogen interaction in M. alligatoris mycoplasmosis. Functional studies directed by genome annotation revealed that M. alligatoris secretes glycosidases -galactosidase and hyaluronidase (Brown et al., 2004). A combination of virulence factors unprecedented among mycoplasmas, it is thought that these enzymes liberate glycans from host extracellular matrix (ECM) and glycosylated cell -surface molecules during nutrient scavenging and allow invasiveness of M. alligatoris infection. An antibody response may not always be protective, but it can potentially have diagnostic value. Since immunodominant antigens elicit the first detectable re sponse in a host, they can serve as diagnostic markers of disease, especially when a rapid diagnosis is desired. For example, immunodominant antigens have been identified and

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22 used in diagnostic applications for acute giardiasis and early onset periodontit is (Califano et al. 1991 ; Weiland et al. 2003 ). It is not clear if immunodominant antigens have a particularly important role in protective immunity. In any case, immunization and challenge studies would be required to validate the protective function o f an immunodominant antigen. The answer may be revealed in the biological function (if any) of the antigen that is to be neutralized or opsonized by its specific antibodies. For example, antibodies against proteins with functions in pathogenicity exposed to host cells later in infection may more likely be protective than those produced against those simply seen by the host immune system in the initial infection process. Antibodies against proteins that aid in adhesion to host cells may also be protective since they would theoretically help prevent pathogen invasion. Though surviving alligators in the M. alligatoris outbreak produced antibodies against particular proteins, it is currently unknown if they contained any protective function to the animals. Antigenic Variation and Immunodominance Many pathogens, including Mycoplasma spp ., Campylobacter fetus, Pneumocystis carinii and Giardia lamblia, as well as vector borne parasites such as Borrelia spp., Anaplasma and related genera, African trypanosomes, Plasmodium spp., and Babesia spp. utilize antigenic variation to evade the immune system. Surface lipoproteins play an important role in the pathogenesis of some Mycoplasma species by allowing escape from immune response ( Citti and Rosengarten, 1997 ). It has been shown that M. arthritidis has an expanded repertoire of variant membrane proteins with expression subject to independent, reversible phase variation ( Droesse et al., 1995) The complete genomes of other pathogenic bacteria, such as Helicobacter pylori

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23 Treponema pallidum and Mycobacterium tuberculosis contain large families of repeated genes with polymorphic sequences that are possibly involved in antigenic variation ( Vinall et al., 2002; Rindi et al. 2007; Radolf and Desrosiers, 2009). While immunodominant antigens can be products of antigenic variation, they can also be highly conserved proteins. For example, the majority of previously defined immunogenic vaccinia virus epitopes are conserved in variola virus and demonstrate the effica cy of using vaccinia virus in the smallpox vaccine (Sette et al., 2009). If M. alligatoris immunodominant antigens are highly conserved, they would serve as potential vaccine components as well as M. alligatoris mycoplasmosis diagnostic tools. Previous S t udies on the Temporal Antibody R esponse of C rocodilians to M. alligatoris To study the relationship between humoral immunity and progression of disease, the temporal patterns of antibody responses of American alligators experimentally inoculated and naturally infected with M. alligatoris were examined. In previous studies, 6 adult alligators were inoculated by instillation into the glottis of 1 mL of broth containing 106 CFU of M. alligatoris Four negative control alligators received either inoculation w ith sterile broth or no treatment. For comparison, caimans and crocodiles (n = 6 infected and 2 negative controls of each species) were also inoculated similarly. Blood samples were obtained at weekly intervals for 4 wk PI, then biweekly, until death or euthanasia 16 wk PI. M. alligatoris was cultured as early as 1 wk PI from peripheral blood of 3 inoculated alligators. Plasma from alligators naturally exposed during the 1995 epizootic was collected opportunistically. Western blots of M. alligatoris whol e cell lysate, as well as a fraction enriched for lipidassociated membrane protein antigens,

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24 were probed with plasma from inoculated caimans and crocodiles (Brown et al. unpublished). A positive immune response in individuals of all species surviving the experimental infection against an immunodominant 64 kDa antigen was detectable as early as 6 wk PI and increased in intensity until euthanasia 16 wk PI (Figure 11 A). For some individuals, a second, approximately 50 kDa antigen evoked a humoral immune re sponse. No difference in the patterns of humoral responses of caimans, crocodiles, and alligators could be discerned. Plasma samples were also obtained opportunistically in 1996, 1997, and 2000 from alligators exposed during the natural epizootic that oc curred in 1995. When M. alligatoris antigens were probed with plasma from these individuals, the same immunodominant bands as well as many others were revealed (Figure 11B ). This result indicated that after the initial acute infection, antibodies were e ventually made against substantially less immunostimulatory antigens. These more complex humoral responses could be due to these individuals having chronic infections or subsequent exposures to sublethal doses of M. alligatoris There is evidence from previous experiments that the immunodominant 64 kDa antigen exists in the LAMP enriched fraction ( Brown et al. unpublished ). However, as techniques that enrich for LAMP fractions can still contain soluble protein contaminati on, more work would need to be done to establish the proteins cellular location. First, its protein identity must be determined by peptide sequencing with a subsequent evaluation for immunodominance of its knockout mutant. Next information on the identified proteins classification, fu nction and cellular location can be collected.

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25 Objectives and Experimental Approaches Long Term Goals of R esearch on M. alligatoris The long term goal of M. alligatoris research is to improve our understanding of certain host pathogen interactions by using the alligator -M. alligatoris model. This goal involves work to (1) explain relative virulence of M. alligatoris, (2) develop predictive models of pathogen emergence and evolution, and (3) to gain insight on virulence of other Mycoplasma s pecies and related organisms. Infectious diseases represent a substantial global threat to human and animal health. Infectious diseases of wildlife should not be overlooked, as many newly recognized infectious diseases stem from zoonotic agents with wildlife r eservoirs. In order to develop new countermeasures such as vaccines and therapeutics, we must first have a better understanding of the mechanisms of pathogenesis, virulence factors, and host susceptibility. Genomics and proteomics provide tools to help us better understand these specialized fields. In turn, we are able to identify new drug targets and vaccine component candidates and improve diagnostics tools. Objectives Some species of Mycoplasma are pathogenic bacteria affecting diverse hosts, rangin g from humans to desert tortoises. Since very little is known regarding M. alligatoris (e.g. the natural reservoir, routes of transmission and host range of the pathogen), outbreaks are potentially very difficult to treat or control. Research to date has shown that crocodilians surviving mycoplasmosis seroconvert around 6 to 8 wk PI, forming specific antibodies against at least two bacterial components of the LAMP fraction. The identities of these antigens are currently unknown.

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26 The objectives of this work were to obtain protein identity candidates for the M. alligatoris 64 kDa immunodominant antigen and to test their respective knockouts for immunodominant antigen expression. These results may provide a basis for future work evaluating candidate knock out virulence in infection experiments. Further characterization such as antigen epitope mapping may facilitate diagnostic and therapeutic efforts. For example, antigen cloning and purification would be performed to test potential vaccine or diagnostic t est components. Results could serve as a model for other pathogenic species with similar characteristics. This work may provide more evidence for specific types of proteins that cause significant host immunostimulation during the acute infection phase. This information may help better predict bacterial cell targets of the vertebrate humoral immune system. Specifically, this could increase our knowledge of the host response of a non mammalian system. Hypotheses 1 Based on previous observations and the general importance o f membrane surface proteins in Mycoplasma pathogenesis, it is hypothesized that the approximately 64kDa -sized immunodominant antigen is associated with the M. alligatoris cell membrane. 2 The approximately 64 kDa -sized immunodominant antig en is a highly conserved protein. Specific A ims The specific aim of the study was to identify the 64 kD a immunodominant antigen of M. alligatori s This could be accomplished through several main approaches: 1 Antigen Isolation Affinity column chromatogr aphy will be used to capture the antigen that binds to specific antibodies in plasma samples collected 8-16 wk PI from exposed alligators. Two methods will be utilized. One column will be covalently bound with monoclonal antibodies against alligator ligh t chain immunoglobulin, which will capture antibodies from alligator plasma. A small

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27 fraction of the captured antibodies should be selective for the immunodominant antigen from M. alligatoris cell lysate. In the second method, a column will be covalently bound with whole seropositive alligator plasma, which will be used to capture the antigen. The alternative approach was isolation by 2DGE or BN -PAGE. Western blotting with plasma collected from infected alligators will be used to confirm isolation of ant igen. 2 Proteomic Analysis Peptide sequencing was performed on the purified putative antigen. Tandem mass spectrometry allowed protein identification with further validation by de novo sequencing. Complete amino acid sequences for the antigen could be determined from the annotated M. alligatoris genome (GenBank NZ_ADNC00000000). 3 Confirmation with Knockout Mutants Mutants (in an existing M. alligatoris knockout library ) corresponding to protein identifications with high confidence scores would be tested for immunodominant antigen expression by Western blot probed with infected alligator plasma. A lack of a reactive protein, or a peptide differing in size from the wild -type M. alligatoris 64 kDa antigen (due to the presence of the inserted transposon) would confirm its identity.

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28 A B Figure 11. Representative Western blots of M. alligatoris antigens probed with plasma from crocodilians exposed to M alligatoris A) Caimans, crocodiles and alligators were experimentally exposed to M. alligatoris Samples were obtained serially from weeks 1 through 16 post inoculation (M = molecular mass standards). B) Samples obtained opportunistically in 1996, 1997, and 2000 from two alligators, C203 and B271, that were exposed during the 1995 n atural epizootic in St. Augustine, FL (M = molecular mass standards).

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29 CHAPTER 2 MATERIALS AND METHODS Plasma s amples Alligator plasma samples were obtained during previous M. alligatoris i nfection studies and stored at 80 C (Brown et al. 2001b). For all affinity chromatography columns and Western blots in this current study, plasma from the following animals were used: accession number AA165099 collected 8 wks and 16 wk post -inoculation (PI), AA165089 collected 10 wk PI and AA165094 collected 10 wk PI. These individuals received 106 CFU of M. alligatoris (ATTC 700619, specimen A21JP2TMycoplasma c ulture. Wild -ty pe M. alligatoris previously frozen at 20 C was cultured at 30 C in ATCC medium 988 SP4 containing 0.5% (wt/vol) glucose and 20% (vol/vol) fetal bovine serum to the early log phase, as detected by acidification of the medium. To verify a MS protein identification, phosphopentomutase knockout and Nan A (irrelevant knockout) mutant cultures were grown in SP4 medium was cultured to the early log phase, as detected by acidification of the medium, at 30 C in SP4 broth containing 0.5% (wt/vol) glucose and 20 % (vol/vol) fetal bovine s erum, supplemented with 10 mg/mL tetracycline. first passage isolate of pure colony) by instillation through the glottis and were euthanized at the end of the study 16 wk PI. Plasma samples used in this study were ELISA positive against M. alligatoris whole cell lysate and shown to detect the 64 kDa band in preliminary experiments. Plasma samples were diluted 1:100 in 1x phosphate buffered saline (PBS) for all experiments. Knockout mutant library construction. A large -scale transposon mutagenesis library of tet R -selected M. alligatoris clones was previously generated by methods identical to Dybvig et al. ( 2008 ). After transformation by electroporation, mutagenization

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30 by random transposon insertion was performed using plasmid pTF20 carrying the minitransposon Tn4001TF2. Approximately 2,000 individual colonies were expanded and filter -cloned to assure h omogeneity. Unique single insertion knockout mutations have been obtained for approximately 650 of 950 clones precisely mapped by direct genomic DNA sequencing in both directions from the point of insertion, via extension from primers complementary to each IS256 arm of the transposon. Affinity Chromatography Antigen P urification Affinity c olumns HiTrap NHS activated HP columns (GE Healthcare Life Sciences, Piscataway, NJ) were prepared according to manufacturers instructions. The columns allow covalent coupling of ligands containing primary amino groups. The medium is based on SepharoseTMWhole cell lysate preparation Whole cell lysate used in the affinity chromatography column (first configuration) was previously prepared. Washed wild type M. alligatoris pellets were disrupted with lysis buffer containing NaHCO (highly cross -linked agarose) beads with 6 atoms spacer arms attached to the matrix by epichlorohydrine and activated by N hydroxysuccinimide. The substitution level i s medium. Nonspecific adsorption of proteins to HiTrap columns is negligible due to the hydrophilic properties of the base matrix. Three different affinity column configurations as described below were tested. After each elution, the column was washed 6 times with 1 mL of 1x PBS to clean and neutralize the column, sealed and stored at 4 C. 3 and Na2CO3 (pH 10.0), and samples w ere diluted to 100 mg protein/mL with 2.2% boric acid (Horowitz and Cassell, 1978). Aliquots were stored at 80 C until use in an affinity column.

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31 Lipid associated m embrane p rotein (LAMP) a ntigen p reparation LAMP antigen used in the affinity chromatography columns (second and third c onfigurations) was previously prepared (Wang and Lo, 1996). Cells were lysed in ice-cold 50 mM Tris -HCl buffer (pH 8.0), 0.15 M NaCl, 1 mM EDTA containing Triton X 114 with vortexing and sonication. The NaCl concentration was adjusted to 0.5 M, followed by centrifugation at 20,840 g for 20 min at 5 C to remove insoluble materials. Triton X 114 phase fractionation was performed by incubating lysates at 37 C, inducing condensation of the detergent. Cloudy and partially biphasic appearing lysates were th en centrifuged at 5,200 g for 10 min at 30 C. After removing aqueous supernatants, the detergent enriched phase was mixed and stored in aliquots at -80 C until use in affinity columns. First Column C onfiguration In the first configuration, a 1 m L column was used to couple 8 wk PI plasma from alligator AA165099 for 6 hours at 4 C. After the coupling procedure, the remaining coupling sites of the column were deactivated and washed. The column was then incubated with 10 mg wild -type M. alligatoris whole cell lysate for 12 hr. After washing wi th 1x PBS, 15 consecutive 200 uL -fractions were eluted with 100 mM glycine HCl (pH 3.0). The fractions were immediately neutralized with 100 mM TRIS buffer (pH 9.0) during the collection process (final pH approximat ely 7.0). Fractions were then pooled into 3 groups based on the order of their elution and microconcentrated with 30 kDa size exclusion centrifugal filters (Millipore, Billerica, MA) in 50 mM TRIS buffer (pH 6.8).

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32 Second C olumn C onfiguration Protein G -pur ified monoclonal mouse (subclass IgG2b kappa) anti alligator IgY light chain (HL1740 5H1-3B6) was previously generated at the ICBR Hybridoma Core Lab, Cancer Genetics Research Complex, University of Florida, Gainesville, FL. In the second configuration, a 1 m L column was used to couple 1.25 mg mouse anti alligator light chain for 16 hr. Coupling was validated as described below. After the coupling procedure, the column was deactivated and washed, and 8 wk PI plasma from alligator AA165099 was injected in to the column and incubated for 12 hr at 4 C. The column was washed with 1x PBS and then incubated with 10 mg wildtype M. alligatoris whole cell lysate for 12 hr. After additional washing wi th 1x PBS, 15 consecutive 200uL fractions were collected. Th e fractions were immediately neutralized, pooled into 3 groups and microconcentrated as previously described. After being coupled with the monoclonal antibody, the affinity column was used for several replicate binding and elution experiments to see if th e column could be reused. Third Column C onfiguration In the third configuration, a 5 mL column was used. To optimize contact with binding sites, samples were recirculated through the column with a variable flow peristaltic pump (Fisher Scientific Pittsbu rgh, PA ) and silicone capillary tubing. Eight wk PI plasma from alligator AA165099 was coupled during 12 hr of circulation at 4 C. The column was washed with 1x PBS and 3.8 mg LAMP antigen was recirculated for 12 hr. After washin g with 1x PBS, 15 conse cutive 1 mL -fractions were collected. The fractions were immediately neutralized, pooled into 3 groups and microconcentrated as previously described.

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33 SDSPAGE Separation of Affinity Chromatography E lutions After microconcentration, pooled elution samples were prepared in 2x Laemmli sample buffer with 350 mM DTT added fresh (Laemmli, 1970) or 4x NuPage LDS sample buffer (Invitrogen Life Technologies, Carlsbad, CA ) and separated by SDS PAGE on 7.5% Tris -HCl Ready gels at 70 V along with pre -stained protein standards. Second Affinity C olumn Monoclonal Antibody Coupling V alidation The following validation was performed to confirm that mouse monoclonal antibodies were covalently bound to the stationary phase beads in the second affinity col umn configuration. After two 1 mL1x PBS washes, the column was injected with 1 mL of alkaline phosphatase conjugate goat anti mouse IgG (H+L) (Promega Corporation, Madison, WI) diluted 1:100 in 1x PBS, sealed and incubated f or about 20 hr at 4 C. After incubation, the column was wa shed 6 times with 1 mL 1x PBS. Twenty consecutive 2 -drop (approximately 80 uL) fractions were collected starting with the first wash, and another series of 20 consecutive 2-drop fractions were co llected starting with the last wash. Next, f orty consecutive 2drop fractions were collected as soon as the f irst 400 uL of the elution buffer was injected into the column. Next, 50 uL aliquots of each fraction (first washes, final washes, elutions, posi tive controls, and negative control) were transferred to wells of a 96well microtiter plate in the same order of their collection from the column. The anti mouse IgG (H+L) alkaline phosphatase conjugate was used in the plate at four dilutions (1:100, 1:5 00, 1:1000, and 1:5000) for positive controls. Th e negative control was 1x PBS. The plate was sealed and stored at 4 C overnight. After rinsing the plate with 1x PBS three times, 0.1 mg substrate (4nitrophenyl phosphate disodium salt hexahydrate) was added to each well. Presence of

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34 antibody was determined by color reaction and absorbance readings of the plate wells at 405 nm. The remaining fraction volumes were then pooled intro groups (first washes, final washes, first elutions and final elutions), prepared in 4x NuPage LDS sample buffer plus 350 mM DTT and separated by SDS -PAGE along with a positive control on a precast 7.5% polyacrylamide Tris -HCl Ready gel (Bio-Rad Laboratories, Hercules, CA) at 70 V along with pre-stained protein standards (Bio Ra d Laboratories). The gel was prepared and subjected to silver staining with the SilverSNAP Stain Kit II (ThermoScientific Pierce, Rockford, IL). If the coupling procedure was successful, the expected outcome was to see at least one band at a molecular weight corresponding to structural components of the goat anti mouse IgG molecule. 2 Dimensional PAGE Antigen S eparation Membrane Protein P reparation. Wild -type M. alligatoris native membrane protein was extracted from approximately 100 mg of 1x PBS washed pelleted cells (wet weight) with the proprietary components of the ProteoExtract Native Membrane Protein Extraction Kit (Calbiochem, EMD Chemicals Inc., La Jolla, CA) according to manufacturer instructions. The purpose of using this kit was to extract in tegral membrane and membraneassociated proteins based on association of proteins with cellular membranes rather than on their hydrophobicity. It was not used specifically to preserve the native state of the extracted proteins, as they were later treated under denaturing conditions in preparation for and during 2DGE. Approximately 50 U Benzonase (Novagen, EMD Chemicals Inc.), a broad -spectrum endonuclease, was added to each approximately 100 mg cell pellet (wet weight) during the protein extraction protoc ol, directly after addition of the protease inhibitor cocktail provided in

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35 the kit. The purpose of adding an endonuclease was to degrade and remove nucleic acids, thus reducing sample viscosity. Samples were incubated at room temperature for an additional 30 minutes with gentle shaking. Protein concentrations were quantified with Bradford or DC assays (Bio -Rad Laboratories). After overnight precipitation with 100% ice -cold acetone, samples were centrifuged at 14,000 g for 15 min at 4 C, supernatant was removed and pellets were dried by vacuum centrifugation for 2-3 min immediately before rehydration and isoelectric focusing. Total Protein P reparation. Total protein was extracted by two different methods. In the first method, wild-type M. alligatoris total prot ein was extracted by adding 1 mL radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.0% IGEPAL CA 630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) (Sigma Aldri ch Co., St. Louis, MO) and 10 uL Halt Protease Inhibitor Single -Use Cocktail (ThermoScientific Pierce) to each approximately 100 mg (wet weight) 1x PBS washed cell p ellet. Samples were incubated at 4 C for 30 minutes with gentle shaking and then centrifuged at 14,000 g for 20 min at 4 C. Protein concentrations were quantified by Bradford or DC assays, precipitated with ice -cold 100% acetone and resuspended to 0.51 ug/uL in 2-D Rehydration/Sample Buffer (Bio-Rad Laboratories) containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG buffer (pH 3-10), 0.04 M DTT, and 0.002% Bromophenol Blue) immediately before rehydration and isoelectric focusing. The ReadyPrepTM total protein extraction kit (Bio-Rad Laboratories) was used according to manufacturer instructions for the second extra ction method. First, wildtype M. al ligatoris cell pellets of approximately 100 mg (wet weight) were washed with ice cold 50 mM Tris buffer (pH 6.8) and centrifuged at 14,000 g for 30 min at 4 C. After

PAGE 36

36 removal of supernatant, the cell pellets were incubated in 50 mM Tris buffer (pH 6.8), 1 x Halt protease inhibitor cocktail and 50 U Benzonase at 4 C for 1 hr with gentle shaking. After incubation, samples were centrifuged again and the supernatant wa s removed. To each pellet, 1 mL of complete 2-D Rehydration/Sample Buffer with 0.2% (w/v) I PG Buffer pH 3-10 NL ampholyte (GE Healthcare Life Sciences) was added as well as an additional 1x Halt protease inhibitor cocktail and 50 U Benzonase. The complete 2D Rehydration/Sample Buffer contains 7M urea, 2M thiourea, 1% (w/v) ASB -14 detergent, 40 mM Tris base, 0.001% Bromophenol Blue, and TBP reducing agent. The samples were chilled on ice and sonicated with an ultrasonic probe with 30 sec bursts 3 times, remaining on ice for 30 sec between each sonication treatment. Samples were then centrifuged at 14,000 g for 30 min at 18 C to pellet cellular debris. Supernatant was transferred to a clean tube and the insoluble pellet was discarded. Protein samples were quantified with CB X Protein assays (G -Biosciences, Maryland Heights, MO ) and diluted in more complete 2-D Rehydration/Sample Buffer 1 and 0.2% (w/v) IPG Buffer pH 310 NL ampholyte to obtain appropriate concentrations for isoelectric focusing (IEF) as described below. Active Rehydration Protein samples (200, 300 or 500 ug) were loaded onto 11 cm, pH 47 linear gradient immobilized pH gradient (IPG) strips (Bio-Rad Laboratories). The proteins were subjected to active rehydration and resolved in the first dimension in a PROTEAN IEF apparatus (Bio-Rad Laboratories). Active rehydration of the IPG gel strip was first carried out at 50 V for 8 hr. Active rehydration allows a more complete uptake of the protein in the sample into the IPG (K inter and Sherman, 2000) The process of active

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37 rehydration occurs most effectively with rehydration buffe r containing the sample to be separated, allowing the proteins to enter the gel along its entire length. As a result, more proteins are detected than in experim ents with passive rehydration. Isoelectric Focusing (IEF) Active rehydration was immediately fo llowed by IEF in the PROTEANSecond Dimensional SDS PAGE IEF apparatus. This procedure separates proteins according to their electric charge differences along a pH gradient. Each protein migrates until it reaches a point in the pH gradient corresponding to its isoelectric point (p I), the pH at which a molecule has a zero net charge. An anode positive end and cathode negative end are created by passing an electric current through the ampholyte medium (sample buffer), causing proteins above their pI, which have a negative net ch arge, to migrate toward the anode. Likewise, proteins below their pI, which have a positive net charge, migrate toward the cathode. IEF was performed under the following conditions: a linear increase of voltage to 250 V for 30 min, a linear increase to 500 for 1 hr, a linear increase 8000 V for 2.5 hr, a rapid increase to 8000 V for 40,000 Vhr, followed by a decrease to 50 V for 20 hr. After IEF, the strips were equilibrated in a solution of 50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue, and 1% DTT, followed by a second equilibration solution of 2.5% iodoacetamide, each for 15 min. Electrophoresis in the second dimension was performed on 816% Tris -Bis Criterion gels (Bio-Rad Laboratories) in 1x SDS running buffer at 60 V for 30 min, then 120 V until the loading dy e front reached the gel bottom. For gels of membrane proteins, a small gel plug containing approximately 20 ug wild-type M. alligatoris total protein was inserted in the gel just prior to electrophoresis to be used as a reference

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38 along with the molecular weight marker. The total protein for the gel plug was extracted with RIPA Buffer plus Blue Native Polyacrylamide Gel Electrophoresis (BN PAGE) Halt protease inhibitor as described above, then mixed with an equal volume of 2% agarose and 0.001% C oomassie Brilliant Blue R -250 powder (Bio Rad Laboratories). BN PAGE BN -PAGE allows isolation of protein complexes from biological membranes and total cell and tissue homogenates without disrupti ng their native state. The anionic dye Coomassie blue G -250 has hydrophobic properties, allowing it to interact with the surface of membrane proteins. Importantly, interacting with the dye makes mem brane proteins water -soluble while preserving their nati ve structure. Thus, no detergent is necessary to solubilize samples. In addition, binding the dye molecules imparts a negative charge on all proteins, making membrane proteins less likely to aggregate (Wittig et al., 2006). Wild -type M. alligatoris nativ e membrane proteins were extracted with ProteoExtractWestern B lotting Native Membrane Protein Extraction Kit as described above, or with the NativePAGE Sample Prep Kit (Invitrogen Life Technologies ) according to manufacturer instructions. Protein concentrations were qu antified with DC assays. Samples extracted by both methods were separated by BN -PAGE on a 4 -16% gradient NativePAGE Novex Bis Tris gel using amperage as low as 1 mA. Duplicate gels were stained and subjected to Western blotting as described below. For each set of samples analyzed b y Western blotting, one gel was stained after PAGE with standard Coomassie Blue stain ( Merril, 1990 ), Bio -Safe Coomassie Stain (Bio -Rad Laboratories), or Colloidal Blue Staining Kit (Invitrogen Life Technologi es ), and

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39 a duplicate gel was electroblotted onto a 0.2 um nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Transfers were performed at 15 V for 30 min on a Semi -Dry Transfer Cell Transblot SD (B io -Rad Laboratories) with 10% methanol 0.01% SDS tr ansfer buffer, or at 100 V for 3 hr by wet transfer with 2 -[ N -morpholino ] ethanesulfonic acid (Sigma-Aldrich Co. ) plus 0.01% SDS transfer buff er. The membranes were blocked with 5% nonfat dried milk in PBS -0.2% sodium azide (PBS/A) -0.5% or SuperBlock (ThermoScientific Pierce) for 1 hr at 25 C or overnight at 4 C, then incubated with either AA165099 8 wk PI or pooled alligator plasma 25 C for 1 hr. Previously prepared (Brown et al. 2001) biotinylated polyclonal rabbit anti alligator immunoglobulins (Ig) light chain and heavy chain antibodies were used for detecting bound alligator in Western blots. Bound specific antibodies were detected by incubation with biotinylated polyclonal rabbit anti alligator Ig. Membranes were incubated in alkaline phosp hatase-conjugated streptavidin diluted 1:1,000 in 1x PBS. The blots were developed in 1 Step NBT/BCIP (ThermoScientific Pierce In some transfer experiments, a second PVDF membrane was inc luded in the gel membrane blot paper sandwich, where it was placed directly on top of the gel. This top ) until bands became visible. To evaluate the validity of the elution samples from the third affinity column, a secondary antibody only control was performed. Affinity column elution samples were prepared in 5x lane marker sample buffer (ThermoScientific Pierce) and separated by SDS-PAGE on a 7.5% Tris -HCl Ready gel. After electroblotting and blocking, the PVDF membrane was incub ated with biotinylated secondary antibodies for 1 hr at 25 C, and then developed as described above. Membranes were washed with 1x PBS 0.05% Tween 20 three times for 5 min in between all steps.

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40 membrane was subjected to Colloidal Gold Total Protein Stain (Bio-Rad Laboratories) after electroblotting according to manufacturers instructions to obtain the protein pattern of the gel. The gold staining was discontinued after appearing unsuccessful. Phosphopentomutase K nockout M utant Evaluation To demonstrate the approach to confirming immunodominant antigen candidates identified by other methods, t otal protein was extracted from a M. alligatoris phosphopentomutase knockout mutant. The control was an irrelevant M. alligatoris sialic acid lyase knockout mutant (NanA). Approximately 100 mg (wet weight) PBS washed cell pellets were briefly boiled in 50 mM TRIS buffer, 2x Laemmli sample buffer and 0.1 % Triton X 100. Samples were kept on ice until separation by SDS -PAGE on a 7.5% Tris HCl Ready gel. Approximately 10 ug of protein was loaded per lane. One gel half was electroblotted to a PVDF membrane and subjected to Western blotting with high infection alligator p lasma as previously described. The other gel half, containing sample duplicates, was stained with Coomassie blue. Peptide S equencing and P rotein I dentification Protein R eduction, Alkylation and E nzymatic D igestion Proteins of interest were excised from gels based on corresponding Western blotting results and submitted for tandem mass spectrometry. Proteins selected for analysis were from eluted LAMP antigen (third affinity column configurat ion) separated by SDS-PAGE as well as total protein and membrane protein 2DGE separation. Tryptic peptides were prepared by ICBR Proteomics Core, Univer sity of Florida, Gainesville, FL for LC MS/MS analysis by the following method. To each excised gel s pot, 200 mL of 50 mM NH4HCO3 in 50% v/v acetonitrile was added and the sample was vortexed. The gel particles were brought to complete dryness with a vacuum centrifuge. Freshly

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41 prepared 45 mM DTT solution was added to the gel pieces for 45 min at 55 C. The DTT solution was removed and an equal volume of freshly prepared 100 mM iodoacetamide was added, followed by 45 min incubation at room temperature. The iodoacetamide solution was removed and the gel pieces were washed with 200 mL of 50 mM NH4HCO3 Approximately 510 uL of 12.5 ng/uL trypsin (Promega Corporation) in 25 mM NH in 50% v/v acetonitrile (pH 8.4) for 30 min while vortexing. The wash procedure was repeated three times. The gel particles were again brought to complete dryness with a vacuum centrifuge. 4HCO3 (pH 8.4) was added to each gel piece and kept at 4 C for 30 min. The enzyme solution as replaced with enough 25 mM NH4HCO3 (pH 8.4) to cover the gel particles and incubated for 1216 hr at 37 C. The react ion was stopped by adding 5% aqueous acetic acid to a final concentration of 0.5% acetic acid. After shaking and centrifugation, supernatant containing tryptic peptides was transferred to clean 0.5 mL tubes. The original gel pieces were treated with an additional 50 L of acetonitrile, agitated by sonication or g entle vortexing for 10 min, and centrifuged to obtain additional supernatant containing tryptic peptides. The pooled extracted peptides were completely dried with a vacuum centrifuge. To each sam ple, 15 L of loading buffer (3% acetonitrile [ACN], 1% acetic acid, and 0.1 % t rifluoroacetic acid [ TFA]) was added, followed by agitation by brief sonication or vortexing. For comparison, a sample was also separately digested with endoproteinase AspN (T hermoScientific Pierce) according to manufacturers instructions to produce different peptide fragments due to cleavage at N -terminally at aspartic acid and cysteic acid.

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42 LC -MS/MS Tandem mass spectrometry was performed with a nanoelectrospray ionization h ybrid quadrupole time of -flight mass spectrometer at the ICBR Proteomics Core, University of Florida, Gainesville, FL. The enzymatically -digested samples were concentrated and desalted onto a capillary trap, an Acclaim a PepMap -Precolumn cartridge (Dionex, Sunnyvale, CA), with 0.1% v/v acetic acid for 5 min with a flow rate of 10 L/min. Samples were next loaded onto an Acclaim PepMap100 Nano HPLC column (Dionex) for reversedphase chromatography. Peptides were eluted over a linear gradient from the c olumn starting at 3% solvent A, 97% solvent B and finished at 60% solvent A, 40% solvent B for 60. Solvent A consisted of 0.1% v/v acetic acid, 3% v/v ACN, and 96.9% v/v H2O. Solvent B consisted of 0.1% v/v acetic acid, 96.9% v/v ACN, and 3% v/v H2Database S earching O. LC MS/MS analysis was carried out on a QSTAR XL or QSTAR Elite (Applied Biosystems, Framingham, MA) mass spectrometer. The focusing potential and ion spray voltage was set to 275 V and 2600 V, respectively. The informationdependent acquisition (IDA) mode of operation was employed, in which a survey scan from m/z 400 1200 was acquired followed by collision-induced dissociation of the three most intense ions. Survey and MS/MS spectra for each IDA cycle were accumulated for 1 and 3 s, respectively. LC -MS/MS spectra were extracted, deisotoping was performed, and charge states were deconvoluted by ABI Analyst version 1.1 or 2.0 software. All LC -MS/MS data were submitted for database searching using Mascot (Matrix Science, London, UK; version 2.0.01). A second, more stringent analysis was performed with Scaffold 2

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43 (version Scaffold 0203 -01, Proteome Software Inc., Portland, OR) software, which uses the two algorithms Mascot and X! Tandem. Only results in Scaffold 2 were examined due to highe r stringency. However, all data had to be first submitted to Mascot before they could be run in Scaffold 2. Scaffold 2 Scaffold 2 i dentifies proteins from MS/MS data by comparing it to a database of known proteins and validating with Mascot*/Sequest*/X! Tandem*/Phenyx* searches. Sequence coverage is automatically calculated within the software. Protein identification results are displayed as probability scores, allowing you to balance the number of proteins and the confidence level of each identificati on. The algorithm for calculating the peptide probabilities from the search engine scores is described by Keller et al. (2002). The algorithm for calculating the protein probabilities from the peptide probabilities is described in Nesvizhskii et al. (2003 ). The algorithm for combining results from multiple searches is described by Searle et al. (2008). The algorithm for grouping proteins across samples is described by Searle (2010). Mascot Mascot is a software package that converts mass spectral data i nto protein identities. Mascot compares the observed spectra to a database of known proteins and determines the most likely matches. Each match is then assigned a P value equaling the likelihood that the match is incorrect (E~PN). Mascot is also one of the algorithms used in the software package Scaffold 2. X! Tandem X! Tandem, the second algorithm in Scaffold 2, is by itself an opensource s oftware package that matches the acquired MS/MS spectra to a model spectrum based on peptides in a specified protein database. Each match is assigned an E value equaling the likelihood that the match is incorrect (E~PN). The model

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44 spectrum is based on the presence or absence of y and b ions. The program considers semi tryptic peptides, sequence polymorphisms and u ses probability based scoring. It automatically searches for missed cleavages, semi tryptic peptides, post -translational modifications, and point mutations. The algorithm for X! Tandem is described in Craig and Beavis ( 2003). An analysis was performed on eluted antigen samples with the anole ( Anolis carolinensis ) protein database available in GenBank ( NC_010972) in Mascot and Scaffold 2. To date, no other r eptilian genome was more complete. The purpose of this analysis was to evaluate the unintended possibility of the eluted antigen being part of the alligator immunoglobulin molecule instead of the desired M. alligatoris antigen. This database, containing o nly 328 proteins, was chosen because of the 276 American alligator ( A. mississippiensis ) protein sequences currently available through GenBank, none were immunoglobulin associated. Trypsin or AspN enzymes were specified in Mascot MS/MS ion searches and Sca ffold 2 searches. Mascot settings included a fragment ion mass tolerance of 0.30 Da and a parent ion tolerance of 0.30 Da. Iodoacetamide derivative of Cys, deamidation of Asn and Gln, oxidation of Met, were specified in Mascot as variable modifications. In Scaffold 2, Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 0.50 Da. Iodoacetamide derivative of cysteine was specified in Mascot and X! Tandem as a fixed modification. Scarbamoylmethylcysteine cyclization (N -terminus) of the n -terminus, deamidation of asparagine and oxidation of methionine were specified in Mascot as variable

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45 modifications. S -carbamoylmethylcysteine cyclization (N terminus) of the n -terminus, deamidati on of glutamine, oxidation of methionine and iodoacetamide derivative of cysteine were specified in X! Tandem as variable modifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by t he Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified unique peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. De N ovo Sequencing Protein Identifications LC -MS/ MS spectra were extracted, deisotoping was performed, and charge states were deconvoluted by ABI Analyst version 1.1 or 2.0 software. Data were converted to XML format and submitted for de novo sequencing using PEAKS Studio 5.1 (Bioinformatics Software, I nc.) PEAKS Studio 5.1 De novo sequencing is a process in which peptide sequences are derived from the masses of their fragments as shown on a tandem mass spectrum without the use of a protein sequence database for reference. This process minimizes false positives, and when combined with database protein identification, offers optimal result validation. PEAKS Studio is a tandem MS program with applications to determine protein/peptide sequences and respective quantitative properties. One major beneficia l feature is that confidence is not only on the peptide sequence, but also on each amino acid assignment. Fragmentation flexibility includes y/b ions or c/z ions. The program also has capabilities to perform an internal database

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46 search engine, meta protein identification (inChorus) tool, peptide sequence tag homology search program/sequence reconstruction method (SPIDER), and post translational modification identification and quantification. According to the PEAKS manufacturer, the algorithm first comput es a y -ion matching score and a b ion matching score at each mass value according to the peaks around it. If there are no peaks around a mass value, a penalty value is assigned. The algorithm then efficiently computes many amino acid sequences that maxim ize the total scores at the mass values of b ions and y ions. These candidate sequences are further evaluated by a more accurate scoring function, which also considers other ion types such as immonium ions and internal -cleavage ions. The problem of ion absence is addressed because the PEAKS model assigns a score (or penalty) for each mass value. The software also computes a positional confidence for each amino acid in the final result by examining the consensus of the top-scoring peptides. PEAKS Criteria S ettings PEAKS data refinement included deisotoping, deconvolution and centroiding. Error tolerance parameters included 0.5 Da for parent ion and 0.5 Da for fragment ion. Deamidation, oxidation, pyro glu from Q, and carbamidomethylation post translati onal modification tolerances were selected. Only results with an ALC (%) score of at least 80 were reported or further evaluated.

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47 CHAPTER 3 RESULTS AND DISCUSSION Affinity C olumns The first affinity column configuration had alligator plasma directly coupled to its SepharoseTM beads and was then incubated with wild-type M. alligatoris whole cell lysate antigen. After multiple attempts to optimize elution, sample solubilization, and SDS-PAGE resolution, protein was visualized on a 7.5% Tris -H Cl gel by Coomassie blue staining. Very faint bands on the gel corresponding to approximately 64 kDa, 250 kDa, and >250 kDa were seen (not shown). However, Western blots were not performed on these elution samples, and thus immunodominance of the 64 kDa p rotein could not be determined. T he very faint stain intensity of the 64 kDa band appr oached the minimum protein concentration detectabl e by basic Coomassie blue staining, or 50 100 ng/ mm3 It is not known why multiple bands were seen in the separated elution samples. After alligator plasma coupling in the first column, PBS washes were collected and separated by SDS -PAGE. The last PBS washes, right b efore elution, were confirmed to be protein-free by SDS -PAGE separation and Coomassie blue staining. Therefore, it gel volume ( Weiss et al., 2009) Nanoelectrospray ionization expe riments on a QSTAR XL MS/MS system have shown that 1 2 fmol of sample digest already in HPLC solvent is sufficient for protein identification concentration, but LC -MS/MS results are improved with higher concentrations (Chen, 2006). W ith in gel digestions, there is significant peptide loss from the gel matrix during sample preparation (Chen, 2006). Though sequencing may have still worked with the low protein concentration of the given sample, efforts were instead made to increase affinity column elution co ncentrations before attempting LC MS/MS.

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48 was thought that all non-specific binding of M. alligatoris proteins were removed prior to elution. However, it is possible that non -specif ic material coincident ally containing a 64 kDa protein was retained in the column until the elution process. For example, the IgY heavy chain has a molecular mass of 65 -70 kDa (Hatta et al., 1993; Sun et al., 2001 ). The 64 kDa protein may have been part of the all igator immunoglobulin molecule or any other plasma protein of a similar molecular weight that leached from the stationary phase Molecular weight alone would not strongly indicate that the larger (250 kDa and >250 kDa) bands from the first affin ity column elutions were from residual immunoglobuli n s. T he whole IgY molecule has been estimated to be approximately 180 kDa b y SDS -PAGE and approximately 167 kDa by MALDI -TOF (Sun et al., 2001; Hatta et al., 1993). F ormations of protein dimers, protein modifications resulting in increased molecular weight, or problems such as poor sample solubility and resolution m ay have also affected the protein separation patterns on the gels. The second affinity column configuration had mouse monoclonal anti alligator IgY light chain coupled directly to it s beads. The column was then incubated with alligator plasma, followed by incubation with wild -type M. alligatoris LAMP antigen. The coupling validation study demonstrated that the monoclonal mouse anti alligator IgY light chain did indeed covalently bind t o the columns beads (Figure 3-1). However, a lack of protein staining on SDS -PAGE-separated elutions indicated unsuccessful antigen capture It is possible that the a ntibodies i n the column were unable to bi nd antigen. For example, once alligator Igs were bound to the monoclonal antibodies by their light chains, there may have been a physical barrier preventing specific antigens access to their respective antibody binding sites. Another likely issue was simply poor antigen

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49 enrichment. For example, perhaps too few M. alligatoris antigen -specific IgY molecules were able to bind to the monoclonal antibodies simply due to naturally low concentrations relative to other plasma protei ns. M aximum binding efficiency would be necessary for all of the antibodies to be captured by the coupled mouse monoclonal antibodies. All of these factors would result in a relatively low number of antigen-specific immunoglobulins in the second column co nfiguration. Assuming that their orientations in the column would allow successful antigen capture, this configuration would still only capture a very small total amount of antigen. Without any previous knowledge of the immunodominant antigen identity, th ere was no accurate way to predict its relative abundance within a certain volume of LAMP or whole cell lysate. Lacking this knowledge, it is uncertain that the column was incubated with the ideal amount of antigen material. IgY concentrations, including those specific for M. alligatoris antigens, were not known for the alligator plasma samples used in the affinity columns. Though plasma concentrations of i mmunoglobul in isotypes are not well documented in reptiles predictions regarding alligator antigen-specific IgY plasma concentrations can be made from other animal models. Total protein values, including globulin values, were measured for the alligator plasma samples as part of a comprehensive biochemistry profile. Most of the alligators, including both normal and infected animals, had total protein and globulin values within the normal ranges for A. mississippiensis : 5.31.3 g/dL and 3.51.0 g/dL, respectively. These A. mississippiensis reference values are also relatively similar to normal total protein and globulin values observed in mammals (Diethelm, 2005; Diethelm and Stein, 2006 ). Total IgG levels can be measured by rate

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50 nephelometry For example, serum levels of IgG, comprising part of the globulin fraction, have been determined in healthy humans (6001,400 mg/dL), mares (1,352.9 1 mg/dL), and dogs (1,560 116 mg/dL) (OReilly, 1993; Ginel et al., 1997; Murphy et al., 2008). IgG subclass concentrations can be determined by immunoradiometric assay (IRMA) using monoclonal antibodies specific to individual subclasses of IgG conjugated to polyglutamated -carboxymethyl se phacryl beads. For example, IgG 1, IgG 2, IgG 3, and IgG 4 subclasses range at about 300 -7,000 mg/L in healthy humans, and all subclasses can increase to varying degrees during processes such as chronic schistosomiasis (Boctor and Peter, 1990). In diseased humans, the same IgG subclasses ranged from 1,400 15,000 mg/L (Boctor and Peter, 1990). These va lues might be used to predict upper limits for IgY subclass concentrations in healthy or infected alligators. S pecific antibody titers after infection are generally lower in reptiles compared to mammals or birds and reptilian secondary responses rarely h ave antibody titers and affinities h igher than in primary responses (Hsu, 1998; Brown, 2002). In addition, only a small percentage of these circulating antibodies might be specific for a single antigen, for example, if multiple antigens are stimulating th e host immune system at the same time. The third affinity column configuration was similar to the first configuration in that alligator plasma was directly coupled to it s beads. However, this column was five times greater in volume, and like the second configuration, wildtype M. alligatoris LAMP was used as a source of antigen Both the alligator plasma and antigen were recirculated with a peristaltic pump to increase overall binding. A protein band from this source (pooled elution aliquots) was submitt ed for LC MS/MS three separate times, each with

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51 slight differences in its treatment. The first submission was a 64 kDa band previously subjected to SDS -PAGE separation on a 7.5% Tris -HCl gel and detected by Coomassie blue staining (Figure 3-2A ), subjected to ingel trypsin digestion prior to LC MS/MS. The protein band was immunoreactive with alligator pl asma by Western blot (Figure 32B ). The second submission was similarly treated and separated by SDS -PAGE, but subjected to ingel AspN (flavastacin) dig estion and LC -MS/MS. This restriction enzyme, a zinc metalloendopeptidase, selectively cleaves peptide bonds N -terminal to aspartic acid residues (Tarentino et al., 1995). The purpose of using a different restriction enzyme was to increase the number and types of peptide fragments available for LC MS/MS analysis. Because fixation with methanol and acetic acid can sometimes interfere with enzymatic digestion of the protein, Bio Safe Coomassie stain was used as a fixative -free stain alternative for other sample aliquots. Therefore, the third submission for LC MS/MS was a 64 kDa band visualized with Bio Safe Coomassie stain and subjected to in -gel trypsin digestion. All affinity column elution samples submitted for LC MS/MS were confirmed by Western blot to have an approximately 64 kDa immunoreactive band as represented in Figure 32B To ensure that these Western blot results were accurately confirming the presence of M. alligatoris antigen, a s econdary antibody only (anti alligator Ig ) control Wes tern blot of the same column elution sample was performed. A valid control result would have no band develop. However, a positive signal on the control was seen at approximately 64 kDa (Figure 3 3 ). Such results indicated possible non-specific binding o r alligator antibodies leached off from the column, which were then recognized by the secondary anti alligator Ig antibody.

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52 Protein matches from the three elution samples were evaluated in Scaffold 2 under specific stringency settings (Table 3 -1). Top mat ches included Lmp3 protein, putative (ORF01306), viral A type inclusion protein, putative (ORF00114), DNA polymerase III subunit, putative (ORF01370) and lipoprotein, putative (ORF00858). DNA polymerase III subunit, putative, from the trypsindigested Bio-Safe Coomassiestained sample, had the highest amino acid coverage (14%). However, the overall quality of spectra from these biological samples was low Quality of spectra was evaluated in Scaffold 2 by X! Tandem and Mascot probability score scatterplots and distributions describing the number of correct and incorrect assigned spectra for the entire biological sample. Matches were evaluated for extent of amino acid coverage, match probability scores, the m/z versus relative intensity spectrum, spectrum /model error data and fragmentation tables. Based on these factors, none of the eluted antigen protein matches were of strong confidence. LC -MS/MS data from elution samples were evaluated for protein matches in the M. crocodyli genome to see if proteins homologous to M. alligatoris would also show up. However, no M. crocodyli matches homologous to any of the M alligatoris matches were found. If the rabbit polyclonal anti alligator Ig dete cted alligator IgY heavy chain, a positive signal for the secondary antibody only -control Western would appear about 67 70 kDa. To investigate the possibility IgY heavy chain leaching off the column and being present in elution samples the LC MS/MS data was also compared to the genome of the green anole, Anolis carolinensis in S caffold. This database, containing only 328 proteins at the time of analysis allowed only a limited evaluation. The only

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53 immunoglobulin heavy chain associated amino acid sequences in the Anolis database were the following: gi|15769508 3|gb|ABV66132.1 immunoglobuli n Y heavy chain constant region gi|157695081|gb|ABV66131.1 immunoglobuli n Y heavy chain constant region gi|157695079|gb|ABV66130.1 immunoglobuli n D heavy chain constant region gi|157695077|gb|ABV66 129.1 immunoglobulin Y delta F c gi|157695075|gb|ABV66128.1 immunoglobulin M heavy chain constant region gi|157380924|gb|ABV46492.1 immunoglobuli n heavy chain constant region The most relevant match was immunoglobulin light chain kappa variable region (gi|171464617). However, Scaffold 2 algorithms indicated that confidence for this match was overall low. In addition, its predicted molecular weight (14 kDa) did not explain control Western blot observations at approximately 64 kDa Standard protein -protein BLAST ( BLASTP) searches were a lso performed using de novo sequences obtained from two affinity column elution sample submissions: the in gel AspN digestion and ingel trypsin digestion, both stained with regular Coomassie. The actual peptide sequences obtained through PEAKS Studio are reported in Tables 3 -3 and 3-4. The best matches found by performing BLASTP searches on the de novo sequences ( M. alligatoris glycosyl hydrolase family 2, ABC transporter permease protein, and M. crocodyli Holliday junction DNA helicase, etc.) were not s ignificant (Table 36). Knockout Mutant Immunodominant Antigen Expression E valuation Phosphopentomutase (deoB) was identified with an initial Mascot search from the in gel trypsin digested Coomassie stained elution sample against the M. alligatoris genome database. This identification was not significant (Mascot score = 43). However, deoB was previously identified as a putative immunogenic S. aureus protein by MALDI -TOF by other researchers (Glowalla et al., 2009). Since a deoB mutant was

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54 available in the M. alligatoris mutant library, it was evaluated for immunodominant antigen expression. Total protein was extracted from the deoB mutant as well f rom as an irrelevant M. alligatoris knockout, sialic acid lyase NanA. Samples were prepared in 2x Laemmli sample buffer and separated by SDS PAGE on a 7.5% Tris HCl gel. A Western blot probed with infected alligator plasma revealed identical immunoreacti vity patterns. A band for both mutants developed at approximately 60 kDa (Figure 34 ). This result indicated that phosphopentomutase was not the correct immunodominant antigen. 2DGE Protein S eparation Both membrane proteins and total protein of M. alliga toris separated by 2DGE and blotted onto PVDF membranes, were further analyzed by immunoblotting. The Western blots were used to select protein spots from their corresponding 2-D gels. 2DGE Membrane Protein S eparation Two immunoreactive areas containing membrane proteins were excised (indicated by circles) from a 2 -D gel (Figure 3 5A ). These areas on the gel best aligned with two positive signals on its corres ponding Western blot (Figure 35B ), which appeared at approximately 50 kDa and 30 kDa. Each protein spot was separately subjected to ingel trypsin digestion and LC MS/MS. Best matches in Scaffold 2 were reported (Table 3 -2). The quality of the MS/MS data was high, and protein matches had high confidence as indicated by various Scaffold paramete rs. However, the likelihood that any of these matches were the a ctual immunodominant antigen seemed low due to s everal issues. A n unstained molecular marker was used for these gels. The marker was visualized in the gel used for selecting the protein spot once it was stained with BioSafe Coomassie. However, the gel used for the Western transfer could not be

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55 stained prior to electroblotting, and thus the membrane itself did not have a visible molecular marker. The gel used for electroblotting was stain ed afterwards to visualize any remaining proteins. This post transfer gel was aligned with the Western blot to estimate where the molecular marker bands should have belonged on the membrane. Other physical references were aligned between the membrane and the gels to excise the two spots. Despite best efforts, accuracy could not be guaranteed. N one of the other 2DGE membrane protein Western blots developed a positive signal. It could not be explained why there were two positive signals instead of one, o r why they appeared at approximately 50 kDa and 30 kDa. 2DGE Total Protein Separation One immunoreactive protein area could be detected by alligator plasma (Figure 3 6B ) on a 2DGE total protein Western blot. Replicate gels and Westerns were run to see i f the same protein identifications could be repeated independently. This band corresponded to a protein approximately 100 kDa in molecular weight (Figure 36A). The results of the immunoreactive protein area that was investigated ( enciricled) by in gel t rypsin digestion and LC MS/MS are elaborated in Table 3 -3. Five protein matches were found using highest stringency settings: Min. Protein = 99.9%, Min # Peptides = 5, Min. Peptide = 95%. One of the top matches from the total protein sample had 33% amino acid sequence coverage with a sequence fragment named lipoprotein from the original FASTA database ( Figure A 1 ). The match had relatively good amino acid coverage in Scaffold 2 compared to other matches. Overall c onfidence in this match was increased since it was identified in two independent 2DGE gels and LC MS/MS sequencing experiments, as well as from de novo analysis. When the complete sequences were

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56 made available in GenBank (after database searc h es were perf ormed in this study), the protein name was changed to c onserved hypothetical protei n (accession no. ZP_06610777.1). A BLASTP search of the lipoprotein sequence had 100% query coverage (E value e-130) with ZP_06610777.1. This full sequence had a theoret ical molecular weight of 89.5 kDa. A lipid addition covalently bound to the protein could result in a molecule with a greater molecular weight. This possibility could explain the immunoreactive protein visualized at 100 kDa. The next best BLASTP matches to the lipoprotein sequence were all lipoproteins or membrane associated in Mycoplasma, including: M. crocodyli lipoprotein (YP_003560325.1), M. fermentans (BAH70134.1), M. pulmonis lipoprotein (NP_326468.1), and M. galliseptic um str. F conserved hypothetical protein (ADC31412.1). Thought some streptococcal proteins were also in the BLASTP match results, their E values were too high to be considered significant. Horizontal streaks were a prevalent problem for the 2DGE experimen ts, especially not iceable in the gels (Figures 3 -5 and 3-6 ). Several modifications were made to address horizontal streaks seen on the 2-D gels and Western blots. Common contaminants include salt, detergents, peptides, nucleic acids, lipids, and phenolic compounds (Chang et al., 2003). Sample preparation modifications included using different protein extraction methods, ensuring that salt concentration was adequately low, and treating protein samples with endonuclease to reduce nucleic acid contamination Different agarose and polyacrylamide overlays were tested to see if a better seal could be made between the IPG strip and the gel before second dimensional electrophoresis. Since no major reductions in horizontal streaks could be seen with

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57 these adjust ments, it is thought that the major problem was in adequate isoelectric focusing. Blue Native PAGE Wild -type M. alligatoris native membrane protein was extracted and separated BN PAGE on a 4-16% gradient NativePAGE Novex Bis Tris gel. Gels were stained an d duplicate gels were subjected to Western blotting. However, Westerns were never successful. The inability to remove an adequate amount of Coomassie stain from the gels prior to electroblotting resulted in extremely high background during immunoblotting

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58 A Figure 31 Coupling validation of affinity column (second configuration). Monoclonal mouse (subclass IgG2b kappa) anti alligator IgY light chain (HL1740 5H1 3B6) was coupled directly to the columns SepharoseTM beads. The column was then incubated with goat anti mouse IgG (H+L) alkaline phosphatase c onjugate. A) Samples were prepared in 4x NuPage sample buffer, separated by SDS -PAGE on a 7.5% Tris -HCl Ready gel and visualized by silver stain. W = first 12 pooled 80ul PBS washes collected from the column before elution, containing excess goat anti -mouse IgG (H+L) Alkaline Phosphatase Conjugate that did not couple to beads. E = first 12 pooled 80ul fractions eluted from the column with glycine HCl elution buf fer. The anti mouse IgG (H+L) alkalin e phosphatase conjugate was used as a positive control. Note: the last PBS wash samples, which did not contain any protein as indicated by absorbance readings at 405 nm, were not run on this gel. B) PBS wash fractions prior to elution as well as elution fractions were consecutively collected from the column. Absorbance of each fraction after treatment was read at 405 nm in a plate reader. PBS washes = 40 consecutive 50ul PBS wash fractions collected Elutions = 40 consecutive 50ul glycine HCl elution fractions collected Positive (+) Controls = anti mouse IgG (H+L) alkaline phosphatase conjugate was used at four dilutions, which were measured in the following order: 1:100, 1:500, 1:1000, and 1:5000. The negative control was 1x PBS.

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59 B Figure 31. Continued

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60 A B Figure 32 M. alligatoris LAMP antigen eluted from affinity chromatography column (third configuration). A) Samples were prepared in 2x Laemmli sample buffer with DTT, separated by SDS PAGE on a 7.5% Tris -HCl Ready gel, and visualized by Coomassie blue staining. E = pooled elution fractions #4-9. M = pre-stained molecular mass standards. Bands indicated by the arrow were excised. Peptides produced by both in-gel trypsin and Asp-N digestion were subjected to LC -MS/MS. B) Western blot of LAMP antigens eluted from affinity chromatography column (third configuration) probed with infected alligator plasma (accession number AAA165099). E = pooled elution fractions #49 from first antigen incubation of third column configuration. M = pre -stained molecular mass standards.

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61 Figure 33 Secondary a ntibody o nly c ontrol. Affinity column e lution samples from the third column configuration were separated by SDS -PAGE on a 7.5% Tris -HCl Ready gel and electroblotted onto a PVDF membrane. After incubation with biotinylated polyclonal rabbit anti alligator Ig antibodies, the membrane was developed to reveal a band at the same molecular weight as that from the third column configuration elutions (approximately 64 kDa). The corresponding band on a Bio -Safe Coomassie -stained duplicate gel (not shown) was excised. Tryptic peptides were subjected to LC -MS/MS, the results of which are elaborated in Table 3-1.

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62 Figure 34 We stern blot of M. alligatoris total protein extracted from a phosphopentomutase knockout mutant (342), and from an irrelevant knockout mutant, NanA (209) as a control. The blot was probed with infected alligator plasma (accession number AA165099) and devel oped, revealing identical band patterns.

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63 A B Figure 3 5 M. alligatoris membrane proteins separated by 2DGE. A) Membrane proteins (300 ug) were resolved in a 4 7 linear pH gradient in the first dimension, a 8 16% gradient SDS -PAGE in the second dimension and visualized by Bio Safe Coomassie staining The results of the proteins that we re identified (encircled) by in gel trypsin digestion and LC MS/MS are elaborated in Table 32. Gel image colors were reversed to allow better visuali zation of the proteins and molecular marker. B) A duplicate gel was electroblotted onto a PVDF membrane and probed with a mix of plasma from three infected alligators: AA165099 8 wk PI, AA165094 10 wk PI, and AA165089 10 wk PI. Two immunoreactive areas w ere observed, as indicated by arrows. The molecular marker in this particular Western blot could not be visualized properly. Note, images in A and B are not to scale.

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64 A B Figure 3 6 M. alligatoris total proteins separated by 2DGE. A) Total proteins (300 ug) w ere resolved in a 4 7 linear pH gradient in the first dimension, and an 8 16% gradient SDS PAGE in the sec ond dimension. The immunoreactive protein area (encircled) was selected for ingel trypsin digestion and LC MS/MS. D e novo seque ncing was also performed. Best protein matches are elaborated in Table 33 B) A duplicate gel was electroblotted onto a PVDF membrane and probed with plasma from infected alligator AA165099 16 wk PI. An immunoreactive area was observed, indicated by th e arrow.

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65 Table 3 1. M. alligatoris proteins identified by LC MS /MS and evaluated in Scaffold (p eptide sources : in gel trypsin or AspN digestion of third column configuration LAMP antigen elutions separated by SDS PAGE ). Putative identifications GenBank Accession Number Theoretical Mol Wt (kDa) Column elution sample submitted Sequence coverage (%) ** Top Mascot sequence Ion Score ** M protein repeat protein ZP_06610681.1 110 3 rd 5 n/a Hypothetical protein MALL_0758 ZP_06610553.1 50.4 3 rd 13 1.5 Hypothetical protein MALL_0396 ZP_06610600.1 35.8 3 rd 14 24.2 Hypothetical protein MALL_0656 ZP_06610235.1 58.9 3 rd 7 14.8 Translation initiation factor IF 2 ZP_06610653.1 66.4 1st 4 26.1 *Protein identifications and related information according to their reference sequences GenBank. Theoretical molecular weight s are based on complete peptide sequences. LC MS/MS data were searched against both M. alligatoris and M. crocodyli, though no M. crocodyli matches were reported due to extremely low confidence scores. ** Sequence coverage % and top Mascot sequence ion scores of protein matches as seen in Scaffold based on LC MS/MS data searched against incomplete FASTA peptide sequences. Criteria s ettings in Scaffold: Min. Protein = 99.9%, Min # Peptides = 2, Min. Peptide = 95%. Column elution sample submitted: (1st) in gel trypsin digestion, Coomassie blue stain; (2nd) ingel AspN digestion, Coomassie blue stain, (3rd) ingel trypsin digestion, Bi o Safe Coomassie stain. Table 3 2. M. alligatoris proteins identified by LC -MS/MS and evaluated in Scaffold (peptide sources: ingel trypsin digestion of M. alligatoris membrane protein separated by 2DGE). Putative identifications GenBank Accession Nu mber Theoretical Mol Wt (kDa) Sequence coverage (%) ** Top Mascot sequence Ion Score ** Conserved hypothetical protein ZP_06610710.1 79.9 27 123.3 T ranslation elongation factor G ZP_06610590.1 76.8 23 86.3 Hypothetical protein MALL_0411 ZP_06610774.1 81.7 25 76.9 Phenylalanyl tRNA synthetase, beta subunit ZP_06610855.1 79.0 10 38.5 ABC transporter, ATP binding protein ZP_06610702.1 79.4 31 71.0 Chaperone protein DnaK ZP_06610514.1 67.1 15 52.9 S1 RNA binding domain protein ZP_06610410.1 79.9 15 63.8 Acyltransferase ZP_06610154.1 27.8 26 87.7 Ribosomal protein L Z P_06610409.1 24.9 26 83.2 Ribosomal protein S5 ZP_06610363.1 24.0 35 64.2 *Protein identifications and related information according to their reference sequences GenBank. Theoretical molecular weights are based on complete peptide sequences. LC MS/MS data were searched against M. alligatoris only. **Sequence coverage % and top Mascot sequence ion scores of protein matches as seen in Scaffold based on LC MS/MS data searched against incomplete FASTA peptide sequences. Criteria settings in Scaffold: Min. Protein = 99.9%, Min # Peptides = 4, Min. Peptide = 95%.

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66 Table 3 3. M. alligatoris proteins identified by LC -MS/MS and evaluated in Scaffold (peptide sources: ingel trypsin digestion of M. alligatoris total protein separated by 2DGE). Putative identifications GenBank Accession Number Theor etical Mol Wt (kDa) Sequence coverage % ** Top Mascot sequence Ion Score ** D xylulose 5 phosphate/D fructose 6 phosphate phosphoketolase ( M. crocodyli ) gi|291600221 89 12 68.7*** C onserved hypothetical protei n ZP_06610777.1 89.5 33 69.8*** Dihydrolipoyl dehydrogenase ZP_06610150 78.3 17 59.6*** Translation elongation factor Tu ZP_06610740.1 43.2 18 94.3 Hypothetical protein MALL_0531 ZP_06610333.1 98.5 15 85.7 Protein identifications and related information according to their reference sequences GenBank. LC MS/MS data were searched against M. alligatoris and M. crocodyli Theoretical molecular weights are based on complete peptide sequences. **Sequence coverage % and top Mascot sequence ion scores of protein matches as seen in Scaffold based on LC MS/MS data searched against incomplete FASTA peptide sequences. All identifications were derived from samples subjected to BioSafe Coomassie staining and ingel t rypsin digestion. Criteria settings in Scaffold: Min. Protein = 99.9%, Min # Peptides = 5, Min. Peptide = 95%. ***Protein identified twice independently (separate 2DGE and LC MS/MS experiments).

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67 Table 3 4. M. alligatoris proteins identifie d by protein-protein BLAST searches of de novo sequence data reported in Appendix Tables A -1, A 2 and A 3 (peptide source: ingel trypsin digestion of third column configuration LAMP antigen elutions separated by SDS PAGE and in gel trypsin digestion of to tal proteins separated by 2DGE). Source Genome Database Searched Digest GenBank Accession No. Protein ID Theoretical Mol Wt (kDa) BLASTP Score (bits) BLASTP Expected Value 2DGE total protein M. alligatoris Trypsin ZP_06610777.1 Conserved hypothetical protein 89.5 21.6 4.8 M. alligatoris Trypsin ZP_06610753.1 RDD family protein 25 21.2 6.5 M. crocodyli Trypsin YP_003559943.1 Putative lipoprotein 137.5 24.3 0.32 M. crocodyli Trypsin YP_003559946.1 Putative lipoprotein 41.3 22.7 1 M. crocodyli Trypsin YP_003560083.1 Phosphoglycerate kinase 42.7 20.8 3.2 M. crocodyli Trypsin YP_003560325.1 Lipoprotein 89.1 20.8 3.9 M. crocodyli Trypsin YP_003559885.1 Hypothetical protein MCRO_0232 26 19.2 10 Affinity column (LAMP) M. alligatoris Trypsin ZP_06610216.1 Glycosyl hydrolase family 2, sugar binding domain protein 95.4 23.9 4.3 M. alligatoris Trypsin ZP_06610133.1 ABC transporter, permease protein 37.7 23.5 5.4 M. alligatoris Trypsin ZP_06610871.1 Glucosamine 6 phosphate deaminase 49.8 23.1 7.4 M. alligatoris AspN ZP_06610601.1 Putative metallophosphoesterase 30.2 23.9 4.1 M. alligatoris AspN ZP_06610215.1 Type I restriction modification DNA specificity domain protein 46.7 23.9 4.4 M. alligatoris AspN ZP_06610729.1 Hypothetical protein MALL_0336 143.5 23.1 6.6 M. crocodyli AspN YP_003559917.1 Holliday junction DNA helicase 35.9 22.3 6 M. crocodyli AspN YP_003560256.1 Thioredoxin disulfide reductase 33.5 21.9 6.8 M. crocodyli AspN YP_003559949.1 Cation transporting P type ATPase 101 21.6 9.6

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68 CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS Conclusions It was initially hypothesized that the approximately 64 kDa immunodominant antigen was (1) associated with the mycoplasma cell membrane and (2) a highly conserved protein. Objectives to test these hypotheses included obtaining candidates for the M. alligatoris 64 kDa imm unodominant antigen and testing their respec tive knockout mutants for immunodominant antigen expression. Despite multiple approaches and persistent efforts to characterize this antigen, empirical evidence did not yield definitive conclusions about its identity. The main accomplishment of the proje ct was establishing several putative proteins that could be considered for subsequent analyses. Preliminary testing of candidates for immunodominant antigen expression was also demonstrated for one (albeit low -confidence) match. A putative lipoprotein, c onserved hypothetical protein was considered to be the most promising result. This identity was obtained from two separate 2DGE gels and LC -MS/MS sequencing experiments as well as from de novo sequencing. This was also the only match found by LC MS/MS t hat was also obtained by independent de novo seq uencing analysis. BLASTP analysi s of its full sequence offered evidence of a conserved protein amongst Mycoplasma species, as functional importance of o rthologous genes did not change. However, based on E v alues, it could not be concluded that this protein was conserved outside of the Mycoplasma genus. Future Directions Results in this study helped characterize a potential immunodominant antigen. However, future work must be done to fully prove that the purified antigen is indeed

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69 immunodominant. Antigen separation and sequencing experiments must be optimized and repeated to make sure results are reproducible. Full M. alligatoris protein sequences now available in GenBank would be utilized to have a stronger database. Next, immunodominance of the candidate(s) must be evaluated in vitro An immediate follow up to obtaining confident matches, possibly including other candidates from these experiments (Tables 3-2 and 33) would include testing their respective M. alligatoris knockout clones for immunodominant antigen expression by Western bl ot. There are at least half a dozen knockout mutants for the c onserved hypothetical protei n (ZP_06610777.1) available in our M. alligatoris knockout mutant library. Alternatively, a systematic screening could be performed on all knockout mutants in the l ibrary by Western blot regardless of preliminary candidate protein identifications. As was done with preliminary results, comparing highly confident MS results to homologous genes in closely related species could reveal biological importance of the immun odominant antigen Further characterization such as epitope mapping may also enhance future diagnostic and therapeutic efforts such as vaccine candidate screenings and design. Analysis of immunogenic epitopes could be valuable for the design of a diagnos tic test. If these experiments prove to be meaningful, other immunodominant antigens such as the 50 kDa antigen should be examined with similar approaches. Biological importance may also be demonstrated in an infection study using the immunodominant anti gen knockout clone to inoculate nave alligators and perform a comprehensive evaluation of its resulting pathogenesis. For example, if the antigen were an important virulence factor, its respective knockout clone might cause a reduction in disease. If th e antigen were vital for the generation of protective antibodies,

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70 infection with its knockout might create a more severe disease. However, if the antigen were vital to initial colonization of the host, its respective knockout clone might not be able to cause any disease. Western blots probed with plasma from naturally -infected alligators 15 years PI displayed 50 and 64 kDa immunodominant bands in addition to many others. Future studies might include identifying the other antigens to see if there is a pr ofound difference between early -response and later -response antigens. As previously discussed, the full ZP_06610777.1 protein sequence had a theoretical molecular weight of 89.5 kDa. A lipid addition covalently bound to the protein could result in a m olecule with a greater molecular weight. This possibility could explain the immunoreactive protein visualized at 100 kDa. Other future work might include determining changes in molecular weight due to lipid additions on mycoplasma membrane associated pro teins or lipoproteins. Technical Improvements 2DGE protein samples were generally of high quality, reflected in good MS/MS data and subsequently reflected in more confident protein matches. Identifications with the highest confidence came from membrane pr oteins separated by 2DGE, followed by total protein separated by 2DGE. However, it could not be ruled out that these preliminary results were exclusively correlated to the 64 kDa antigen as 2DGE immunoreactive band results were obtained without excluding other potential immunodominant antigens of other molecular weights (e.g. at 50 kDa). There was always possibility of mechanical or human error in selecting bands from gels, highlighting the importance of repeating experiments. Therefore, the likelihood o f selecting the correct immunodominant antigen, at least initially, is suspected to be low. One reason for this is that there was no prior enrichment of the immunodominant

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71 antigen when separating total protein by 2DGE. If the immunodominant antigen is in deed a membrane protein, then there was only slight enrichment when separating membrane proteins. Another reason was that horizontal streaking patterns indicated imperfect resolution of individual proteins. Therefore, one protein spot could potentially contain multiple or aggregated proteins. This possibility was also suggested by the observation that multiple, dissimilar protein matches were found in Scaffold 2, even with high stringency settings. It is known that 2DGE of total protein might not be s ufficient to separate the complete proteome at one time, even for bacteria with a limited number of ORFs (Regula et al., 2000). Limiting factors include the dynamic range of 2DGE, which prevents detection of the low abundance proteins, solubilization of m embranes, and the poor resolution of basic proteins, proteins that are too large (>150 kDa) or too small (<10kDa) (Regula et al. 2000; Chang et al., 2003). Even if proteins appear to be adequately resolved, it is still possible to pick up more than one pr otein in an area that appears to correspond to Western blot patterns. 2DGE does not always have high reproducibility, which also decreases the chances of selecting the right gel spots for analysis. Developing an optimal IEF method for mycoplasmas would t ake time but would be extremely useful for future research. This technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate spots. The standard IEF program steps used in this experiment w ere originally developed with E. coli. Thus, optimal settings for mycoplasmas could be quite different. In a study of the immunoproteome of Mycoplasma mycoides subsp. mycoides small

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72 colony type, horizontal streaking problems were not apparent (Jores et al., 2009). It m ight be worthwhile following this studys methods as closely as possible for sample preparation and 2DGE with special regard to IEF steps. The number of 2-D gel protein spots identified in this study could not be accurately measured due t o significant horizontal streaking issues seen in the gels. Therefore, it c ould not be confirmed that the protein patterns seen for the total protein gels were within the expected range. Genome sequencing and annotation of the type strain A21JP2TA strong point of this study was that two approaches were used to identify protein matches: LC MS/MS and de novo sequencing. Ideally, de novo sequencing should always be included to have higher confidence in protein matc hes in Scaffold or similar software, as well as the in the quality of the MS/MS spectra themselves. De novo sequencing is useful because it can help validate matches obtained by LC/MS/MS The method is a good control because amino acid sequences are obtained without a protein database, helping eliminate false positives. However, once the sequences obtained de novo are co mpared to a protein database, the quality of the protein match results will depend on the quality of the given database. has revealed 803 open reading frames (ORFs). Therefore, with ideal 2DGE of total proteins it would have been expected to see a similar number of protein spots. Even a higher number of total protein s pots could be expected if by chance, there are any p roteins remaining to be annotated. Alternative Imm unodominant A ntigen C andidate S creening T echniques One way to identify immuno dominant proteins for potential development of improved control measures is 2DGE combined with tandem MS analyses. In mycoplasmas, this method is especially appealing since high A+T content and using

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73 TGA as a tryptophan codon instead of a stop codon can interfere with traditional genetic approaches such as using E. coli expression libraries. With the genome sequence of M. alligatoris available, the essential prerequisite for this approach already exists. Phage display based identification has also been used for finding novel antigens with potential diagnostic applications. Mycoplasma mycoides subsp. mycoides small colony type, which causes contagious bovine pleuropneumonia, has been evaluated by this method (Naseem et al., 2010). Furthermore, researchers found that best performing antigens for diagnostic applications included a conserved hypothetical protein ( Naseem et al., 2010). Candidates were expressed as GST -fusion proteins in E. coli, purified and used as solid phase antigens in ELISAs. Theoretically, the same method could be applied to M. alligatoris and the diagnostic potential of recombinant antigens could be tested usi ng the sera of experimentally infected animals and control animals. A rigorous evaluation of candidate diagnostic sensitivity and specificity would be necessary. A complement fixation test could also be used to screen candidates for diagnostic tests No n laboratory based methods to identify possible immunodominant antigens include sorting all known M. alligatoris proteins by theoretical molecular weight and focus on candidates that are approximately 64 kDa in molecular weight An alternative, possibly m ore practical approach would be to use Gene Ontology, a collaborative bioinformatics project, to sort proteins by cellular location and/or function. However, Gene Ontology is not currently available for M. alligatoris Vaccine D evelopment Live attenuated vaccines are already available against various pathogenic Mycoplasma species (May et al., 2009). Similarly designed vaccines co uld potentially be effective in alligators against M. alligatoris However, reptile vaccines have not been

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74 developed and will n ot likely be developed due to lack of a significant economic incentive. However, candidates could still theoretically be tested as targets of opsonizing antibodies in vitro and as vaccine candidates in a crocodilian model of sepsis. Antigen over express ion and purification would need to be performed to test potential vaccine or diagnostic test components. A PCR strategy could be developed to generate a synthetic construct, which could be cloned into a prokaryotic expression vector. A new strategy for r apidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage express vector containing prokaryotic promoters upstream of the insertion site (March et al., 2006). The phage library is plat ed on E. coli cells, immunoblotted, and probed with hyperimmune and/or convalescent -phase antiserum to rapid ly identify vaccine candidates. Algorithm Based Evaluations of Diagnostic Value To find potential protein epitopes of diagnostic value, various algo rithms can first be employed. Prediction of antigenic epitopes can be performed using the algorithm of Hopp and Woods (1981), Zimmerman et al (1968), and Levitt (1978) (from www.expasy.ch/cgi bin/protscale.pl ). The secondary structure of polypeptides ca n be determined by the methods of Garnier Robson (Garnier and Robson, 1989) and Chou Fasman (Chou and Fasman, 1978). Surface properties of the structural proteins, such as hydrophilicity, can be analyzed by the methods of Kyte Doolittle (Kyte and Doolittle, 1982), Karplus Schulz (Karplus and Schulz, 1985), Emini (Emini et al., 1985) and Jameson Wolf (Jameson and Wolf, 1988), respectively. Peptides resulting from these methods with good hydrophilicity, high

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75 acces sibility and strong antigenicity could be chosen for further investigation such as testing purified antigens for immunoreactivity with infected animal sera. Alternatives to H ypotheses The immunodominant antigen protein has been proposed to be a membrane associated protein or lipoprotein. However, the alternative to this hypothesis (that the antigen is cytosolic) is still possible. In another study, staphylococcal proteins like enolase and EF -Tu, normally characterized as predominantly cytosolic proteins, were shown to be located on the surface of S. aureus and other bacterial pathogens (Glowalla et al., 2009). A certain number of the proteins identified are enzymes that play a crucial role in the biogenesis of the bacterial cell wall, supporting their surface location. The exact mechanism of the extracellular translocation of these proteins is unknown. However, secretion followed by noncovalent, receptor mediated reassociation with t he cell wall was suggested for pneumococcal enolase. Therefore, it is not surprising that some of the proteins identified in this screen were also found in secreted or cytosolic protein fractions. The possibility that the 64 kDa immunodominant antigen is in fact a cytosolic protein could be tested with the same methods already discussed. Responses to F uture P otential M. alligatoris O utbreaks Ideal antibiotic targets of M. alligatoris have not yet been identified. Work to characterize such targets at th e molecular level could be done. Treatment of mycoplasmoses usually involves antibiotics that inhibit DNA replication or protein synthesis. Drugs targeting spreading factors such as sialidases might be effective. In vitro drug susceptibility for M. alli gatoris has been previously studied by determining the minimum inhibitory concentration (MIC) for multiple antibacterial agents. The MIC for

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76 doxycycline, enrofloxacin, sarafloxacin, oxytetracycline, tilmicosin, and tylosin were < 1 g/mL (Helmick et al., 2002). High efficacy in vivo has not yet been demonstrated. In response to the 1995 outbreak, oxytetracycline was administered to all alligators intramuscularly at approximately 10 mg/kg once per week for five weeks, then once every two weeks for 3 months (Clipppinger et al., 2000). However, control of the disease was not ideal, as many animals still died. Autologous vaccinations might be explored as a mode of therapy. In an effort to treat farmed crocodiles in Zimbabwe afflicted with M. crocodyli infe ction, it was found that use of an autogenous vaccine was more effective in treating symptoms than antibiotic therapy (Mohan et al., 2001). Because M. alligatoris pathogenesis is so rapid, survival of an alligator would likely depend mostly on innate immunity and the present health status of the affected individual. Processes would likely include phagocytosis of M. alligatoris cells, which relies on opsonization of bacteria by antibodies and complement. Recognition of opsonizing antibodies bound to the s urface of the bacterial cell via Fc receptors of neutrophils is necessary for the induction of the oxidative burst and thus for killing of the phagocytosed bacteria and induction of a long-term immune response. Based on ELISA seroprevalence of M. alligat oris and lack of reoccurring outbreaks, it is assumed that formation of anti mycoplasma antibodies offers adequate longterm protection against reinfection. However, experimental studies involving the reinoculation of previously infected crocodilians have not been done. The original cause of the 1995 M. alligatoris outbreak is still unknown. Understanding the pathology of and prevention of the pathogen is important for protecting the health of both captive and non-captive crocodilians. Further work is

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77 necessary to better understand virulence of M. alligatoris and to gain insight on virulence of other Mycoplasmas and related microorganisms.

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78 APPENDIX DE NOVO SEQUENCES AND FASTA ZP_06610777 FRAGMENT PEPTIDE SEQUENCE Table A-1. De novo sequence data obtained from LC MS/MS spectra using PEAKS Studio software (p eptide so urce: in gel trypsin digestion of third column configuration LAMP antigen elutions separated by SDS PAGE). Sequence TLC ALC(%) Rank m/z Z Mass TTLPQ[2]VPGK 7.697482109 85.52757899 1 471.2416687 2 940.52295 TTLPEVPGK 7.697482109 85.52757899 2 471.2416687 2 940.52295 MLSGM[4]APLK 7.667612553 85.19569503 2 482.2719727 2 962.49286 MLSGFAPLK 7.667612553 85.19569503 1 482.2719727 2 962.5259 SSGMLLNNALK 9.341050148 84.91863771 1 574.2905884 2 1146.6067 LSSPATLNSR 8.448849678 84.48849678 1 523.3428345 2 1044.5564 SSGMLLNNALK 9.178469658 83.44063325 1 574.3457031 2 1146.6067 LSSPATLDSR 8.305230141 83.05230141 2 523.826416 2 1045.5403 LSSPATLN[2]SR 8.305230141 83.05230141 1 523.826416 2 1045.5403 SSGMLLDNALK 9.036511421 82.15010383 2 574.8052368 2 1147.5906 SSGMLLN[2]NALK 9.036511421 82.15010383 1 574.8052368 2 1147.5906 SSLSNALPALK 9.031148911 82.10135373 1 550.8555908 2 1099.6238 VATVSPLR 6.554719925 81.93399906 1 421.7575684 2 841.50214 AVGYLVSGYKR 9.01044178 81.91310709 1 606.8172607 2 1211.6663 AVGYLVSGYQR 9.01044178 81.91310709 5 606.8172607 2 1211.6299 SSMALVRPSK 8.176656723 81.76656723 1 538.2861938 2 1074.5854 SKAVGYLVSGYKR 10.51771927 80.90553284 1 714.3775635 2 1426.7932 SQAVGYLVSGYKR 10.51771927 80.90553284 3 714.3775635 2 1426.7568 TTLPEVPGK 7.233291149 80.36990166 2 471.2472839 2 940.52295 TTLPQ[2]VPGK 7.233291149 80.36990166 1 471.2472839 2 940.52295 Note: Only results with an ALC (%) score of 80 or higher was allowed. Sequence = the sequence of the peptide (including modifications if present) as determined by de novo sequencing. TLC = Total local confidence (the confidence that we have in the peptide sequence), calculated by a dding the positional confidence for each amino acid in the peptide sequence. ALC = Average local confidence (the confidence that we have in the peptide sequence), calculated by adding the positional confidence for each amino acid in the peptide sequence and dividing by the total number of amino acids. Rank = the sequences for a particular spectrum (ID) as sorted by score (TLC). m/z = Measured mass/charge value, in Daltons, for the peptide. Z = Calculated charge value for the peptide. Mass is calculated using the measured m/z and calculated z, used as the experimental mass of the peptide.

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79 Table A-2. De novo sequence data obtained from LC MS/MS spectra using PEAKS Studio software (p eptide source: ingel Asp -N digest ion of third column configuration LAMP antigen elutions separated by SDS PAGE). Sequence TLC ALC(%) Rank m/z Z Mass DAASAPSPVSL 10.04759789 91.34179896 1 1014.758728 1 1013.5028 SVVP 3.622637272 90.5659318 1 401.2255859 1 400.23218 DVVNGAPSH 7.526432037 83.62702264 1 448.2688599 2 894.4195 DPLLL 4.110112667 82.20225334 1 570.4898071 1 569.3424 DGGLGPGGGGN[2] GGLH 12.30580235 82.0386823 1 611.6010132 2 1221.5376 DGGLGPGGGGDG GLH 12.30580235 82.0386823 2 611.6010132 2 1221.5376 Q[2]AVP 3.277987957 81.94969893 1 415.2221375 1 414.21143 EAVP 3.277987957 81.94969893 2 415.2221375 1 414.21143 DVGAY 4.094697952 81.89395905 1 524.3009033 1 523.2278 DLTLGAFPAGSLSY LPPV 14.72895813 81.82754517 1 606.6923828 3 1816.9609 DLTLGAM[3]PAGSL SYLPV 14.72895813 81.82754517 5 606.6923828 3 1816.928 DVHGTTAGMT 8.15039444 81.5039444 1 495.3058167 2 988.42834 DLLGTAM[3]HGGL H 9.773213387 81.44344489 1 619.3223877 2 1236.5923 DLLGTAFHGGLH 9.773213387 81.44344489 2 619.3223877 2 1236.6252 DGGMHNLH 6.502185345 81.27731681 1 440.7293701 2 879.36566 DVKMLSGM[3]APL AK 10.51620102 80.89385399 1 688.9056396 2 1375.7205 DVKMLSGM[3]APL AQ 10.51620102 80.89385399 4 688.9056396 2 1375.6841 DVQMLSGM[3]APL AK 10.51620102 80.89385399 3 688.9056396 2 1375.6841 DVKMLSGFAPLAK 10.51620102 80.89385399 2 688.9056396 2 1375.7534 DLLGTAMH 6.45478344 80.684793 1 429.2340698 2 856.41125 Note: Only results with an ALC (%) score of 80 or higher was allowed.

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80 Table A-3. De novo sequencing data obtained from LC MS/MS spectra using PEAKS Studio software (p eptide source: ingel trypsin digest ion of total protein separated by 2DGE ). Sequence TLC ALC(%) Rank m/z Z Mass VRHK 3.717977762 92.94944406 1 538.2775269 1 538.334 VLNVVTSTK 7.769430637 86.32700708 1 480.7709045 2 959.5651 QATVSLPR 6.852394581 85.65493226 2 435.7689819 2 870.49225 KATVSLPR 6.852394581 85.65493226 1 435.7689819 2 870.5286 LSSPATLNSR 8.360575676 83.60575676 1 523.2537231 2 1044.5564 VATVSLPR 6.68298769 83.53734612 1 421.717926 2 841.50214 NTDLAKR 5.774003029 82.48575756 1 408.7219543 2 816.4454 NTDLAQR 5.774003029 82.48575756 2 408.7219543 2 816.409 NTN[2]LAKR 5.774003029 82.48575756 3 408.7219543 2 816.4454 Note: Only results with an ALC(%) score of 80 or higher was allowed. ORF01382 lipoprotein {Mycoplasma alligatoris A21JP2T (AS)}: MNKTKKLVITGLASASLLSALAVISCGSSTNGGSDNGSNTGTGSQSGQIDKNKEILLAVDGPQQGMYDHV VAEFAKSESYKLGYRIRLIKKDVFGALDTFVGHTDRNVVPDLFYAPQDRITDLAQRNVVSDLDTFDKSLFD DVLAVTGASSDEKTQARTFGTVVGANEGDKTFSPVRKLFGIRHNQEAIVLASTKELAGVKADMANDKTNT LEDLVKSGEAFIRLQDF Figure A 1. P eptide fragment sequence of immunodominant antigen candidate ZP_06610777 as seen in the FASTA database used for Scaffold 2 evaluations A protein -protein BLAST search of this sequence had 100% query coverage (E value 3e -130) with fully sequenced M. alligatoris conserved hypothetical protein (ZP_06610777)

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90 BIOGRAPHICAL SKETCH Niora J. Fabian was born in Boston, Massachusetts. After graduating from Boston Latin School in 2002, she attended Boston University. In 2006, she received a B.A. cum laude in bi ology with a specialization in marine b iology. Throughout her undergraduate career, s he gained research experience in various fields of biology. During the summer of 2005, Niora completed a research fellowship at the Conservation and Research for Endangered Species (CRES), in San Diego, California. Working in a molecular diagnostics and pathology laboratory at CRES, she was a contributing author to her third publication. After college graduation, she worked as a veterinary technician at a mixed species animal hospital She also helped rehabilitate wildlife as a volunteer with Project Wil dlife in San Diego. From 2008 to 2010, Niora worked as a graduate assistant at the University of Floridas College of Veterinary Medicine in the Department of Infectious Dis eases and Pathology, earning a masters degree in veterinary medical s ciences. At the same time, she was employed as a technician at an animal hospital in Gainesville, FL, furthering her experience in veterinary emergency medicine. Additionally, she has worked as a research animal care technician from 2008 until the present. In this p osition, she was responsible for intensive neonatal medical treatment and behavioral data collection for dogs affl icted with metabolic diseases. A member of the class of 2014, Niora will attend vet school at the University of Floridas College of Veterina ry Medicine.