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Characterization of fimbriae of Actinomyces naeslundii N16 using monoclonal and polyclonal antibodies

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Characterization of fimbriae of Actinomyces naeslundii N16 using monoclonal and polyclonal antibodies
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Bragg, Sandra L., 1947-
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English
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vi, 137 leaves : ill. ; 28 cm.

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Actinomyces ( jstor )
Antibodies ( jstor )
Antigens ( jstor )
Antiserum ( jstor )
Chromatography ( jstor )
Epitopes ( jstor )
Fimbriae ( jstor )
Gels ( jstor )
Molecular weight ( jstor )
Rabbits ( jstor )
Actinomyces ( lcsh )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Microbiology and Cell Science thesis Ph.D
Periodontal disease ( lcsh )
Pili (Microbiology) ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references (leaves 127-136).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Sandra L. Bragg.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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CHARACTERIZATION OF FIMBRIAE
USING MONOCLONAL AND
OF ACTINOMYCES NAESLUNDII N16
POLYCLONAL ANTIBODIES
By
SANDRA L. BRAGG
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988


ACKNOWLEDGMENTS
The author wishes to express her sincere gratitude to Dr. Arnold S.
Bleiweis, chairman of her supervisory committee, for his valuable
guidance and encouragement throughout this study. She would also like
to thank the other members of her committee for their advice, and
especially Dr. Paul A. Klein, for his advice and moral support.
This study was supported by the Centers for Disease Control,
Atlanta, GA. The author would like to thank the Division of Mycotic
Diseases, especially Dr. Libero Ajello, Dr. Leo Kaufman, and Dr. Errol
Reiss for their infinite patience and cooperation.
The electron microscopy would not have been possible without the
expertise of Dr. Greg Erdos and Mary Lane Martin. The assistance of Ray
Simons and Don Howard for their excellent scientific photography is
gratefully acknowledged.
Special thanks go to Marianna Wilson; without her support and
expert assistance, completion of this dissertation would not have been
possible.
11


TABLE OF CONTENTS
paRe
ACKNOWLEDGMENTS ii
ABSTRACT v
INTRODUCTION 1
MATERIALS AND METHODS 8
Antigen Preparation 8
Cultures 8
Culture Conditions 9
Preparation of Crude Soluble Antigens 9
Preparation of Fimbriae for Purification 11
Sonication 11
French press shearing 12
Gel Filtration Chromatography 13
Treatments of Fimbriae by Physical/Chemical Means 14
Antibody Preparation 17
Immunization of Mice 17
Monoclonal Antibodies 17
Isotyping 18
Polyclonal Antibodies 18
DEAE Chromatography 19
Protein A-Sepharose Chromatography 20
Radiolabeling 20
Immunoaff inity Chromatography 20
Dissociation Experiment 21
Assays 23
Electron Microscopy 23
Hemagglutination 24
Coaggregation 25
Bacterial Agglutination 26
Radioimmunoassay 26
Indirect Enzyme Immunoassay 27
Enzyme Immunodot Assay 28
Immunodiffusion 28
Laurell Rocket Immunoelectrophoresis 29
Crossed Immunoelectrophoresis with Autoradiography 29
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis... 29
Immunoblot 30
iii


RESULTS AND DISCUSSION
31
Monoclonal Antibodies 31
Evidence for the Presence of Both Types
of Fimbriae on A. naeslundii Serotype 3 Strains 35
Evidence for the Presence of Type 1 Fimbriae 35
Evidence for the Presence of Type 2 Fimbriae 43
Antigen Preparation: Results of Preliminary Experiments 46
Identification of N16 Type 1 and Type 2 Fimbriae
in Crude Antigen Extracts by XIEP-A 49
Purification of N16 Type 2 Fimbriae from a Crude Sonicate 51
Batch A 51
Batch B 54
Purification of N16 Fimbriae from the
French Press Supernatant 60
Effects of Various Physical and Chemical Treatments
on N16 Fimbriae 64
Effects of Temperature and Reduction on N16 Fimbriae 77
Assessment of the Purity
of Fimbrial Samples by SDS-PAGE-Immunoblot 83
Antigenic Relatedness of Actinomyces Fimbriae 90
Ouchterlony Analysis 90
XIEP-A 92
Bacterial Agglutination 106
Immunoblot Analysis 115
SUMMARY AND CONCLUSIONS 119
REFERENCES 127
BIOGRAPHICAL SKETCH
137


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION OF FIMBRIAE OF ACTINOMYCES NAESLUNDII N16
USING MONOCLONAL AND POLYCLONAL ANTIBODIES
By
Sandra L. Bragg
August 1988
Chairman: Dr. Arnold S. Bleiweis
Major Department: Microbiology and Cell Science
Two populations of fimbriae, which differ both in antigenicity and
biological activity, have been identified on Actinomyces viscosus T14V
cells. Although A. naeslundii serotype 1 isolates possess only one of
these fimbrial populations (type 2 fimbriae), there was functional
evidence to suggest that A. naeslundii serotype 3 strain N16 had both
types of fimbriae. The purpose of this study was to characterize the
fimbriae of A. naeslundii N16 immunologically by using both monoclonal
and polyclonal antibodies.
Three monoclonal antibodies (MAbs) to N16 were produced; all three
bound to N16 fimbriae as determined by immunoelectron microscopy. In a
solid-phase radioimmunoassay MAb 3B5.A1 reacted with 100% of the
A. naeslundii serotype 3 isolates tested, but it did not react with any
heterologous isolates. Type 1 and type 2 fimbriae were detected in
Lancefield extracts of N16 cells by crossed immunoelectrophoresis (XIEP)
125
using rabbit antiserum against N16 whole cells. When I-MAb 3B5.A1
was also incorporated into the gel, autoradiography indicated that MAb
v


3B5.A1 was specific for type 2 fimbriae. The N16 type 2 fimbriae were
purified by gel filtration and immunoaffinity chromatography on a MAb
3B5.A1 column.
Polyclonal antisera specific for N16 type 1 or type 2 fimbriae were
produced by immunizing rabbits with fimbrial immunoprecipitins excised
from XIEP gels. These antisera were used to identify fimbrial bands on
immunoblots of SDS-PAGE resolved N16 fimbrial antigens. Although N16
fimbriae could not be completely dissociated, type 1 fimbrial subunits
at 65 kd, 57-60 kd, and a weaker doublet at 53-54 kd and type 2 subunits
at 63 kd and a doublet at 39-40 kd were identified in fimbrial extracts
reduced at 100C.
Fimbriae-specific polyclonal and monoclonal antibodies were used in
various immunological assays to determine that (a) N16 type 1 fimbriae
are not related antigenically to type 2 fimbriae, (b) each type of
fimbriae has epitopes that are present on the corresponding fimbriae of
certain heterologous strains, and (c) MAb 3B5.A1 recognizes a
serotype-specific epitope residing on the type 2 fimbriae of
A. naeslundii serotype 3 strains.
vi


INTRODUCTION
Many structures found on the cell surfaces of microorganisms
exhibit properties that enable microorganisms to cause disease. These
properties include (a) adherence to host surfaces and other microbes,
(b) evasion of host defenses, and (c) destruction of host tissues. In
addition, surface molecules carry antigenic determinants that form the
basis of many serological and immunological identification
procedures. The fimbriae of certain species of Actinomyces are one
example of surface components that not only mediate adherence both to
host surfaces and to other microbes but also possess important
antigenic determinants.
Actinomyces are gram-positive, nonacidfast, nonsporeforming,
nonmotile bacteria that are highly variable in morphology, most
characteristically diphtheroidal, or filamentous and branched. They
ferment carbohydrate with production of acid but no gas, and except
for A. viscosus, all species are catalase-negative. Their cell walls
do not contain diaminopimelic acid, a characteristic that
differentiates them from Arachnia propionica, a morphologically
similar organism. Their natural habitat is the oral cavity of man and
other animals (96). All Actinomyces species, as well as Arachnia
propionica. are potential agents of actinomycosis (45).
Classical actinomycosis is a chronic granulomatous disease
characterized by the formation of abscesses and draining sinuses. It
is an endogenously acquired infection that can involve the soft tissue
1


2
and bone in any area of the body. Clinically, actinomycosis is
usually categorized as cervicofacial, thoracic, or abdominal. Of
these categories, cervicofacial actinomycosis is the most common, and
Actinomyces israelii is the most important etiological agent of
actinomycosis in humans. However, the extraoral infections of
classical actinomycosis are not as prevalent as intraoral periodontal
infections, and it has been suggested that the clinical classification
scheme be broadened to include a periodontal category (56).
During the last twenty-five years, most of the research efforts
dealing with Actinomyces species have been directed toward trying to
delineate the role that Actinomyces species play in the formation of
dental plaque and the development of periodontal disease. In the
early 1960s A. viscosus was found to be the etiologic agent of a
transmissible periodontal disease in Syrian hamsters (59,65). That
discovery initiated an explosion of research activity focused on the
pathogenicity of Actinomyces species in the oral cavity of man and
other animals because periodontal disease, unlike classical
actinomycosis, was and still is a major public health problem.
Virtually all humans and many animals accumulate dental plaque on
their teeth, and the formation of plaque may be followed by the
development of both caries and periodontal disease (49).
Periodontal disease is a collective term for a variety of chronic
inflammatory diseases of the structures that support the teeth.
Periodontal disease can range from mild inflammation of the marginal
gingiva (gingivitis) to severe forms of periodontitis, in which
extensive destruction of soft tissues and resorption of alveolar bone
can result in loss of the teeth. Although the microbial etiology and


3
pathology of the different types or stages of periodontal disease may
vary (98) the formation of dental plaque is the common first step in
the development of periodontal disease (49). However, successful
colonization of the tooth surface or other surfaces of the oral cavity
depends upon the ability of oral microbes to anchor themselves, either
directly or indirectly, to these surfaces to avoid being swept away by
the flow of saliva (47,104). Thus, an understanding of the mechanisms
by which microbes attach to surfaces in the oral cavity and to other
microbes is crucial to understanding the sequence of events leading to
periodontal disease.
Studies with experimental animals indicated that both A. viscosus
and A. naeslundii could form dental plaque and initiate the
pathological changes associated with periodontal disease
(55,58,59,60,97). However, most of the attention has been focused on
A. viscosus, particularly in studies on mechanisms of attachment to
oral surfaces and other bacteria (6,13,14,16,19,21,22,27,48,72,81,82,
92,106,107) and mechanisms for tissue destruction (1,8,9,10,39,40,51,
53,74,78,94).
There were two factors that contributed to the placing of more
emphasis on A. viscosus than A. naeslundii as a subject for
periodontal research. One was the recognition that these two species
tended to be distributed differently in the human oral cavity in that
A. viscosus preferentially colonized the teeth, whereas A. naeslundii
was associated with mucosal epithelial surfaces (34,35). The second
was the existence of the T14V-T14AV model system for comparative
studies of the properties that enable T14V to be virulent (i.e., cause
periodontal disease) in experimental animals and T14AV to be avirulent


4
(6,51). Although it was soon determined that the virulence-
associated differences in the antigens of T14V and T14AV were
quantitative rather than qualitative (19,22,90), T14V became the focus
of intensive study because it was found to have two different
adherence-related properties. These were the ability to coaggregate
with Streptococcus sanguis via a lactose-inhibitable lectin (82) and
the ability to adsorb well to saliva-coated hydroxyapatite (24).
A. naeslundii, on the other hand, coaggregated streptococci (36) but
did not bind well to saliva-coated hydroxyapatite (24). Some of these
early studies established an association between the presence of
fibrils or fimbriae on the surface of A. viscosus cells and
adherence-related functions (19,22,82).
Fimbriae are proteinaceous surface appendages that are found on
many gram-negative and gram-positive bacteria and that often mediate
attachment to host surfaces. Fimbriae from five strains of
A. viscosus and two strains of A. naeslundii have been isolated and
characterized as to their amino acid compositions (21,76,77,106).
Polar uncharged and nonpolar amino acids together made up 62-74% of
the total amino acids, whereas basic amino acids accounted for only
10-17% of the total; aspartic and glutamic acid generally comprised
20-24% of the total amino acids (76). The minimum molecular weight of
a fimbrial subunit as calculated from amino acid data was determined
to be approximately 25 kd for T14V (type 1) fimbriae (106) or 30 kd
for A. viscosus WVU627 fimbriae (77). However, a method for
completely dissociating Actinomyces fimbriae into subunits has not
been discovered (12).


5
Over the last decade, the fimbriae of A. viscosus and
A. naeslundii have been the subject of intensive study. A great deal
of progress has been made in identifying the adherence-related
functions of the fimbriae and defining the molecular basis of the
interactions of certain Actinomyces cells with other microorganisms
and surfaces within the oral cavity. Nearly all of the studies have
focused on only two strains, A. viscosus serotype 2 strain T14V and
A. naeslundii serotype 1 strain WVU45, and spontaneously occurring
mutants derived from these two strains. Two populations of
Actinomyces fimbriae, which differ both in antigenicity and biological
activity, have been identified (20,84). Type 1 fimbriae mediate
adherence to saliva-coated hydroxyapatite in vitro and to the salivary
pellicle on teeth in vivo (23). Type 2 fimbriae via their
lactose-inhibitable lectin activity mediate adherence to other oral
bacteria and mammalian cells (11,12,15,44,68,80,93,103). The
functional and molecular properties of the fimbriae from these two
strains have been recently reviewed (84).
Both types of fimbriae are present on T14V and other serotype 2
isolates of A. viscosus, whereas WVU45 and other A. naeslundii
serotype 1 isolates possess only type 2 fimbriae (20). In 1974 Jordan
et al. (57) described a new serotype of A. naeslundii based on their
studies of strain N16 and 20 similar isolates. These strains had been
isolated from cervical plaque in a sampling of 59 Down's syndrome
patients who had moderate to severe periodontal disease (64). In
studies with gnotobiotic rats and hamsters, Jordan and co-workers have
shown that N16 can cause heavy plaque deposits and severe periodontal
pathology, including alveolar bone loss, root surface caries,and even


6
enamel caries in some animals (57,60). Strain N16 and isolates that
are biochemically and serologically identical to it have been
designated A. naeslundii serotype 3 (46).
There is both functional and immunological evidence to suggest
that A. naeslundii serotype 3 strain N16 has both types of fimbriae.
The N16 isolate exhibits lactose-reversible coaggregation with
Streptococcus sanguis 34 (18). It produces its own neuraminidase and
agglutinates human erythrocytes in a reaction that is reversed by
lactose (29). It possesses numerous surface fibrils or fimbriae and
adsorbs to saliva-treated hydroxyapatite (SHA) in vitro as avidly as
T14V (26). In fact, Clark et al. (25) demonstrated that on average
A. naeslundii serotype 3 adsorbed better to SHA than any other species
or serotype of Actinomyces tested. Strain N16 agglutinated with MAbs
against T14V type 2 fimbriae (14). It reacted with rabbit IgG against
T14V type 1 fimbriae in a dot enzyme immunoassay (28), and its
adsorption to SHA was partially inhibited by the antibody (23,28).
Although most of our knowledge of Actinomyces fimbriae has been
based on studies of A. viscosus serotype 2 T14V and A. naeslundii
serotype 1 WVU45, A. naeslundii serotype 3 was the Actinomyces species
most frequently isolated from subgingival dental plaque from sites
with moderate, severe, or juvenile periodontitis (87), and in another
study A. naeslundii serotype 3 was considered to be one of the most
likely etiological agents of gingivitis (86). Thus, A. naeslundii
serotype 3 strain N16 appears to be a logical choice for a study of
surface antigens of a periodontopathic actinomycete.


7
The goals of this study were to (a) produce monoclonal antibodies
against surface antigens of A. naeslundii serotype 3 strain N16, (b)
select at least one MAb that exhibited serotype specificity, and (c)
use the MAb as a tool to isolate and characterize the surface
component carrying the serotype-specific epitope.


MATERIALS AND METHODS
Antigen Preparation
Cultures
A. naeslundii N16 (WVU820), WVU1267, WVU1468, WVU1527, and
WVU1528 were obtained from M. A. Gerencser, West Virginia University.
W1629, W2273, and W2821 were obtained from the Centers for Disease
Control, Atlanta, GA. UF92 and UF524 were fresh clinical isolates
provided by J. E. Beem, University of Florida, Gainesville, FL. The
identity of each of these ten isolates was confirmed by biochemical
tests and by direct staining with serotype-specific fluorescent
antibody reagents; all ten isolates were designated A. naeslundii
serotype 3. Approximately 50 additional isolates, including
representatives of all the recognized serotypes of A. naeslundii,
A. viscosus, A. israelii, A. odontolyticus, Arachnia propionica,
Rothia dentocariosa, and Bacterionema matruchotii were obtained from
culture collections at the Centers for Disease Control for use as
heterologous organisms in evaluating the specificity of monoclonal
antibodies. Other strains used in this study were kindly provided by
P. E. Kolenbrander, National Institute of Dental Research, Bethesda,
MD, and by W. B. Clark and P. J. Crowley, University of Florida.
Stock cultures of all isolates used in this study were maintained by
. . . o
lyophilization in skim milk and by storage at -20 C in Trypticase
soy broth (TSB; BBL Microbiology Systems, Cockeysville, MD) containing
20% (v/v) glycerol.
8


9
Culture Conditions
Cells for all procedures were obtained by culturing the isolates
in TSB supplemented with 2.5 g of K^HPO^ per liter in flasks that
could be chemically sealed to provide the atmospheric environment
appropriate for each isolate. After inoculation of a TSB-containing
flask with an actively growing TSB culture (2% v/v), the flask was
plugged with a rubber stopper through which an open-ended screw-capped
tube filled with cotton had been inserted (7). Prior to tightening of
the screwcap, the cotton was saturated with equal volumes of 10%
Na^CO^ and 1 M KH2P0^ to generate an aerobic 4- C02
environment or with 10% Na^O^ and pyrogallol solution to generate
an anaerobic + C02 environment (45). The cultures were incubated
without shaking at 37C. Cells were harvested from cultures in mid
exponential to early stationary phase by centrifugation (10,000 x g,
10 min), washed twice, and stored in buffer at 4C. The buffers
used for washing and making cell suspensions varied depending on the
procedure for which the cells were intended; they are noted in each
specific method.
Preparation of Crude Soluble Antigens
Several different methods were used to obtain crude soluble
antigens from whole cells.
Ammonium sulfate fractionation of French pressure cell
supernatants. N16 cells harvested from 8.5 1 of 36 h TSB cultures
were suspended in 0.05 M phosphate buffer, pH 7.2, to form a thick
paste; then the cell suspension was passed once through a French
pressure cell (Aminco Model J4-3337, American Instrument Co., Silver
Springs, MD) at 10,000 psi to remove the fimbriae (106). The


10
pressed cell suspension was centrifuged (48,300 x g, 20 min) to pellet
intact cells and cell walls; then the crude supernatant was
ultracentrifuged at 160,000 x g for 24 h to pellet the fimbriae. The
pellet was partially resuspended in 0.1 M Tris-HCl, pH 7.5, by
sonicating for 1 min at full power with a Kontes microultrasonic cell
disruptor (Kontes, Vineland, NJ). After centrifugation at 23,700 x g
for 10 min, the fimbriae-containing supernatant was processed by
ammonium sulfate fractionation to obtain precipitates at 10, 20, 30,
40, and 50% (w/v) (NH^)^SO^. The precipitates were collected by
centrifugation (30,900 x g, 15 min), dissolved in and dialyzed against
deionized water, and lyophilized. Lyophilized antigens were dissolved
in 0.5% Triton X-100 by adding 100 yl to 2 mg dry weight and
sonicating for 10 sec. The lyophilized material was highly resistant
to solubilization, so lyophilization was not used on subsequent
batches of fimbriae.
Continuous flow sonication. N16 cells (10% packed cells by
volume) in Tris-buffered saline (TBS; 0.025 M Tris-HCl, 0.15 M NaCl,
4 4
10 M CaCl2, 10 M MgCl2, and 0.02% NaN3, pH 7.6) were
passed at a flow rate of 5 ml/min three times through a continuous
flow sonifier cell disruptor (Heat Systems Ultrasonics, Inc.,
Farmingdale, NY) set at 100 W (14).
Virtis homogenization. A suspension of N16 cells in buffer was
agitated twice at 23,000 rpm for 5 min in a Virtis Model 45
homogenizer (The VirTis Company, Inc., Gardiner, NY) (77).
Extraction with 8 M lithium chloride. N16 cells were suspended
in 8 M LiCl, pH 7.0, (10% packed cells by volume) and stirred
o
continuously for 1 h at 25 C (82).


11
Lancefield extraction. N16 cells were suspended in 0.04 N HC1 in
saline (5 ml per g wet weight of cells); the suspension was heated for
15 min in a boiling water bath, cooled, and neutralized (90).
For the last four methods described above, the cells and
supernatants were separated by centrifugation (10,000 x g, 10 min),
and the supernatants were concentrated by ultrafiltration (PM10
membrane; Amicon Corp., Danvers, MA). The protein content of each
sample was determined by the Bio-Rad Protein Assay, a Coomassie blue
dye binding assay (3) (Bio-Rad Laboratories, Richmond, CA). Total
carbohydrate was estimated by the phenol-sulfuric acid procedure (33).
Preparation of Fimbriae for Purification
Sonication
Batch A. N16 cells were harvested from 12.5 1 of 36 h TSB
cultures, washed, and resuspended in TBS to a total volume of 240 ml
(10-207 v/v). Fimbriae were removed by sonicating 30 ml aliquots in
15 sec bursts at 100 W. After 0.5, 1, 2, 3, 4, 5, and 6 min of
sonication, cells and supernatants were separated by centrifugation
(10,000 x g, 10 min). The cells were resuspended in fresh buffer, and
a few drops of the suspension were set aside for subsequent
examination by hemagglutination (HA) and electron microscopy (EM)
before the next cycle of sonication was started. All the supernatants
were pooled and centrifuged (27,000 x g, 30 min) to remove
particulates. The crude 6 min sonicate was concentrated to 10 ml by
ultrafiltration (Amicon PM10 membrane) and by addition of dry Sephadex
G-25 prior to chromatography on Bio-Gel A-5m.
Batch B. N16 cells were harvested from 27.5 1 of 24-29 h TSB
cultures, washed, and resuspended in TBS to a total volume of 750 ml


12
(10-20% v/v) Fimbriae were removed from 720 ml of cell suspension by
sonicating 30 ml aliquots in 30 sec bursts at 100 W. After sonication
for a total of 2 min, the cell suspensions were centrifuged at 10,000
x g for 10 min to separate intact cells from supernatants. The
sonicated cells were resuspended in fresh buffer for examination by
HA. The supernatant or crude 2 min sonicate was further clarified by
centrifugation (27,000 x g, 30 min), then concentrated by
ultrafiltration (Amicon PM10 membrane) to 180 ml. The Amicon
concentrated sonicate was mixed with an equal volume of saturated
. o
(NH^)2^4 to give 50% saturation. After incubating at 4 C
overnight, the precipitate was collected by centrifugation (10,000 x g,
30 min), dissolved in water, and re-precipitated with (NH^J^SO^
at 35% final saturation. The precipitate from the 35% (NH^J^SO^
saturation was collected by centrifugation, resuspended in water, and
dialyzed against TBS. This was the starting material for gel
filtration chromatography.
French press shearing
N16 cells were harvested from 30 1 of 16-24 h TSB cultures,
washed once in TBS, and resuspended in TBS (5 ml of TBS per 1 of
culture harvested). The cell suspension was passed once through a
French pressure cell (Model J4-3337; American Instrument Company,
Silver Spring, MD) at 10,000 psi.
The amount of lysis caused by the French press was estimated to
be 1.2%, as determined by the following equation:
% lysis = (A260/A280 of untreated) (A260/A280 of treated) X 100
A260/A280 of untreated


13
Intact cells were removed from the pressed cell suspension by
centrifugation at 10,000 x g for 10 min. The French press supernatant
was clarified further by centrifugation at 48,300 x g for 20 min; the
clear supernatant was stored at -80C until processed.
Gel Filtration Chromatography
Columns were packed with Bio-Gel A-5m (exclusion limit 5 x 106
daltons for globular proteins; Bio-Rad Laboratories). The void and
total volumes were determined with blue dextran 2000 and potassium
ferricyanide, respectively. Crude sonicates were applied to the
columns and eluted with TBS. A Marriotte flask was used to maintain a
constant operating pressure. For all chromatographic techniques,
effluents and eluates were monitored by A with a Uvicord II UV
analyzer and recorder, and fractions were collected with an Ultrorac
fraction collector (LKB Instruments Inc., Rockville, MD). The Batch A
sonicate was run on a 12 x 750 mm column packed to a bed volume of
77.5 ml; the flow rate was 8 ml/h, and 3 ml fractions were collected.
The Batch B sonicate, a 35% saturated (NH^^SO^ fraction, was
applied in several aliquots (20 ml each at 3.9 mg protein per ml) to a
25 x 1000 mm column packed to a bed volume of 410 ml; the flow rate
was 25 ml/h, and 5 ml fractions were collected. Column fractions were
assayed for (a) total protein by the Bio-Rad Protein Assay with bovine
albumin as the standard, (b) total carbohydrate by the phenol-sulfuric
acid method with glucose as the standard, and (c) fimbriae reactive
with MAb 3B5.A1 by an enzyme immunodot assay. Some fractions were
also examined by SDS-PAGE.


14
Treatments of Fimbriae by Physical/Chemical Means
Acetone precipitation. Five volumes of cold acetone were added
to the sample. After having been mixed thoroughly, the sample was
. o
incubated at -20 C for at least 10 min. Precipitates were collected
by centrifugation (10,000 x g, 5 min) and dried by evaporation under
vacuum. Precipitates were reconstituted in an appropriate buffer.
Magnesium chloride precipitation. Four parts of 1 M MgCl^ were
mixed with one part sample to obtain a final concentration of 0.1 M
MgCl^. The sample was incubated at 4C for 24 h, then examined
for evidence of precipitation.
Freon extraction. Equal volumes of sample and Freon 113
(trichlorotrifluoroethane) were mixed thoroughly by vortexing. After
centrifugation (2000 x g, 30 min), the upper aqueous phase was
collected.
Heat. Samples were placed in a waterbath at 37C for 60 min,
at 65C for 30 min, or at 100C for 5 min or 60 min.
Sonication. Samples in capped microfuge tubes were placed in an
ice slurry in a cup horn sonicator (Heat Systems-Ultrasonics, Inc.,
Plainview, NY) and sonicated continuously at full power for 1, 5, or
10 minutes.
Urea. An acetone precipitate of the sample was dissolved in a
volume of 8 M urea in 0.05 M Tris-HCl, pH 8.0, equal to the original
volume of sample; then it was incubated at 37C for 1 h.
Guanidine hydrochloride. An acetone precipitate of the sample
was dissolved in saturated (8.6 M) guanidine HC1 at a final
concentration of 1 ml of guanidine HCl per mg total protein. After
. o
incubation at 37 C for 1 h, the sample was diluted with and dialyzed
against 10 mM EDTA, 0.05 M Tris-HCl, 0.15 M NaCl, 0.1% NaN^, pH 7.5.


15
Acid. One aliquot was treated by Lancefield extraction; it was
o
adjusted to 0.04 N HC1 in saline and heated at 100 C for 15 min.
o
Two others were adjusted to 0.1 N HC1 and heated at 100 C for 5 min
o
or at 37 C for 1 h. All were neutralized by addition of NaOH after
treatment.
Base. The sample was adjusted to 0.1 N NaOH and incubated at
37C for 1 h; then it was neutralized with HCl.
Periodate oxidation. The sample was adjusted to 0.1 M sodium
metaperiodate in 0.05 M acetate buffer, pH 4.5, and incubated in the
dark at 4C for 24 h; then ethylene glycol was added to 0.3 M final
concentration to consume excess periodate.
Enzymatic digestions. Solutions of lysozyme (Cat. No. 36-324,
Miles Laboratories, Naperville, IL), mutanolysin (M-3765, Sigma
Chemical Co., St. Louis, MO), papain (Sigma P-4762), and Pronase (Cat.
No. 537088, Calbiochem, San Diego, CA) were prepared in 0.1 M sodium
phosphate, pH 6.2. Solutions of a-chymotrypsin, trypsin (Sigma
T-8253, Type III), Staphylococcus aureus V8 protease (Sigma P-8400,
Type XVII), and proteinase K (Sigma P-0390, Type XI) were prepared in
0.05 M Tris-HCl, pH 8.0. Acetone precipitates of crude N16 French
press supernatants were dissolved in 0.1 M phosphate-buffered saline,
pH 6.2, or 0.05 M Tris-buffered saline, pH 8.0, depending on the
enzyme to be used.
Antigen samples containing 25 yg of total protein were
incubated (37C, 30 min) with serial dilutions of lysozyme ranging
from 0.1 yg to an upper limit of 7 yg (175 units) of lysozyme per
sample. Likewise, samples were treated with mutanolysin in amounts
ranging from 0.3 Units to 17.5 Units. To ensure that the lysozyme and


16
mutanolysin were active, a suspension of A. naeslundii PK19 cells was
treated with each enzyme, and muramidase activity was detected as a
decrease in turbidity (A ) of the cell suspension.
6 00
Samples that were to be treated with proteases were heated at
o
100 C for 2 min in the presence of 0.5% SDS prior to the addition of
enzyme solutions in order to make the proteins more susceptible to
digestion and to inactivate any endogenous proteases that might be
present in the crude antigen sample. Samples containing 500 vig of
total protein were mixed with 50 pg of enzyme (chymotrypsin,
trypsin, papain, Pronase, proteinase K, or V8 protease) for a 10:1
ratio of protein:enzyme, and the appropriate buffer was added to bring
the final enzyme concentration to 100 yg per ml. The
. o
protease-treated samples were incubated in a 37 C waterbath for 1 h.
At the end of the incubation period each sample was prepared
immediately for SDS-PAGE and stored at -80C until it could be
evaluated.
Aliquots of bovine serum albumin were treated exactly like the
N16 samples as a positive control for activity of the proteases and
for detection of protease contamination in the muramidases. Samples
that received buffer in place of enzyme served as untreated controls.
Samples containing enzyme only were also used as controls.
Treated samples were subjected to SDS-PAGE; the SDS-PAGE resolved
proteins were transferred to nitrocellulose. These blots were reacted
with either R10 rabbit IgG against N16 type 1 fimbriae or R2P rabbit
IgG against N16 type 2 fimbriae in an indirect EIA to visualize
immunoreactive fimbrial bands.


17
Antibody Production
Immunization of Mice
The cell suspensions for immunizations were formalin-killed whole
cells suspended in phosphate-buffered saline (PBS; 0.01 M phosphate,
0.85% NaCl, pH 7.4) containing 0.3% formalin. Cell aggregates were
dispersed by sonication, and the turbidity of each cell suspension was
adjusted to a No. 8 McFarland Standard. To obtain antigen-primed
spleen cells for fusions, 10-week old female BALB/c mice were
immunized with A. naeslundii N16 cells. Two mice that received 0.1 ml
intravenously on days 1, 8, and 16 were sacrificed on day 20 for
fusion of their spleen cells with X63Ag8.653 myeloma cells. Two
additional mice received 0.1 ml mixed with an equal volume of Freund's
complete adjuvant intraperitoneally on day 1 and 0.1 ml without
adjuvant intravenously on day 19 and were sacrificed on day 22 for a
fusion with P3X63Ag8 cells.
Monoclonal Antibodies
Hybridomas were produced, using the method of Simrell and Klein
8 7
(95), by fusing 10 spleen cells from N16-immunized mice with 10
myeloma cells in the presence of 50% polyethylene glycol-1000. For
one fusion myeloma cell line P3X63Ag8, which secretes immunoglobulin
molecules with gamma-1 heavy chains and kappa light chains (66), was
used. For another fusion, the Kearney myeloma cell line X63Ag8.653,
which does not express any immunoglobulin chains (63), was used.
Hybrid cells were selected by growth in Dulbecco's modified Eagle's
medium (DMEM) containing hypoxanthine, aminopterin, and thymidine (HAT
medium). Culture supernatants were screened for specific antibody by
solid-phase radioimmunoassay (RIA). Hybridomas secreting antibody to


18
N16 were cloned by limiting dilution by seeding a 96-well tissue
culture plate at a density of one-half cell per well; each well
4
contained 5 x 10 mouse peritoneal exudate cells as a feeder layer.
Three different clones were selected for further evaluation. They
were propagated in tissue culture and as ascites tumors in female
BALB/c mice primed by intraperitoneal injection of 0.5 ml pristane
(2,6,10,14-tetramethylpentadecane; Aldrich Chemical Company, Inc.,
Milwaukee, WI). Culture supernatants and sera or ascites fluids from
hybridoma-bearing mice were stored at -20C. Hybridoma cells in
DMEM containing 30% fetal calf serum and 10% dimethylsulfoxide were
stored in liquid nitrogen.
Isotyping
The class and subclass of immunoglobulin secreted by each
hybridoma were determined initially by immunodiffusion in which
subclass-specific anti-heavy chain antisera (Meloy Laboratories, Inc.
Springfield, VA) were reacted with the hybridoma culture
supernatants. These results were confirmed subsequently by testing
hybridoma culture supernatants or ascites fluids with an enzyme
immunoassay isotyping kit (Mouse Typer, Bio-Rad Laboratories).
Polyclonal Antibodies
Polyclonal antisera monospecific for type 1 and type 2 fimbriae
of A. naeslundii N16 were produced in female New Zealand white rabbits
by immunization with immunoprecipitins cut from crossed
immunoelectrophoresis (XIEP) gels (71). Type 1 and type 2 fimbriae
from partially purified samples of N16 fimbriae were separated and
precipitated by XIEP versus rabbit antiserum R29 raised against N16
whole cells. Sections of gel containing the immunoprecipitate were


19
cut from several XIEP patterns. The appropriate segments (a minimum
total of 10 cm per immunogen) were pooled, washed exhaustively with
saline to remove excess soluble reactants, and solubilized in 1 ml of
6 M KI, One-fourth of the immunogen was mixed with an equal volume of
complete Freund's adjuvant and injected subcutaneously into multiple
sites. The remainder of the immunogen was emulsified with an equal
volume of incomplete Freund's adjuvant and administered in 3
subcutaneous injections at approximately weeks 3, 5, and 7. Three
rabbits received type 1 fimbrial immunoarcs, and three were immunized
with type 2 fimbrial immunoarcs. Antibody responses were monitored by
microtiter plate EIA, and additional injections of immunogen were
given as needed to boost or maintain antibody titers. The rabbits
were bled periodically from the central artery of the ear throughout
the schedule and were exsanguinated by cardiac puncture 3-9 months
after the first injection.
RIO against N16 type 1 fimbriae and R2P against N16 type 2
fimbriae were the two rabbit antisera used most often in this study.
J. 0. Cisar, National Institute for Dental Research, kindly provided
the following samples of monospecific rabbit IgG: R59 against
A. viscosus T14V type 1 fimbriae, R55 against T14V type 2 fimbriae,
and R70 against A. naeslundii WVU45 type 2 fimbriae. Rabbit antiserum
raised against immunoarcs of Histoplasma capsulatum was kindly
provided by P. Standard, Division of Mycotic Diseases, Centers for
Disease Control, for use as a negative control.
DEAE Chromatography
IgG was purified from rabbit antisera or hybridoma culture
supernatants by chromatography on DEAE Bio-Gel A (Bio-Rad


20
Laboratories). Antisera and supernatants were precipitated with
(NH4)2S04 at 50% saturation. The precipitates were dissolved
and equilibrated with 0.01 M phosphate buffer, pH 7.8. Samples were
applied to DEAE Bio-Gel A columns, and IgG was eluted with
equilibrating buffer. Purified IgG was concentrated by
ultrafiltration and stored at 4C with 0.1% NaN^ as preservative.
Protein A-Sepharose Chromatography
Protein A-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ)
was packed in a column with flow adaptors. Hybridoma culture
supernatants or rabbit antisera were precipitated with ammonium
sulfate at 50% saturation. The precipitates were dissolved in water
and equilibrated against starting buffer. Samples were applied and
the unbound fraction eluted in 0.05 M Tris-HCl, 0.25 M NaCl, 0.1%
NaN^, pH 8. IgG was eluted with 7 M urea in 0.05 M Tris-HCl, pH 8.
Radiolabelins
125
Purified immunoglobulins in PBS and Na I (5 pCi per pg
protein) were placed in glass tubes coated with IODO-GEN
(1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril; Pierce Chemical
Co., Rockford, IL). The samples were incubated for 10 min at 25C,
and then removed from the reaction vessel to terminate the iodination.
125 125
I-labeled Igs were separated from free Na I by gel filtration
on PD-10 columns (Pharmacia) equilibrated with PBS containing 1%
125
bovine serum albumin and 0.1% NaN^. All I-labeled samples
contained 90-100% TCA-precipitable counts per minute.
Immunoaffinity Chromatography
MAb 3B5.A1, purified from hybridoma culture supernatants by
protein A-Sepharose chromatography, was coupled to Affi-Gel 10


21
(Bio-Rad) and packed in a K16/20 column with flow adaptors
(Pharmacia); bed volume was 9.5 ml. Samples were applied by
continuously cycling them through the column in an ascending direction
at a flow rate of 10 ml/h. Unbound and bound fractions were eluted at
a flow rate of 40 ml/h in an ascending or descending direction,
respectively. The unbound fraction was eluted with 0.05 M Tris-HCl,
0.25 M NaCl, 0.1% NaN^, pH 8. The bound fraction was eluted with 7
M urea in 0.05 M Tris-HCl, pH 8.
Rabbit IgG, 250 mg in 0.1 M HEPES, pH 7.5, purified by DEAE and
protein A-Sepharose chromatography from antisera monospecific for N16
type 1 or type 2 fimbriae, was coupled to 50 ml of Affi-Gel 10 and
packed in a K26/40 column with flow adaptors. The coupling efficiency
for each gel was >90%. Initially samples were eluted as described for
the MAb immunoaffinity column; however, on the basis of results of a
dissociation experiment, 6 M NaSCN was substituted for 7 M urea in the
buffer for elution of the bound fimbriae.
Dissociation Experiment
For protein A-Sepharose chromatography and initial experiments
with immunoaffinity chromatography, 7 M urea was used as the
dissociating buffer for release of bound molecules from the ligand.
However, to experimentally determine the most effective dissociating
buffer to use with each immunoaffinity column, various dissociating
buffers were evaluated in a microtiter plate EIA for their efficacy in
releasing antibody bound to antigen.
The wells of a 96-well Immulon 2 plate (Dynatech Laboratories,
Inc., Alexandria, VA) were coated with a crude fimbriae-containing
sonicate of N16 cells. Aliquots of the antibody samples used to


22
prepare the three imntunoaf f inity columns, i.e., MAb 3B5.A1, rabbit IgG
anti-N16 type 1 fimbriae, and rabbit IgG anti-N16 type 2 fimbriae,
were the antibody samples tested in the dissociating experiment; the
optimal dilution of each antibody had been determined by indirect EIA
versus the fimbriae-coated plates. An indirect EIA was performed as
described below, except that prior to the addition of the enzyme
conjugate, each well received 200 ul of a dissociating or control
buffer for 1 h at 25C. Buffers were evaluated in triplicate, and
the average A of the triplicate samples was used in the following
formula for measuring the efficacy of each buffer in dissociating the
antigen-antibody bonds:
% release of Ab 1 (A490 with dissociating buffer)
from fimbriae = (A490 with control buffer) X 100
The control buffer was the buffer normally used to elute the unbound
fraction during imntunoaffinity chromatography, i.e., 0.05 M Tris-HCl,
0.25 M NaCl, 0.1% NaN3, pH 7.5 at 25C.
In order to rule out the possibility that the dissociating
buffers themselves were removing the fimbriae from the plate or
adversely affecting the fimbriae in some other manner rather than
merely dissociating the antibody, an indirect EIA was performed on a
fimbriae-coated plate that was first incubated with dissociating
buffers at maximum concentration overnight at ambient temperature. If
the dissociating buffers were releasing fimbriae from the plates or
irreversibly denaturing fimbrial epitopes, the A^gQ of wells that
were incubated with dissociating buffers would be lower than that of
controls.


23
Assays
Electron Microscopy
For electron microscopy (EM) of whole cells, the specimens were
prepared by the pseudoreplica technique as described by Martin et al.
(75), using 0.5% uranyl acetate for negative staining. For
immunoelectron microscopy (IEM), N16 cells were incubated with a 1:100
dilution of rabbit antiserum overnight at 4C, washed with pH 7.2
PBS, and incubated (2 h, 25C) with a 1:10 dilution of
gold-conjugated goat anti-rabbit IgG (Auroprobe EM GAR G10, Janssen
Life Sciences Products, Piscataway, NJ). Then the gold-labeled cells
were prepared for EM and negatively stained as described above.
For thin section IEM colloidal gold (diameter, approximately 15
nm) was prepared by citrate reduction of chlorauric acid (43) and
coupled to goat anti-mouse IgG by the method of Horisberger and Rosset
(54).
N16 cells were grown in brain heart infusion broth (48 h,
37C), then harvested and washed with pH 7.2 PBS by vacuum
filtration on Nucleopore filters (45 p pore size). The cells were
reacted with monoclonal antibody by floating the filters on hybridoma
culture supernatant (immune sample) or on myeloma cell line P3X63Ag8
culture supernatant (negative control) for 1 h at 25C. The filters
were washed extensively with PBS, and then floated on gold-conjugated
goat anti-mouse IgG for 1 h at 25C. Filters were washed in PBS,
fixed for 1 h in 2.5% glutaraldehyde in PBS with 0.5% tannic acid,
post-fixed in 1% OsO^, dehydrated, embedded in Spurr's resin, and
sectioned. Thin sections were stained with uranyl acetate and lead
citrate and examined on a JEOL 100-CX electron microscope.


24
Hemagglutination
The assay described below, as performed with unstabilized
erythrocytes (RBC), is basically that of Costello et al. (29).
Preparation of neuraminidase-treated RBC. Human type 0 blood was
drawn in anticoagulant and diluted with four volumes of pH 7.2 PBS.
RBC were collected by centrifugation at 750 x g for 10 min and washed
twice more in the same manner. A 15% (v/v) suspension of RBC in
neuraminidase (30 pg per ml; Cat. No. N2876 type V neuraminidase
from Clostridium perfringens, Sigma Chemical Co.) in pH 5.0 PBS was
incubated in a 37C waterbath for 2 h. Neuraminidase-treated RBC
(NTRBC) were washed three times and suspended in TBS containing 0.4%
BSA. NTRBC had to be used within a day or two, unless they were
stabilized by treatment with formaldehyde or glutaraldehyde.
Formaldehyde stabilization of NTRBC. After neuraminidase
treatment an aliquot of NTRBC was washed in pH 7.2 PBS to remove the
enzyme and acid buffer. Then a 10% (v/v) suspension of NTRBC in PBS,
pH 7.2, was mixed with an equal volume of 3.7% formaldehyde. The
suspension was incubated at 25C with occasional stirring for 4-6 h,
then at 37C with continuous stirring for 14-18 h. The
formaldehyde-stabilized NTRBC were washed 4X with 10 volumes of TBS
and 3X with TBS containing 0.47 BSA. They were stored at 4C in
TBS-BSA. These cells were evaluated versus freshly prepared and aged
preparations of NTRBC that had not been treated with formaldehyde to
determine whether formaldehyde stabilization prolonged the "life" of
the NTRBC.
Glutaraldehyde stabilization of NTRBC. A 1-27 (v/v) suspension
of RBC in cold 17 glutaraldehyde in pH 8.2 PBS was incubated in an ice


25
bath for 30 min with intermittent mixing (2). The glutaraldehyde-
treated RBC (G-RBC) were collected by centrifugation (750 x g, 10 min)
and washed once in saline. To block potentially reactive free
aldehyde groups, a 10% (v/v) suspension of G-RBC in 0.1 M glycine was
incubated at 25C for 1 h. Then G-RBC were washed 5-10 times in
TBS. They were stored in TBS with 0.4% BSA at 4C. Glutaraldehyde
stabilization could be used either before or after neuraminidase
treatment.
Test procedure. Starting with a bacterial cell suspension
equivalent to A = 2.0, serial two-fold dilutions in TBS with
6 50
0.4% BSA were made in a U-bottom microtiter plate, leaving 25 pi per
well. Then 25 ul of a 1% (v/v) suspension of NTRBC in TBS-BSA were
added to each well and mixed for 1 min. The reactions were read
immediately and after overnight incubation at ambient temperature. To
test for lactose-reversibility, 50 pi of 0.04 M lactose (0.02 M
final concentration) were added. Alternatively, the effects of
various inhibitors were tested by adding 50 pi of inhibitor to the
bacterial cells prior to the addition of NTRBC.
CoaRRregation
The protocol described below is based on the methods of Mclntire
et al. (82) and Cisar et al. (18).
Tube assay. Bacterial cell suspensions in TBS were adjusted to
A650 = 2.0-2.1 (1-cm cuvette, Beckman Model 25 spectrophotometer).
In a 10 x 75 mm test tube 0.2 ml of an actinomycete cell suspension
was mixed with an equal volume of a streptococcal cell suspension.
For controls, 0.2 ml of buffer was added in place of one of the cell
suspensions. The suspensions were mixed by vortexing, then incubated


26
overnight at ambient temperature. They were mixed again before the
reactions were graded on a scale of 0 to 4+ : 0 = no visible
aggregates; 1+ = small uniform aggregates in suspension; 2+ = definite
aggregates that did not settle immediately; 3+ = large aggregates that
settled rapidly, leaving some turbidity in the supernatant; 4+ = large
aggregates that settled immediately, leaving a clear supernatant.
Microtiter plate assay. A microtiter plate coaggregation assay
was developed to replace the standard tube assay. The procedure was
similar to that described for the tube assay, except that 25 pi of
each cell suspension were mixed in a U-bottom microtiter plate.
Bacterial Agglutination
In a U-bottom microtiter plate serial dilutions of the antibody
samples were made in TBS containing BSA (4 mg/ml). Normal sera and
diluent were used as negative controls. To 25 pi of antibody were
added 25 pi of a suspension of bacterial cells in TBS adjusted to
A^^0 = 1.0 (1 cm cuvette, Beckman Model 25 spectrophotometer), and
the plate was shaken for 1-2 min. Reactions were read after overnight
. o
incubation at 25 C (14).
Radioimmunoassay
A solid-phase radioimmunoassay (RIA) was developed as a rapid,
sensitive screening assay for detection of monoclonal antibodies to
surface antigens of N16 and other isolates. Polyvinyl chloride
microtiter plates (U-bottom, Dynatech Laboratories, Inc.) were coated
with formalin-killed whole cells by placing 25 pi of a No. 4
McFarland cell suspension in each well for 1 h at 25C. Excess
antigen was rinsed out, and free sites were blocked by the addition of
a few drops of 10% agamma horse serum to each well. The antigen-


27
coated wells were incubated (1 h, 25C) with 25 yl of undiluted
hybridoma culture supernatant. The wells were washed three times with
o
PBS containing 1% fetal calf serum and incubated (1 h, 25 C) with 25
125
y 1 of I-labeled rabbit anti-mouse IgG (heavy and light chain
specific) containing 30,000-50,000 counts per minute (cpm). After the
unbound radiolabeled second antibody had been washed away, individual
wells were cut out, and the bound radioactivity was measured in a
125
gamma counter. Reactions in which I-cpm were at least twice that
of the negative control were considered to be positive. Culture
supernatants from the myeloma lines used for fusion served as negative
controls. Murine antisera were used as positive controls if positive
culture supernatants or ascites fluids were not available.
Indirect Enzyme Immunoassay
Indirect enzyme immunoassays (EIA) were performed in Immulon 2
96-well flat-bottomed polystyrene microtiter plates (Dynatech
Laboratories, Inc.). Wells were coated with antigens by placing 50
yl of whole cells or soluble antigens optimally diluted in TBS in
each well and drying at 37C. Antigen-coated plates were stored at
ambient temperature until used. Plates were washed once with blocking
buffer (PBS with 1% BSA), then 3 times with PBS with 0.05%
polyoxyethylene sorbitan monolaurate (Tween 20) (PBST). Antibodies
diluted in PBST were added to the wells (25 yl per well) and
incubated at 25C for 1 h. Plates were washed 3 times with PBST.
Then 100 yl of horseradish peroxidase-labeled goat anti-rabbit or
mouse IgG optimally diluted in PBST were added and incubated at 25C
for 1 h. After the excess conjugate had been removed by washing, 200
yl of substrate solution (0.1 mg of ortho-phenylenediamine and


28
2 yl of 3% ^22 Per citrate-phosphate buffer, pH 5.0) were
added and incubated at 25C for 15-30 min. Reactions were stopped
with 25 yl/well of 4 M H SO and the absorbance at 490 nm was
2 4
read on a Dynatech MR 600 Microplate Reader (Dynatech Laboratories,
Inc.). Absorbance readings >0.2 above the negative control were
considered positive.
Enzyme Immunodot Assay
Antigens were affixed to strips of nitrocellulose by applying
2-yl drops at 5-mm intervals and allowing them to dry at 25C
(79). The antigen-coated strips were processed by enzyme immunoassay
essentially as described by Tsang et al. (99). The strips were washed
4 times for 5 min each time with PBS with 0.37 Tween 20 (PBSTW) with a
quick rinse in deionized water after each PBSTW wash. The
antigen-coated strips were immersed in antibodies diluted in PBSTW and
incubated with gentle agitation for 1 h at 25C. Then the strips
were washed as before to remove unbound antibodies. The strips were
covered with the appropriate peroxidase-conjugated anti-Ig and
incubated for 1 h at 25C. Unbound conjugate was removed by washing
as described above, followed by a final wash with PBS. The strips
were incubated with substrate (50 mg of 3,3'-diaminobenzidine and 10
yl of 30% 1^02 per 100 ml of PBS) for 10 min or until the spots
had developed the desired degree of intensity. The reaction was
stopped by rinsing the strips thoroughly with water.
Immunodiffusion
Immunodiffusion was performed in 17. agarose in PBS with wells
formed by use of a microimmunodiffusion template (89).


29
Laurell Rocket Immunoelectrophoresis
Laurell rocket immunoelectrophoresis (LRI) was performed as
described by Powell et al. (90) in 0.75% agarose in 0.043 M sodium
barbital buffer, pH 8.3, at a constant current of 8 mA per 50 mm x 75
mm slide. Gels were dried and stained with 0.57 Coomassie brilliant
blue R-250 in ethanol:glacial acetic acidrwater (4.5:1.0:4.5).
Crossed Immunoelectrophoresis with Autoradiography
Glass slides (50 mm x 75 mm) were coated with 7 ml of 0.757
agarose in barbital buffer (0.0375 M barbital, 2 mM calcium lactate,
0.057 NaN^, pH 8.6). After the antigen wells were cut and filled,
the gels were electrophoresed at 8 mA constant current for 1-3 h for
separation of antigens in the first dimension. Then the gel above the
antigen wells was replaced with 3 ml of agarose containing an
unlabeled rabbit antiserum raised against whole cells and an
125
I-labeled Ig (50). After electrophoresis in the second dimension
overnight at 4 mA constant current, gels were washed, dried, and
exposed to Kodak XAR-2 film with a Dupont Cronex Lightning Plus
intensifying screen at -70C. Gels were stained with Coomassie
brilliant blue R-250 and photographed.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Socium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed as described by Tsang et al. (102). Samples
were prepared to obtain final concentrations of 17 SDS, 0.9 M urea,
0.057 bromophenol blue, 87 glycerol, and 0.25 mg total protein per
ml. Some samples also received dithiothreitol (DTT) or
2-mercaptoethanol for reduction of disulfide bonds. Samples were
heated at 37C for 30 min, at 65C for 15 min, or at 100C for


30
5 min. Gels consisting of a 5-20% linear gradient resolving gel and a
3% stacking gel 0.75 mm thick were cast using the Pharmacia GSC-2 177
mm x 200 mm vertical slab system (Pharmacia). The gels were
electrophoresed in the Pharmacia GE 2/4 LS electrophoresis chamber
using a discontinuous buffer system with 0.424 M Tris-HCl, pH 9.18, in
the lower reservoir and 0.04 M boric acid, 0.04 M Tris, 0.17 SDS in
the upper reservoir. Generally 0.25 pg of protein per mm width of
sample lane was applied. Either high molecular weight markers
(Bio-Rad) and low molecular weight markers (Pharmacia) or prestained
protein molecular weight standards (Bethesda Research Laboratories,
Gaithersburg, MD) were included in each gel (101). After
electrophoresis protein bands were visualized by the silver stain as
described by Tsang et al. (100), which is a combination of the methods
of Merril et al. (85) and Morrissey (88).
Immunoblot
SDS-PAGE resolved proteins were transferred electrophoretically
to nitrocellulose (0.2 p, Schleicher and Schuell, Keene, NH); then
antigens were visualized by an indirect enzyme immunoassay, using
peroxidase conjugates (Bio-Rad Western blotting grade) and
diaminobenzidine/ ^02 as the substrate. These methods have been
described in detail by Tsang et al. (99,102). For detecting unstained
molecular weight markers or other proteins on blots, nitrocellulose
strips were stained with India ink (52) or Aurodye forte (Janssen Life
Sciences Products, Piscataway, NJ).


RESULTS AND DISCUSSION
Monoclonal Antibodies
Three different hybridomas secreting antibodies to A. naeslundii
serotype 3 strain N16 were produced. Hybridomas 3B5.A1 and 2A3.B3,
both obtained from the fusion with the non-Ig-producing Kearney
myeloma cell line, secreted IgG and IgG monoclonal antibodies,
2a 3
respectively. The third hybridoma, 2B5.B6, which was a product of the
fusion with an IgG^(k)-secreting myeloma cell line, secreted IgM
monoclonal antibodies along with the Ig chains of the myeloma parent.
Preliminary indications of differences in specificity and the
range of reactivity of each of the three monoclonal antibodies were
obtained by radioimmunoassay, using whole cells as antigens. When
tested against a panel of ten isolates of A. naeslundii serotype 3,
monoclonal antibodies 3B5.A1 and 2A3.B3 reacted with all 10 isolates,
whereas monoclonal antibody 2B5.B6 reacted with only A of the 10
isolates (Table 1). On the basis of these results, monoclonal
antibodies 3B5.A1 and 2A3.B3 appeared to have potential as serotyping
reagents, provided that they did not also react with heterologous
serotypes; monoclonal antibody 2B5.B6 had too narrow a range of
reactivity to be useful for serotyping, unless it could be used as an
epidemiological tool for fine typing below the serotype level. To
determine whether or not the monoclonal antibodies would cross-react
with other organisms, the panel of cells for the RIA was expanded to
include 51 heterologous isolates: 17 isolates of A. naeslundii that
31


32
TABLE 1. Summary of RIA results
for isolates within the N16 serogroup
Isolate
Monoclonal antibody
3B5.A1 2A3.B3 2B5.B6
N16
+a
+
+
WVU1267
+
+
-
WVU1468
+
+
-
WVU1527
+
+
+
WVU1528
+
+
+
W1629
+
+
-
W2273
+
+
-
W2821
+
+
+
UF92
+
+
-
UF524
+
+

a + = ^25I_Cpm > 125j_Cpm 0f negative control x 2;
- = l^I-cpm < 125j_Cpm 0f negative control x 2 for the average
1^^I-cpm of samples run in duplicate. 12^I-cpm of negative
controls ranged from 53 to 78, whereas L2^I-cpm of + reactions
ranged from 1919 to 6283.


33
did not belong to the N16 serogroup (serotype 3), 13 isolates of
A. viscosus, 8 isolates of A. israelii, and 13 isolates of less
closely related species. When assayed using polyclonal antisera,
A. naeslundii and A. viscosus are closely related antigenically, and
antisera raised to whole cells of N16 could be expected to cross-react
with A. viscosus serotype 2 in particular. However, monoclonal
antibody 3B5.A1 did not react with any heterologous species or
serotypes (Table 2). On the other hand, monoclonal antibody 2A3.B3
cross-reacted with 7 of 17 non-serotype 3 A. naeslundii isolates, as
well as 7 of 11 A. viscosus serotype 2 isolates. However, the
125
I-cpm for all but one of these cross-reactions were less than 34%
125
of the I-cpm of the least reactive A. naeslundii serotype 3
isolate. Monoclonal antibody 2B5.B6 cross-reacted with 4 of 17
isolates representing other serotypes of A. naeslundii, and these
cross-reactions were as strong as the homologous reactions. None of
the three monoclonal antibodies reacted with isolates of A. israelii.
A. odontolyticus, A. pyogenes, A. propionica, R. dentocariosa,
P. avidum, or Bacteroides gingivalis. These results suggested that
monoclonal antibody 3B5.A1 recognizes a serotype-specific antigenic
determinant but that monoclonal antibodies 2A3.B3 and 2B5.B6 see
epitopes common to other species or serotypes.
The location of the epitopes to which the monoclonal antibodies
were binding was presumed to be the cell surface because whole cells
were used as immunogens and as antigens in immunoassays to measure
monoclonal antibody activity. The most immunogenic and biologically
significant surface components of A. naeslundii and A. viscosus
isolates are their fimbriae. Electron microscopy of A. naeslundii N16


34
TABLE 2. Summary of the specificities
of three monoclonal antibodies to N16 as determined by RIA
Species/serotype
No. isolates
tested
Monoclonal antibody
3B5.A1 2A3.B3 2B5.B6
Actinomyces naeslundii/3
10
10a
10
4
A. naeslundii/1, 2, 4
17
0
7
4
A. viscosus/1
2
0
0
0
A. viscosus/2
11
0
7
0
A. israelii/1
6
0
0
0
A. israelii/2
2
0
0
0
A. odontolyticus
3
0
0
0
A. pyogenes
1
0
0
0
Arachnia propinica
3
0
0
0
Rothia dentocariosa
2
0
0
0
Propionibacterium avidum
3
0
0
0
Bacteroides Rinfcivalis
1
0
0
0
a Number of isolates that gave positive reactions, as defined in
Table 1. The range of l^I-cpm for positive reactions was as
follows: 2547-6283 for 3B5.A1 against homologous isolates; 1919-6025
for 2A3.B3 against homologous isolates; 2097-2628 for 2B5.B6 against
homologous and heterologous isolates. Except for 3207 125I-cpm
against A. naeslundii W1250, 2A3.B3 gave 161-649 -*-2^I-cpm against
heterologous serotypes of A. naeslundii and 180-328 12^I-cpm against
A. viscosus serotype 2 isolates.


35
cells revealed that they possess abundant fimbriae (Figure 1). The
location and density of the epitopes recognized by each monoclonal
antibody, as determined by EM of N16 cells immunolabeled with colloidal
gold, are shown in Figure 2. All three monoclonal antibodies bound to
epitopes residing on the fimbriae, but based on the far greater number
of gold particles seen on cells immunolabeled with MAb 3B5.A1, the
3B5.A1 epitope appeared to be more abundant than those recognized by
MAbs 2A3.B3 and 2B5.B6.
All three of the MAbs against N16 fimbriae were used on occasion
in later experiments. However, most of this study focused on the use
of MAb 3B5.A1 because preliminary evaluations indicated that (a) it
reacted with all 10 A. naeslundii serotype 3 strains by RIA, (b) it
appeared to recognize a unique, serotype-specific epitope, and (c) on
the basis of the density of immunogold labeling, it appeared to
recognize an epitope that was more abundant than those of the other
two MAbs.
Evidence for the Presence of Both Types of Fimbriae
on A. naeslundii Serotype 3 Strains
Evidence for the Presence of Type 1 Fimbriae
Although this study did not include any functional assays to
demonstrate the presence of type 1 fimbriae on N16 or other
A. naeslundii serotype 3 isolates, Clark et al. (23,25,26,28) have
presented evidence that 3 of the 10 strains listed in Table 1 possess
type 1 fimbriae. In one study they showed by EM that N16 and UF92
cells had fimbriae; both strains adsorbed very well to saliva-coated
hydroxyapatite (SHA) relative to the adsorption of T14V, 100% for N16
and 84% for UF92; and their adherence to SHA could be blocked by
purified T14V fimbriae (26). In another study N16 and G1468 (WVU1468)


36
Figure 1. Electron micrograph of A. naeslundii N16 cells negatively
stained with 0.5% uranyl acetate. Note abundant fimbriae
on the cell surface. Bar, 1 ym.


Figure 2. Electron micrographs of thin sections of immunogold labeled
A. naeslundii N16 cells demonstrating the binding of the
monoclonal antibodies to fimbrial epitopes. The cells were
incubated with MAb-containing culture supernatants of
myeloma or hybridoma cell lines and then with goat anti
mouse IgG conjugated to colloidal gold (diameter, 15 nm).
Bar, 0.25 ym.
A) P3X63Ag8, negative control; B) MAb 2B5.B6; arrows
indicate gold particles; C) MAb 2A3.B3; D) MAb 3B5.A1;
E) MAb 3B5.A1; F) MAb 3B5.A1


38


Figure 2.
continued


40


Figure 2.
continued




43
adsorbed to SHA better than T14V, despite the fact that each had a
lower hydrophobic index than T14V (25). N16 and WVU1468 reacted in an
immunodot EIA with rabbit IgG specific for the T14V type 1 fimbriae
(28), and rabbit IgG anti-T14V type 1 fimbriae was able to partially
inhibit adsorption of N16 cells to SHA and give 917 inhibition for
WVU1468 (23,28). The ability of N16, WVU1468, and UF92 to adsorb well
to SHA and the fact that T14V fimbriae or antibodies to T14V type 1
fimbriae can inhibit the adsorption suggests that these A. naeslundii
serotype 3 strains possess type 1 fimbriae. Immunological assays
performed in this study (data to be presented later) showed that all
of the A. naeslundii serotype 3 strains listed in Table 1 have type 1
fimbriae.
Evidence for the Presence of Type 2 Fimbriae
Hemagglutination. Many human strains of A. viscosus and
A. naeslundii can agglutinate human types A, B, 0, and AB erythrocytes
(29,37). With untreated RBC, hemagglutination occurs immediately at
4C, but the reaction takes longer at room temperature or 37C.
With neuraminidase-treated RBC, hemagglutination occurs immediately at
all three temperatures (29). Hemagglutination by A. viscosus and
A. naeslundii cells can be completely reversed by lactose. The
mechanism proposed for this reaction involves two steps: 1) the
unmasking of G-galactoside-containing receptors on the RBC when
terminal sialic acid residues are removed by neuraminidase released
from Actinomyces cells, and 2) the multivalent binding of these
receptors by multiple low affinity, lactose-reversible lectin sites on
the surface of the actinomycete (29,37). Since the lactose-reversible
lectin activity is a function of type 2 fimbriae (20), HA can be used


44
to determine whether or not Actinomyces cells have type 2 fimbriae.
Costello et al. (29) reported that N16 and WVU820 (N16) produced
neuraminidase and agglutinated human RBCs as described above. On the
other hand, Ellen et al. (37) reported that strain 820, which is
presumably WVU820 (N16), did not agglutinate non-sialidase-treated
horse, sheep, guinea pig, or human RBCs. Since Ellen et al. measured
HA after only a 5 min incubation, the inability of WVU820 to
agglutinate RBCs was probably a result of not allowing sufficient time
for the WVU820 neuraminidase to remove the terminal sugar blocking the
receptor.
In this study the 10 A. naeslundii serotype 3 strains listed in
Table 1 were tested for HA activity. Preliminary experiments were
performed with unfixed NTRBC, but unfixed NTRBC were found to be so
unstable that they generally had to be used within 24 h of
preparation. Even then they often gave weak HA reactions or lysed
during assays. In an effort to improve their shelf-life, NTRBC were
fixed with formaldehyde or glutaraldehyde and compared to unfixed
NTRBC. HA reactions with fixed NTRBC were stronger than with unfixed,
and glutaraldehyde-fixed RBC were superior to formaldehyde-fixed
cells. Not only did glutaraldehyde-fixed NTRBC agglutinate better
than unfixed cells, but they also had a shelf-life at 4C that could
be measured in years instead of hours. The characteristics of the
agglutination reactions between N16 cells and human RBC (in terms of
the effects of temperature, incubation time, etc. as reported by
Costello et al.) were the same for glutaraldehyde-fixed RBC or NTRBC
as for unfixed RBC or NTRBC, except that HA with fixed cells was
stronger and, therefore, more difficult to reverse. All 10


45
A. naeslundii serotype 3 isolates agglutinated G-NTRBC, and all of the
HA reactions were reversed by lactose. Twelve sugars and EDTA were
tested for their ability to inhibit HA by N16 cells. Glucose,
fructose, mannose, sucrose, xylose and a-methyl galactoside
exhibited no inhibition of HA at concentrations of inhibitor < 25 mM.
Of the substances that did inhibit at concentrations < 25 mM, the
relative potency of the inhibitors was as follows: EDTA > lactose >
fi-methyl galactoside > talse > fucose > galactose > N-acetyl
galactosamine. These results on the relative potency of inhibitors of
the N16 HA reaction are similar to the results of Ellen et al. (37)
for the inhibition of HA by A. naeslundii serotype 2 W752 cells and
also the results of Mclntire et al. (81,82) for inhibition of
coaggregation between T14V and Streptococcus sanguis strain 34 cells.
N16 cells also agglutinated sheep and guinea pig RBC, and HA was
completely inhibited with EDTA or lactose at a final concentration of
25 mM.
Coaggregation. Many human strains of A. viscosus and
A. naeslundii can agglutinate certain strains of oral streptococci
(18.19.36.69.81.82). These coaggregation reactions involve different
mechanisms for interaction, one of which is the binding of a
carbohydrate moiety on S. sanguis 34 cells by a lectin on Actinomyces
cells in a reaction that can be completely reversed by lactose
(18.69.81.82). Coaggregation, like hemagglutination, is a function of
type 2 fimbriae (20,92), so it also can be used to determine whether
or not Actinomyces cells have type 2 fimbriae.
Cisar et al. (18) showed that WVU820 (N16) and W1527 (WVU1527)
exhibited lactose-reversible coaggregation with S. sanguis 34 cells.


46
Their observations were confirmed in this study, using both the tube
and microtiter plate coaggregation assays (data not shown).
In summary, there was ample evidence from our preliminary studies
to suggest that N16 and other A. naeslundii serotype 3 strains had
both type 1 and type 2 fimbriae. The next step was to find a good way
to remove the fimbriae from the cells and obtain crude soluble
fimbrial extracts suitable for purification of fimbriae and for use as
antigens in immunological assays.
Antigen Preparation: Results of Preliminary Experiments
In the early stages of this research several different methods
for preparing soluble fimbrial extracts of N16 cells were tried.
First, the method of Wheeler and Clark (106) for purifying VA-1
fibrils (type 1 fimbriae) from A. viscosus T14V cells by ammonium
sulfate fractionation of French pressure cell supernatants was
attempted. Laurell rocket immunoelectrophoresis of the N16 fractions
prepared in this manner, i.e. 10, 20, 30, 40, and 50% (w/v) ammonium
sulfate saturated samples, revealed multiple antigens in each fraction
when antiserum to N16 whole cells was employed. When these fractions
125
were used to coat wells in the RIA, MAb 3B5.A1 gave I-cpm with
the 20% and 30% ammonium sulfate fractions that were 5-8 times higher
than those obtained with the 10%, 40%, or 50% fractions; thus the 20%
and 30% fractions appeared to be enriched for the antigen that 3B5.A1
recognizes. Each fraction was examined by transmission electron
microscopy for the presence of fimbriae, but the 20% fraction was the
only one in which fimbriae were readily observed.
Although the French pressure cell was an effective means for
removing fimbriae from cells with minimal cell lysis, access to this


47
equipment was not readily available. So, several other methods for
extracting fimbrial antigens were investigated: Lancefield extraction,
sonication, Virtis homogenization, and extraction with 8 M lithium
chloride. N16 cells, before and after being treated by these various
procedures, were tested for lectin activity by hemagglutination.
Since type 2 fimbriae of Actinomyces exhibit lectin activity, the
efficacy of each treatment of the cells in removing type 2 fimbriae
could be compared in this manner; presumably, type 1 fimbriae would be
removed also. Sonication and Lancefield extraction reduced the lectin
activity of N16 cells from 2+ to 0, whereas Virtis homogenization and
extraction with 8 M lithium chloride had little, if any, effect on the
lectin activity. Electron microscopy confirmed that sonication and
Lancefield extraction were much more effective than Virtis
homogenization or lithium chloride in removing fimbriae from N16
cells. The observation that treatment of N16 cells with 8 M LiCl had
no detectable effect on the fimbriae is contrary to the observation of
Mclntire et al. (82) that A. viscosus T14V cells treated in such a
manner lost their fimbriae and, consequently, their ability to
coaggregate with S. sanguis 34.
The total protein and total carbohydrate extracted per gram wet
weight of cells by these procedures are compared in Table 3.
Sonication released greater than 15 times more protein than either
Virtis homogenization or lithium chloride extraction, and the protein
to carbohydrate ratio was greater than 1.0 in each case. For
Lancefield extraction the protein to carbohydrate ratio was only
0.14. Lancefield extraction was eliminated from consideration as a
method for extracting fimbriae for subsequent purification because it


48
released 7 times more carbohydrate than protein and because of the
likelihood of protein denaturation resulting from acid hydrolysis.
However, it was the preferred method for preparing soluble antigens
for immunoelectrophoresis because (a) it was the most convenient way
to extract antigens from a large number of strains, and (b) it
invariably gave strong, symmetrical type 1 and type 2 fimbrial rockets
in LRI and XIEP.
TABLE 3. Total protein and total carbohydrate
released from N16 cells by several methods for extracting fimbriae
Method of extraction
CHOa
Protein*3
Continuous flow sonication
5.0
11.2
Virtis homogenization
0.5
0.6
8 M LiCl
0.3
0.4
Lancefield extraction0
22.8
3.1
mg total carbohydrate released per g wet weight of cells.
b
mg total protein released per g wet weight of cells.
c A different method was used to determine the wet weight of
cells subjected to Lancefield extraction, so the quantity of CHO and
protein extracted by that method cannot be compared directly to the
quantities extracted by the other methods.
Although French pressure cell shearing and continuous flow
sonication were my first and second choices, respectively, for
obtaining crude extracts for purification of N16 fimbriae, lack of
access to the appropriate equipment at the proper time ultimately
dictated that the initial crude fimbrial extracts be obtained by a
batch method of sonication. Later in the study, a French pressure
cell extract was also processed.


49
Identification of N16 Type 1 and Type 2 Fimbriae in
Crude Antigen Extracts by XIEP-A
Immunoelectron microscopy had been used to demonstrate that the
3B5.A1 epitope was located on N16 fimbriae, but whether those fimbriae
were type 1 or type 2 fimbriae had not yet been established. Analysis
of N16 crude sonicates by XIEP against rabbit antiserum to whole cells
revealed the presence of two major antigens that were thought to
correspond to type 1 and type 2 fimbriae (Figure 3, upper panels).
Cisar et al. (14) had shown that in XIEP Ag 1 (type 1 fimbriae) of
T14V migrated closer to the anode than Ag 2 (type 2 fimbriae).
Whether the type 1 and type 2 fimbriae of all Actinomyces strains
maintained the same relative electrophoretic mobilities was not
known. In fact, since crude sonicates of N16 cells contained other
antigens besides fimbrial antigens, and the antiserum used to
precipitate the antigens was not specific for fimbriae, the two N16
rockets did not necessarily have to be fimbrial antigens.
In order to establish the identity of the two N16 rockets and to
determine which type of fimbriae MAb 3B5.A1 recognized,
125
fimbriae-specific I-Ab was incorporated into the Ab-containing
portion of the gel along with the non-radiolabeled polyspecific
125
antiserum. When I-Ab specific for T14V type 1 fimbriae was used,
autoradiography of the gel demonstrated that the N16 rocket closer to
the anode corresponded to type 1 fimbriae (Figure 3A). When the gel
125
contained I-MAb 3B5.A1, autoradiography revealed that MAb 3B5.A1
bound to the other rocket (Figure 3B). Since (a) MAb 3B5.A1 binds to
fimbriae, (b) it did not bind to type 1 fimbriae, and (c) only two
types of Actinomyces fimbriae have been identified, by a process of
elimination it was determined that the antigen that migrated more


50
Figure 3. Identification of A. naeslundii N16 type I and type 2
fimbriae by XIEP-A. N16 cell surface antigens extracted by
sonication were separated by electrophoresis in the first
dimension with the anode to the left. Then they were
precipitated by electrophoresis (at a right angle to the
first dimension) into gel containing R29 rabbit antiserum
against N16 whole cells and ^25i-labeled IgG specific for
fimbriae. Two rockets precipitated by R29 are seen in the
Coomassie-stained gels (upper panel); the corresponding
autoradiographs (lower panel) indicate that the rocket
closer to the anode represents type 1 fimbriae, whereas the
other rocket represents type 2 fimbriae. In this and all
subsequent figures of XIEP patterns, the anode is on the
left for electrophoresis in the first dimension and at the
top for electrophoresis in the second dimension. The
type 1 fimbrial rocket is always on the left and the type 2
fimbrial rocket on the right.
A) 1-25i-R59 specific for the type 1 fimbriae of
A. yiscosus T14V binding to the type 1 fimbriae of N16;
B) -*-25I-MAb 3B5.A1 binding to the type 2 fimbriae of N16.


51
slowly toward the anode was type 2 fimbriae and that MAb 3B5.A1 was
specific for type 2 fimbriae.
Immunological detection of type 2 fimbriae throughout various
purification procedures and purification of type 2 fimbriae by
immunoaffinity chromatography were made possible by the availability
of MAb 3B5.A1. Also, XIEP-A proved to be a useful technique for
monitoring antigen fractions for the presence of both types of
fimbriae, as well as non-fimbrial antigens.
Purification of N16 Type 2 Fimbriae From a Crude Sonicate
Batch A
Aliquots of the untreated control cells and the sonicated cells
collected at different time intervals were assayed for lectin activity
by HA to monitor the removal of type 2 fimbriae as measured by the
decline in HA activity. Cells sonicated for only 30 sec had the same
HA titer as the untreated control cells. With cells sonicated from 1
to 6 min total, the HA titers of the cells declined as the total
sonication time increased. Most of the decrease in lectin activity
had occurred after 2 min of sonication, but even after sonication for
a total of 6 min, the cells still retained weak HA-positive activity.
Electron microscopy on the cells sonicated for 6 min revealed that
nearly all the cells had lysed and the cell walls appeared to be
devoid of fimbriae.
When the crude 6 min sonicate was applied to the Bio-Gel A-5m
column and eluted with TBS, two major protein-rich peaks were eluted,
one just after the void volume and the other near the total volume
(Figure 4). When the column fractions were dotted on nitrocellulose
and reacted with MAb 3B5.A1 in an indirect EIA, fractions 11-24 gave


Figure 4. Elution profile for chromatography of A. naeslundii N16 crude
sonicate on Bio-Gel A-5m. The hatched bar represents fractions
strongly reactive with MAb 3B5.A1 as determined by immunodot EIA.


2.2
Ln
LO


54
3-4+ reactions, but the intensity of the immunodots decreased sharply
beyond fraction 24. Although fractions 18-24 also contained type 2
fimbriae, only fractions 11-17 (designated A-5m peak 1) were pooled,
because fractions beyond the first major protein peak contained
increasing levels of carbohydrate. The relatively high carbohydrate
content of all the fractions in this batch probably was a reflection
of the substantial amount of cell breakage that occurred when the
cells were sonicated. When A-5m peak 1 was examined by XIEP against
rabbit antiserum to N16 whole cells, both types of fimbriae and some
nonfimbrial antigens were detected. A-5m peak 1 from Batch A was not
purified further because another batch, less contaminated with
cytoplasmic components, was prepared.
Batch B
Two minutes of sonication caused a 16-fold decrease in the 2+ HA
endpoint relative to the untreated cell suspension, suggesting
significant removal of type 2 fimbriae from the cells. Electron
microscopy of negatively stained cells confirmed that sonication
removed some but not all fimbriae from the cells. When the crude
sonicate was precipitated with (NH ) SO at 50% saturation and
again at 35% saturation, the supernatant fractions after dialysis and
concentration were examined for the presence of fimbriae by XIEP. The
results indicated that some of both types of fimbriae were lost to the
supernatant fraction with each precipitation; however, no attempt was
made to quantitate the amounts of fimbriae or total protein not
precipitated.
The precipitate from 35% (NH^)2SO^ saturation of the crude
2 min sonicate contained 390 mg total protein. This sample was


55
chromatographed in 5 separate aliquots on a Bio-Gel A-5m column; a
representative elution profile is shown in Figure 5. This elution
profile is similar to that obtained with Batch A (Figure 4), except
that the Batch B fractions contained much lower levels of total
carbohydrate. The first of two major protein-rich peaks that were
eluted from the column contained most of the MAb 3B5.A1 reactive
(type 2) fimbriae as determined by immunodot EIA on the column
fractions. All of the fractions representing the first peak (peak 1)
from each run, i.e., fractions 14-32 in Figure 5, were pooled; the
A-5m peak 1 pool contained 159 mg total protein.
A 5 mg aliquot of the peak 1 pool was chromatographed on the MAb
3B5.A1 immunoaffinity column. Analysis of the column fractions by
XIEP-A indicated that the unbound fraction resembled the starting
material, which suggested overloading of the column, whereas the bound
fraction appeared to contain only type 2 fimbriae.
The unbound fraction from the first immunoaffinity run was
rechromatographed on the MAb column. This resulted in a reduction in
type 2 fimbriae relative to type 1 in the unbound fraction and
recovery of type 2 fimbriae in the bound fraction with only minor
contamination with type 1 fimbriae (Figure 6). Thus, MAb 3B5.A1,
which is specific for the type 2 fimbriae of N16, is an effective tool
for the immunochromatographic purification of this structural entity.
The fimbrial immunoprecipitin arcs that were used to immunize
rabbits for the production of polyclonal antibodies specific for N16
type 1 or type 2 fimbriae were obtained from these fractions, as shown
in Figure 7 (refer also to Materials and Methods).


Figure 5. A representative elution profile for Bio-Gel A-5m chromatography of
aliquots of the fraction obtained by precipitating A. naeslundii
N16 crude sonicate with (NH^^SC^ at 357 saturation. The
hatched bar represents fractions strongly reactive with MAb 3B5.A1
as determined by immunodot EIA.


mg/ml


58
Figure 6. Results of immunoaffinity chromatography of A-5m peak 1 on
the MAb 3B5.Al-Affi-Gel 10 column as demonstrated by
XIEP-A. The antibody-containing portion of each gel
included R29 antiserum against N16 whole cells and
125I-labeled MAb 3B5.A1 specific for type 2 fimbriae.
The Coomassie-stained gels (upper panel) show all the
antigens precipitated by the polyspecific rabbit antiserum,
whereas their corresponding autoradiographs (lower panel)
identify the type 2 fimbriae.
A) A-5m peak 1, the starting material for immunoaffinity
chromatography, contained both types of fimbriae; B) The
unbound fraction also contained both types of fimbriae, but
there was a reduction in type 2 fimbriae relative to type 1;
C) The bound fraction contained mostly type 2 fimbriae with
a barely detectable quantity of type 1 fimbriae (arrow).


59
Figure 7. Source of immunogens for production of rabbit antisera
monospecific for N16 type 1 or type 2 fimbriae. Segments
of fimbrial immunoprecipitates were excised from the areas
indicated on the XIEP gels. R29 against N16 ceils was the
precipitating antiserum in both gels.
A) Type 1 fimbrial immunoarcs for production of RIO
antiserum;
B) Type 2 fimbrial immunoarcs for production of R2P
antiserum.


60
When RIO antiserum against N16 type 1 fimbrial immunoprecitins or
R2P against N16 type 2 fimbriae were examined by immunoelectron
microscopy against N16 cells, both antisera reacted with epitopes
located on the fimbriae (Figure 8; RIO reaction not shown). These
antisera were used to make polyclonal rabbit IgG immunoaffinity
columns for the purification of both types of fimbriae from the N16
French press supernatant.
Purification of N16 Fimbriae from the French Press Supernatant
In addition to the purification of N16 type 2 fimbriae from a
crude sonicate by sequential chromatography first on Bio-Gel A-5m and
then on the MAb immunoaffinity column, an attempt was made to purify
N16 type 1 fimbriae and type 2 fimbriae directly from a crude French
press supernatant by immunoaffinity chromatography on rabbit IgG
(RIgG) anti-N16 type 1 fimbriae and rabbit IgG anti-N16 type 2
fimbriae columns, respectively. When a 90 ml aliquot (252 mg protein)
was chromatographed on the polyclonal anti-N16 type 2 fimbriae
immunoaffinity column, the unbound fraction contained both types of
fimbriae, and the bound fraction, desorbed with 7 M urea buffer,
contained type 2 fimbriae with perhaps a trace of type 1 fimbriae, as
determined by immunodot EIA. Since the unbound fraction still
contained plenty of type 2 fimbriae, approximately half of it was
rechromatographed and eluted as before. The quantity of bound protein
desorbed on each run was so low that it was virtually undetectable by
A280; also, the recovery from the second run appeared to be lower
than the first, suggesting that the bound antigen was not being
desorbed completely with 7 M urea.


61
Figure 8. Electron micrograph of A. naeslundii N16 cells showing
indirect immunogold labeling of type 2 fimbriae. The cells
were incubated with R2P antiserum (rabbit anti-N16 type 2
fimbrial immunoarcs) and then with goat anti-rabbit IgG
conjugated to colloidal gold (diameter, 10 nm). The
gold-labeled cells were prepared for EM by the
pseudoreplica technique and were negatively stained with
0.5% uranyl acetate. Bar, 1 pm.


62
A dissociation experiment was undertaken to assess the efficacy
of various buffers in dissociating the antigen-antibody complexes
formed between N16 fimbriae and the anti-fimbrial antibodies used to
prepare the immunoaffinity columns; the results are summarized in
Table 4. Although 8 M urea buffer released 85% of Ag-bound MAb
3B5.A1, it released only 28.8% of rabbit IgG anti-N16 type 1 fimbriae
and 18.5% of rabbit IgG anti-N16 type 2 fimbriae. Thus, the 7 M urea
buffer normally used as the dissociating buffer for immunoaffinity
chromatography would be ineffective at desorbing bound fimbriae from
either of the polyclonal rabbit IgG anti-N16 fimbriae immunoaffinity
columns. The most effective dissociating buffer for the polyclonal
immunoaffinity columns was 6 M NaSCN, which gave 95.4% release of
rabbit IgG anti-N16 type 1 fimbriae and 91.0% release for rabbit IgG
anti-N16 type 2 fimbriae. There was no evidence that any of the
dissociating buffers irreversibly denatured fimbrial epitopes or
released fimbriae from the plate during these assays.
Based on the results of the dissociation experiment, the
desorbing buffer was changed to 6 M NaSCN, 0.05 M Tris-HCl, pH 7.5,
and the rabbit IgG anti-N16 type 2 fimbriae immunoaffinity column was
desorbed again to release the fimbriae that were not released by 7 M
urea. The bound fractions recovered from the type 2 immunoaffinity
column were pooled, concentrated, and dialyzed versus TBS by
ultrafiltration (Amicon YM10). Crystalline (NH^^SO^ was added
to 100% saturation, and the precipitated sample was stored at 4C
until further processing.
The unbound material recovered from the rabbit IgG anti-N16 type
2 fimbriae immunoaffinity column was chromatographed on the anti-N16


63
TABLE 4. Summary of the efficacy of various dissociation buffers in
disrupting binding between antibodies to N16 fimbriae and N16 fimbriae
coated on Immulon 2 microtiter plates
Antibody samples
Dissociation buffer RIgG a-1 RIgG a-2 MAb 3B5.A1
NaSCN, 6 M
NaSCN, 3 M
NaSCN, 1 M
GuHCl, 6 M
GuHCl, 3 M
GuHCl, 1 M
Urea, 8 M
Urea, 4 M
Urea, 2 M
Urea, 1 M
Glycine-HCl, pH 2.5
Glycine-HCl/10% C2H602 b, pH 2.5
NH4OH, pH 11.5
NH40H/107. C2H602, pH 11.5
95.4a
91.0
85.6
47.8
23.4
83.6
16.9
6.4
78.0
93.0
84.5
ND
46.5
30.2
82.2
17.1
8.6
76.6
28.8
18.5
85.0
5.3
3.9
73.9
3.1
2.1
33.2
12.1
0.0
7.7
81.7
64.9
86.7
72.3
54.0
85.6
16.8
7.4
82.8
26.8
10.8
73.6
Percent release of antibody from fimbriae-coated plates.
Ethylene glycol.


64
type 1 fimbriae immunoaffinity column. Since the column did not bind
all the type 1 fimbriae on the first pass, the unbound fraction was
rechromatographed for a total of three runs. The bound fractions were
eluted with 6 M NaSCN, pooled, concentrated, and dialyzed versus
saline by ultrafiltration. Examination of the unbound and bound
fractions by immunodot EIA revealed that rabbit Ig was leaching from
the column during desorption of the bound fimbriae with 6 M NaSCN.
Crystalline (NH^) SO4 was added to the unbound and bound
fractions, and they were stored as 75% and 100% saturated solutions at
4C.
Fimbrial samples from the French press supernatant, as well as
samples obtained from the Batch A and Batch B sonicates, were examined
by SDS-PAGE and immunoblot. However, before one can understand the
results of those experiments, an explanation of the nature of
Actinomyces fimbriae and the patterns they exhibit on immunoblots is
essential.
Effects of Various Physical and Chemical Treatments
on N16 Fimbriae
The fimbriae of E. coli and other gram-negative bacteria are
polymers of smaller subunits, and under the appropriate conditions,
they can be dissociated into their constituent monomers. The
following experiments were undertaken to (a) determine whether or not
N16 fimbriae had a subunit architecture or exhibited other properties
similar to those reported for the fimbriae of E. coli, (b) provide
additional evidence that N16 type 1 fimbriae differed from type 2
fimbriae, (c) see whether certain procedures caused changes in the
molecular weight or antigenicity of fimbrial bands.


65
Aliquots of the N16 crude French press sample were subjected to a
variety of physical and chemical treatments. The first was addition
of MgCl^ Unlike E. coli fimbriae (83), N16 fimbriae did not
precipitate with a final concentration of 0.1 M MgCl^.
Since the N16 crude French press supernatant contained both types
of fimbriae, as well as non-fimbrial components, the effects of the
other treatments on each type of fimbriae were demonstrated by
SDS-PAGE immunoblot analysis, using antibodies monospecific for type 1
or type 2 fimbriae. For some of these experiments a convenient method
for exchanging buffers and concentrating samples was needed. Acetone
precipitation appeared to be the ideal choice, but first it had to be
established that acetone precipitation would give total recovery of
fimbriae without adversely affecting their immunological reactivity.
When acetone precipitates of the crude fimbrial sample were compared
to the original sample, they gave identical patterns on immunoblots.
Acetone precipitation was thus employed whenever necessary to
accomplish the objectives cited above.
The effects of some of the physical and chemical treatments on
N16 type 1 and type 2 fimbriae are shown in Figures 9 and 10,
respectively. Exposure to Freon appeared to have no effect on N16
fimbriae; therefore, if extensive lysis were to occur during
sonication or French press shearing of cells to remove fimbriae,
lipids could be extracted from the crude fimbrial sample with Freon
before applying it to gel filtration or immunoaffinity columns.
Treatment with mutanolysin or lysozyme (lysozyme not shown) did
not change the fimbrial patterns observed on immunoblots. This
suggested that the ladderlike series of bands >100 kd represented


Figure 9. Immunoblot analysis of the effects of various physical and
chemical treatments on N16 type 1 fimbriae. Aliquots of
the N16 antigen sample were treated as indicated, then
prepared for SDS-PAGE by heating at 100 C for 5 min without
reduction. Each lane was loaded with 1 pg of total
protein, based on the protein concentration prior to
treatment. Prestained molecular weight standards were
mixed with two of the samples; their positions on the blot
were marked with a ballpoint pen, and approximate molecular
weights are expressed in kilodaltons. The blot was
developed with RIO IgG (anti-N16 type 1 fimbriae) at 1 pg
per ml as the primary antibody.


4S 00
I I
to
ON
4S
U>
00
v£>
III
44 4
m
ta
: 1
111
tit
Periodate-oxidized
Untreated
Lancefield-extracted
0.1 N HC1, 37 C, 1 h
0.1 N NaOH, 37 C, 1 h
8 M urea
Mutanolysin
Untreated
0.1 N HC1, 100 C, 5 min
0.1 N HC1, 100 C, 5 min
Untreated
Freon-extracted
Sonicated 10 min
Sonicated 5 min
Sonicated 1 min
100 C, 60 min
100 C, 5 min
65 C, 30 min
37 C, 60 min


Figure 10. Immunoblot analysis of the effects of various physical and
chemical treatments on N16 type 2 fimbriae. This
experiment was identical to that described in Figure 9,
except that the blot was developed with R2P IgG (anti-N16
type 2 fimbriae) as the primary antibody, and the lane
order of the samples is reversed.


1 J
"1
N>
O' OJ
.£> 00
O'
oo
37 C, 60 min
65 C, 30 min
100 C, 5 min
100 C, 60 min
Sonicated 1 min
Sonicated 5 min
Sonicated 10 min
Freon-extracted
Untreated
0.1 N HC1, 100 C, 5 min
0.1 N HC1, 100 C, 5 min
Untreated
Mutanolysin
8 M urea
0.1 N NaOH, 37 C, 1 h
0.1 N HC1, 37 C, 1 h
Lancefield-extracted
Untreated
Periodate-oxidized
O'
£>


70
fimbriae of different lengths rather than a fimbrial protein attached
to different lengths of peptidoglycan.
Sonication generated some fragments that were not evident in the
untreated control. The longer the sample was sonicated, the more
fragments were generated and the greater the intensity of the
immunological reaction. However, the generation of different
molecular weight bands by sonication did not appear to be completely
random. The highest molecular weight material was broken down into
smaller fragments, but the breakdown products generally were the same
molecular weight as fragments already present in the untreated
control. The fimbriae were fairly resistant to breakage into
fragments smaller than 100 kd; after 10 min of continuous sonication,
most of the fimbrial bands were still greater than 100 kd, even though
the most immunodominant bands were in the 35-65 kd range.
N16 fimbriae were not affected by 8 M urea or saturated guanidine
hydrochloride, treatments that would dissociate some types of E. coli
fimbriae into subunit monomers (61,62).
Another method that has been reported to disaggregate E. coli
fimbriae, i.e. 0.1 N HCl at 100C for 5 min (31), did not have a
similar effect on N16 fimbriae. Instead of causing dissociation into
monomers, hot acid caused loss of band resolution, probably as a
result of acid hydrolysis. This also occurred with Lancefield
extraction (0.04 N HCl at 100C for 15 min). However, 0.1 N HCl at
37C for 1 h had little, if any, effect on N16 fimbriae; and heating
o
at 100 C in the absence of a pH<2 did not cause loss of band
resolution. So, it was the combination of a very acidic pH and
o
100 C that was destructive to the N16 fimbriae.


71
Base was even more destructive than acid, as judged by the total
loss of band resolution and substantially reduced antigenicity when
N16 fimbriae were incubated with 0.1 N NaOH at 37C for 1 h.
Sodium periodate oxidation also caused some loss of band
resolution but not to the extent seen with acid or base hydrolysis.
There was some loss of antigenicity with increasing temperature
and, as might be expected, heating at 100C for 1 h was the most
destructive.
N16 type 1 fimbriae could be distinguished from type 2 fimbriae
by their different patterns on immunoblots, i.e. the different
molecular weight distribution of the unreduced immunodominant bands.
Disregarding their intrinsically different patterns, the two fimbrial
types generally behaved similarly in response to the various
treatments. For example, neither was affected by high concentrations
of urea or guanidine HC1, but both were most adversely affected by
incubation with 0.1 N NaOH. The decrease in antigenicity of N16
fimbriae when incubated with NaOH or heated at 100C for 60 min is
consistent with the findings of Masuda et al. (77) for A. viscosus
WVU627 fimbriae. Although they reported that WVU627 fimbriae were
also labile when incubated with 0.1 N HC1 at 37C for 60 min, a
similar effect was not observed with N16 fimbriae. However,
immunoblot analysis of SDS-PAGE-resolved proteins may not have been
the best way to examine the effects of the various physical and
chemical treatments because denaturation of the proteins by heating in
SDS-PAGE sample buffer may mask the true effects of the other
treatments. It might have been better to use an assay that could
quantitate the amount of immunoreactive fimbriae remaining after


72
treatment, perhaps by using LRI, XIEP, EIA, or radioimmuno-
precipitation. On the other hand, SDS-PAGE immunoblots were probably
the best way to see the effects of sonication.
A. naeslundii N16 fimbriae were not disaggregated by any of the
methods that would dissociate E. coli fimbriae, pili, or flagella
(31,61,62,70,83). These results are consistent with the observation
that A. viscosus T14V fimbriae cannot be completely dissociated either
(12,84).
Immunoblots were also used to assess the effects of various
proteases on the molecular weight distribution and immunological
reactivity of N16 type 1 and type 2 fimbrial bands. All the proteases
eliminated the immunoreactive bands >200 kd for both types of
fimbriae. Samples treated with papain, Pronase, or proteinase K
exhibited a total loss of immunoreactive type 1 and type 2 fimbrial
bands. However, since papain caused only limited digestion of the BSA
control as determined from the presence of multiple bands in the range
below 35 kd on a silver-stained SDS-PAGE gel, it is possible that
papain and perhaps the other enzymes destroyed the fimbrial epitopes
without completely digesting the fimbriae.
The effects of chymotrypsin, trypsin, and V8 protease on N16 type
1 and type 2 fimbriae are shown in Figure 11 and Figure 12,
respectively. Each of these enzymes caused limited digestion of both
types of fimbriae, but the resulting immunoblot patterns were
different for each type of fimbriae. Trypsin caused a total loss of
band resolution for type 1 fimbriae; except for two discrete bands at
<14 kd, trypsin-treated type 1 fimbriae gave an immunoreactive smear
from 40-200 kd on immunoblot. On the other hand, the immunoreactive


Figure 11. Immunoblot analysis of the effects of digestions with
different proteases on N16 type 1 fimbriae. The lanes
containing untreated or enzyme-treated N16 antigens were
loaded with 2 pg of N16 protein, based on the
concentration of the N16 sample prior to treatment,
whereas the lanes containing enzyme only (lanes 3, 5, and
8 from left to right) were loaded with quantities of
enzyme equivalent to those in the enzyme-treated samples.
All samples were unreduced, except for the one marked R.
Prestained molecular weight standards were mixed with the
untreated control in lane 1. The blot was developed with
RIO IgG (anti-N16 type 1 fimbriae) at 2.5 pg per ml as
the primary antibody.


200
Untreated
Trypsin-treated
Trypsin
Chymotrypsin-treated
Chymotrypsin
V8 protease-treated, R
V8 protease-treated
V8 protease


Figure 12. Immunoblot analysis of the effects of digestions with
different proteases on N16 type 2 fimbriae. This
experiment was identical to that described in Figure 11,
except that the blot was developed with R2P IgG (anti-N16
type 2 fimbriae) as the primary antibody, and the lane
order reads right to left.


'W
ii K :
1 OBBI
ii i m nm
E l V
II I III
4>* 00
hO
p'
co
ON
00
vO
'-J
V8 protease
V8 protease-treated
V8 protease-treated, R
Chymotrypsin
Chymotrypsin-treated
Trypsin
Trypsin-treated
Untreated


77
type 2 fimbrial bands after trypsin treatment were mostly between the
14 and 43 kd markers, and for the most part, the bands were discrete
rather than unfocused. The effect of chymotrypsin on type 1 fimbriae
was very similar to that of trypsin, whereas for type 2 fimbriae, the
pattern obtained by digestion with chymotrypsin was quite different
from that obtained with trypsin. V8 protease-treated samples were
less immunoreactive than the trypsin- or chymotrypsin-treated samples,
even though V8 protease appeared to be less efficient than the other
two enzymes at digesting the higher molecular weight fragments.
Effects of Temperature and Reduction on N16 Fimbriae
On immunoblots the most immunoreactive N16 type 1 and type 2
fimbrial bands undergo a shift in apparent molecular weight in
response to increases in the temperature for SDS-PAGE sample
preparation, as demonstrated in Figure 13. In unreduced samples
heated at 37C for 30 min there are a series of type 1
immunoreactive fimbrial bands at or slightly below the 43 kd molecular
. o
weight marker, whereas after heating at 100 C for 5 min, the
immunodominant bands are in the 57-65 kd range. Both sets of bands
are present in the sample heated at 65C for 15 min, so the
immunoblot pattern at this intermediate temperature shows the
"transitional state of proteins switching from their lower molecular
weights at 37C to higher molecular weights at 100C. Reduction
with 0.01 M DTT (or 1% 2-mercaptoethanol) further simplifies the
pattern of bands so that with samples reduced at 100C the primary
type 1 fimbrial bands on immunoblots are as follows: the uppermost
band has an apparent molecular weight of about 65 kd; there is a broad
area of immunoreactivity in the 57-60 kd range, which represents at


Figure 13. Immunoblot analysis of the effects of temperature and
reduction on the type 1 and type 2 fimbriae of
A. naeslundii N16. Aliquots of the N16 antigen sample
were treated with SDS-PAGE sample buffer, with or without
0.1 M dithiothreitol (DTT), by heating at the temperature
and times indicated. Each lane of the SDS-PAGE gel was
loaded with 1 pg of protein. Approximate molecular
weights are expressed in kilodaltons. The left half of
the blot was developed with RIO IgG at 5 pg/ml, and the
right half was developed with R2P IgG at 5 pg/ml.


I I
H-
00
37
c,
30
min,
-
DTT
37
c,
30
min,
+
DTT
65
c.
15
min,
-
DTT
65
c,
15
min,
+
DTT
100
c,
5
min,
-
DTT
100
c,
5
min,
+
DTT
100
c,
5
min,
+
DTT
100
c,
5
min,
-
DTT
65
c,
15
min,
+
DTT
65
c,
15
min,
-
DTT
37
c,
30
min,
+
DTT
37
c,
30
min,
-
DTT
ho
I
ro
ON
C* 0> vO
LO 00
I
KJ
O
o
vO
Anti-Type 1 Anti-Type


80
least two bands, including a component at 57 kd that reacts with
normal RIgG; then there is a weaker doublet at about 53-54 kd.
In unreduced samples heated at 37C, the immunoblot pattern for
type 2 fimbrial bands shows a series of bands near the 43 kd marker,
o
whereas unreduced samples heated at 100 C show a very reactive
doublet at about 62 and 63 kd and several closely spaced bands at
o
about 39-40 kd. As with the type 1 pattern, the 65 C type 2 pattern
o o
showed the transition from the 37 C to the 100 C pattern. Type 2
fimbriae were also affected by reduction in that the prominent doublet
at 62-63 kd in the nonreduced sample is replaced by a single 63 kd
band, and a minor doublet is still evident at 39-40 kd. On type 2
blots there was a very weakly immunoreactive band at about 23 kd that
was present in reduced and nonreduced samples prepared at any
temperature. A similar band at 18 kd (not visible in Figure 13) was
sometimes visible on type 1 blots. These low molecular weight bands
might represent fimbrial subunits (E. coli fimbrial subunits are
approximately this size); however, they may just be products of
degradation.
The observation that A. naeslundii N16 fimbrial proteins are heat
modifiable is consistent with the report of Yeung et al. (108) that
A. viscosus T14V type 1 fimbriae have a heat modifiable subunit with
an apparent molecular weight of about 50 kd at 37C or 65 kd at
100 C. Similarly, Yeung et al. reported that the fimbrial subunit of
A. naeslundii WVU45 type 2 fimbriae has an apparent molecular weight
of about 48 kd at 37C or 60 kd at 100C (109, M. K. Yeung,
personal communication).
It is likely that the observed shifts in molecular weights of
fimbrial proteins in response to heat are the result of changes in


81
conformation. Strong intramolecular interactions at the lower
temperature may prevent complete unfolding of the protein and
saturation with SDS, thereby altering the mobility in gels. The
results obtained with the cloned T14V type 1 fimbrial subunit (108)
tend to support such an explanation. Intermolecular interactions of a
noncovalent or covalent nature could also prevent complete
denaturation of fimbrial complexes and account for some of the
observed differences in the 37C and 100C immunoblot patterns.
The observation that N16 fimbriae were susceptible to reducing
agents differs from all previous reports on fimbriae from other
strains of Actinomyces (76,77,108). However, Masuda et al. (76) were
unable to get many of their fimbrial samples to migrate into 5 or 7%
SDS-PAGE gels, and their single percentage gels would not have the
resolving power of the 5-20% gradient gels used in this study. Thus,
solubility problems and inadequate resolution of proteins with very
similar molecular weights might have prevented them from detecting the
effects of reduction on fimbrial bands. This is especially likely in
light of the finding of Yeung et al. (108) that the cloned T14V type 1
fimbrial subunit migrated as a single band in 10 or 12% gels but as a
doublet in 5-12% gradient gels.
If one were examining the effect of reduction on a purified
protein complex that migrated as a single band on SDS-PAGE gels, one
would expect that a reduction would cause a single higher molecular
weight band to dissociate into one or more lower molecular weight
bands, depending on whether the complex was composed of identical or
dissimilar polypeptide chains held together by disulfide bonds.
However, fimbrial immunoblot patterns are too complex to allow such a


82
simple interpretation. A comparison of the differences between the
patterns of type 2 fimbriae prepared with or without reduction at
100C indicates that reduction caused changes in the visible bands
at virtually every molecular weight level. There are too many bands
present to speculate on whether any particular band was derived from
another, but the fact that the reduced samples appear to have fewer
and better focused bands than nonreduced samples suggests that the
reduction of intra- or intermolecular disulfide bonds allows complete
denaturation and SDS saturation of the proteins, thereby overcoming
conformational variations that affect the mobilities and apparent
molecular weights of fimbrial fragments.
Another possible explanation for the presence of fewer reactive
bands in reduced samples is that reduction may destroy the fimbrial
epitopes on some bands so that they are no longer visible on
immunoblots. This explanation would be more plausible if the blots
were developed with monoclonal antibody; but since they were developed
with polyclonal antibodies, it is not likely that destruction of
epitopes could explain these results.
In summary, since complete dissociation of N16 fimbriae was not
possible, immunoblot patterns exhibited a multitude of fimbrial
bands. The simplest pattern of bands was obtained with fimbrial
samples heated at 100C in the presence of DTT or
2-mercaptoethanol. In addition to the ladderlike series of bands in
the high molecular weight range, there were several strongly
immunoreactive bands (referred to as fimbrial subunits) in the 35-65
kd range. A. viscosus T14V type 1 and type 2 fimbriae have been
reported to give immunoblot patterns (32,108) similar to those


83
obtained in this study with N16 fimbriae. The fimbrial subunit
patterns for unreduced and reduced A. naeslundii N16 type 1 and type 2
fimbriae are summarized in Figure 14.
Assessment of the Purity of Fimbrial Samples by SDS-PAGE-Immunoblot
When aliquots of the type 1 and type 2 fimbriae purified from the
polyclonal antibody immunoaffinity columns were examined by SDS-PAGE
and immunoblot, it was apparent from the presence of immunoglobulin
heavy and light chains on the silver-stained SDS-PAGE gel (not shown)
that both samples were contaminated with RIgG that had leached off the
immunoaffinity columns. Consequently, immunoblot analysis of these
two fractions was complicated by the reaction of the peroxidase
conjugate with RIgG present in the fimbrial samples. The immunoblot
patterns of these two samples as well as those for the crude French
press supernatant, Batch A A-5m peak 1, Batch B 35% saturated
(NH^)^SO^ precipitate of the crude sonicate, and two samples of
the MAb-purified type 2 fimbriae, are shown in Figure 15.
The fimbrial subunits for both type 1 and type 2 fimbriae appear
to be present in all the samples except the two MAb-purified
fractions, although the weak type 1 bands in the type 2 fimbriae
purified from the RIgG column may actually be a reaction of conjugate
with immunoglobulin heavy chains contaminating the preparation. The
65 kd type 1 fimbrial subunit is readily apparent only in the type 1
fimbriae recovered from the RIgG column, indicating that this fraction
was enriched for type 1 fimbriae. That band is also present, but not
as obvious, in the French press supernatant, sonicate, and A-5m peak 1
samples, all of which should contain both types of fimbriae. The 63
kd type 2 fimbrial subunit is present in the type 2 fimbrial samples,


84
Type 1 Type 2
R NR R NR
43
26
Figure 14. Line drawing summarizing the "fimbrial subunit" patterns
obtained on immunoblots for A. naeslundii N16 type 1 and
type 2 fimbriae for samples heated at 100 C for 5 min.
R = reduced with 0.01 M DTT; NR = unreduced. Approximate
molecular weights are expressed in kilodaltons.


Figure 15. Immunoblot analysis of several preparations of N16
fimbriae at different stages of purification. Crude
French press (FP) supernatant was the starting material
for immunoaffinity purification of type 1 and type 2
fimbriae from the RIgG anti-type 1 and anti-type 2
columns, respectively. The middle lane contains the
Batch B 35% precipitate of the crude 2 min
sonicate. Also shown are A-5m peak 1 from Batch A and two
samples of type 2 fimbriae purified on the MAb 3B5.A1
column. All samples were reduced with DTT (0.01 M final
concentration). The blot was developed with a combination
of RIO IgG at 5 pg/ml and R2P IgG at 2.5 yg/ml, so
both types of fimbriae are evident.


FP supernatant
Type 1 fimbriae/RIgG
Type 2 fimbriae/RIgG
Sonicate/35% ppt
A-5m peak 1
Type 2 fimbriae/MAb-1
Type 2 fimbriae/MAb-2


87
but the 39-40 kd doublet is not apparent. In fact, the 39-40 kd
doublet was present only in the crude French press supernatant, so
there is some question as to whether or not it is a type 2 fimbrial
subunit.
An attempt was made to remove the RIgG contaminating the
immunoaffinity purified type 1 and type 2 samples by adsorption with
protein A-Sepharose, but SDS-PAGE analysis of the samples after
protein A adsorption indicated that immunoglobulin chains were still
present in the fimbrial samples.
The protein A-adsorbed fimbrial samples and several of the other
samples were examined again by immunoblot analysis as shown in Figure
16. Duplicate blots were developed with either RIO against N16 type 1
fimbriae or R2P against N16 type 2 fimbriae. Also included for
comparison to N16 fimbriae were samples of purified T14V type 1 and
type 2 fimbriae. The T14V fimbrial samples appeared to be pure since
type 2 fimbriae did not react with RIgG against N16 type 1 fimbriae or
vice versa. On the other hand, the N16 type 2 fimbriae recovered from
the MAb immunoaffinity column exhibited minor contamination with type
1 fimbriae, as determined previously by XIEP (Figure 6C). The subunit
pattern for T14V type 1 fimbriae on the immunoblot developed with RIgG
against N16 type 1 fimbriae differs from the N16 type 1 fimbrial
subunit pattern, whereas the type 2 patterns for each appear to be
more similar. Another interesting observation is that the 63 kd N16
type 2 subunit (in the French press supernatant reacted with anti-type
2) appears as a doublet. On all the other immunoblots it appeared to
be a single band, but this sample was electrophoresed longer than
usual, and better resolution was obtained.


Figure 16. Iiranunoblot analysis of A. naeslundii N16 and A. viscosus
T14V fimbrial samples. All samples were reduced with DTT
at 100 C. The left half of the blot was developed with
RIO IgG anti-N16 type 1 fimbriae, and the right half was
developed with R2P IgG anti-N16 type 2 fimbriae.


N)
on
LO
ON
00
VO
N)
O
o
I
1.
1
*T-'
* I1--
FP supernatant
T14V type 1 fimbriae
Type 1 fimbriae/RIgG
RI&G
Type 2 fimbriae/RIgG
Type 2 fimbriae/MAb
T14V type 2 fimbriae
A-5m peak 1
FP supernatant
T14V type 1 fimbriae
Type 1 fimbriae/RIgG
RIgG
Type 2 fimbriae/RIgG
Type 2 fimbriae/MAb
T14V type 2 fimbriae
, A-5m peak 1
M
00
lO
Anti-Type 1 Anti-Type


90
In summary, the N16 fimbrial samples obtained by immunoaffinity
chromatography were enriched for one of the fimbrial types, but none
of the samples were completely pure. The MAb immunoaffinity column
appears to offer the most promise for purification of type 2 fimbriae,
but it might be necessary to incorporate a detergent into the starting
buffer to prevent nonspecific adsorption of type 1 fimbriae. As for
the RIgG columns, it was probably too much to expect that they could
produce pure fimbriae from totally crude starting material. However,
since they required such harsh conditions to elute bound fimbriae that
some RIgG also was stripped from the columns, it is unlikely that
these immunoaffinity columns would be as useful as MAb columns.
Antigenic Relatedness of Actinomyces Fimbriae
Ouchterlony Analysis
Like the type 1 and type 2 fimbriae of A. viscosus T14V (20), the
type 1 and type 2 fimbriae of A. naeslundii N16 were found to be
unrelated antigenically, as demonstrated by the reactions of
non-identity between type 1 and type 2 fimbrial precipitins in
immunodiffusion (Figure 17A). A reaction of partial identity between
the type 2 fimbrial bands precipitated by MAb 3B5.A1 and the
polyclonal reference antiserum (Figure 17B) provided additional
evidence that MAb 3B5.A1 is specific for an epitope on the type 2
fimbriae; however, the presence of a spur suggests that there are
other epitopes on N16 type 2 fimbriae that elicit the formation of Abs
in a polyclonal response to the antigen. MAbs 2A3.B3 and 2B5.B6 did
not precipitate either type of fimbriae, perhaps because (a) the
proportions of antigen and antibody were not at equivalence or (b) the


91
Figure 17. Immunodiffusion reactions of antibodies to N16 fimbriae.
The center wells contained N16 fimbriae partially purified
from a French press supernatant by precipitation at 20%
(w/v) (NH4)2SO4. Rabbit antiserum R29 against N16
whole cells precipitated both type 1 and type 2 fimbriae.
A) A reaction of non-identity between type 1 and type 2
fimbriae. Antisera were RIO against N16 type 1 fimbriae
and R2P against N16 type 2 fimbriae.
B) A reaction of partial identity between the type 2
fimbrial bands precipitated by MAb 3B5.A1 and the
polyclonal reference antiserum R29. MAb 2A3.B3 and
MAb 2B5.B6 did not precipitate the fimbriae.


92
density of fimbrial epitopes was too low, as suggested previously by
the sparse distribution of gold particles in immunoelectronmicroscopy.
XIEP-A
The antigenic relatedness among fimbriae from different isolates
of A. naeslundii and A. viscosus was examined by XIEP-A. Unlabeled
homologous antisera raised against whole cell antigens were
incorporated into gels to precipitate fimbrial and sometimes non-
125
fimbrial antigens; in addition, an I-labeled antibody specific
for one type of fimbriae was incorporated into the gel so that
autoradiography would reveal which type of fimbriae reacted with the
monospecific antibody. Six fimbriae-specific antibodies were examined,
and the results of reactions with A. naeslundii antigens are
summarized in Table 5.
RIO rabbit IgG anti-N16 type 1 fimbriae reacted strongly with the
type 1 fimbriae of all 10 A. naeslundii serotype 3 isolates but not
with the type 2 fimbriae (Figure 18). It cross-reacted with the type
1 fimbriae of A. naeslundii serotype 2 W1544, but it did not react
with A. naeslundii serotype 1 W826 (WVU45), a serotype that has been
reported to lack type 1 fimbriae (17).
R2P rabbit IgG anti-N16 type 2 fimbriae reacted strongly with the
type 2 fimbriae of the 10 A. naeslundii serotype 3 isolates, but it
was essentially negative for type 1 fimbriae and the fimbriae of other
serotypes of A. naeslundii. Heterologous fimbriae were sometimes
visible on autoradiographs, but the intensity was so weak that it was
difficult to determine whether the reactions were the result of
nonspecific binding or very low levels of cross-reactivity; normal
rabbit IgG gave similar results.


Table 5. Summary of the reactions of fimbriae-specific 125I-labeled antibodies
with Lancefield-extracted type 1 and type 2 fimbriae of A. naeslundii isolates
Antigen
RIO
R2P
125i-Antibodya
MAb R59
R55
R70
Type
1
Type
2
Type
1
Type
2
Type
1
Type
2
Type
1
Type
2
Type
1
Type
2
Type
1
Type
2
N16
4b
0
0
4
0
4
4
0
1
1
1
1
WVU1267
4
0
0
4
0
4
4
0
1
4
1
2
WVU1468
4
0
0
4
0
4
4
0
1
4
1
2
WVU1527
4
0
0
4
0
4
4
0
1
1
1
1
WVU1528
4
0
0
4
0
4
4
0
1
1
1
1
W1629
4
0
0
4
0
4
3
1-2
1
1
1
2
W2273
4
0
0
4
0
4
4
0
1
1
1
1
W2821
4
0
0
4
0
4
4
0
1
1
1
1
UF92
4
0
0
4
0
4
4
0
1
4
1
1
UF524
2
0
0
4
0
4
4
1
1
1
1
1
W826 (WVU45)
_c
0
-
0
-
0
-
0
-
0
-
4
W1544
2-3
0
0
0
0
0
3
1
0
4
0
0
a RIO =
rabbit IgG anti-N16
type 1
fimbriae
R2P =
rabbit IgG anti-N16
type 2
fimbriae
MAb =
MAb 3B5.A1 anti-N16
type 2
fimbriae
R59 =
rabbit IgG anti-T14V
type 1
. fimbriae
R55 =
rabbit IgG anti-T14V
type i
! fimbriae
R70 = rabbit IgG anti-WVU45 type 2 fimbriae
b Reactions of l^I-antibodies with fimbrial rockets in XIEP-A were graded on a scale of
0 to 4 relative to the intensity of reaction with the homologous control. The homologous
reaction for R59 was 4,1 (type 1, type 2 fimbriae, respectively), and the homologous reaction for
R55 was 1,4.
c
W826 (WVU45) does not have type 1 fimbriae (17).
o


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FILES



CHARACTERIZATION OF FIMBRIAE OF ACTINOMYCES NAESLUNDII N16
USING MONOCLONAL AND POLYCLONAL ANTIBODIES
By
SANDRA L. BRAGG
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988

ACKNOWLEDGMENTS
The author wishes to express her sincere gratitude to Dr. Arnold S.
Bleiweis, chairman of her supervisory committee, for his valuable
guidance and encouragement throughout this study. She would also like
to thank the other members of her committee for their advice, and
especially Dr. Paul A. Klein, for his advice and moral support.
This study was supported by the Centers for Disease Control,
Atlanta, GA. The author would like to thank the Division of Mycotic
Diseases, especially Dr. Libero Ajello, Dr. Leo Kaufman, and Dr. Errol
Reiss for their infinite patience and cooperation.
The electron microscopy would not have been possible without the
expertise of Dr. Greg Erdos and Mary Lane Martin. The assistance of Ray
Simons and Don Howard for their excellent scientific photography is
gratefully acknowledged.
Special thanks go to Marianna Wilson; without her support and
expert assistance, completion of this dissertation would not have been
possible.
li

TABLE OF CONTENTS
paRe
ACKNOWLEDGMENTS ii
ABSTRACT v
INTRODUCTION 1
MATERIALS AND METHODS 8
Antigen Preparation 8
Cultures 8
Culture Conditions 9
Preparation of Crude Soluble Antigens 9
Preparation of Fimbriae for Purification 11
Sonication 11
French press shearing 12
Gel Filtration Chromatography 13
Treatments of Fimbriae by Physical/Chemical Means 14
Antibody Preparation 17
Immunization of Mice 17
Monoclonal Antibodies 17
Isotyping 18
Polyclonal Antibodies 18
DEAE Chromatography 19
Protein A-Sepharose Chromatography 20
Radiolabeling 20
Immunoaff inity Chromatography 20
Dissociation Experiment 21
Assays 23
Electron Microscopy 23
Hemagglutination 24
Coaggregation 25
Bacterial Agglutination 26
Radioimmunoassay 26
Indirect Enzyme Immunoassay 27
Enzyme Immunodot Assay 28
Immunodiffusion 28
Laurell Rocket Immunoelectrophoresis 29
Crossed Immunoelectrophoresis with Autoradiography 29
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis... 29
Immunoblot 30
iii

RESULTS AND DISCUSSION
31
Monoclonal Antibodies 31
Evidence for the Presence of Both Types
of Fimbriae on A. naeslundii Serotype 3 Strains 35
Evidence for the Presence of Type 1 Fimbriae 35
Evidence for the Presence of Type 2 Fimbriae 43
Antigen Preparation: Results of Preliminary Experiments 46
Identification of N16 Type 1 and Type 2 Fimbriae
in Crude Antigen Extracts by XIEP-A 49
Purification of N16 Type 2 Fimbriae from a Crude Sonicate 51
Batch A 51
Batch B 54
Purification of N16 Fimbriae from the
French Press Supernatant 60
Effects of Various Physical and Chemical Treatments
on N16 Fimbriae 64
Effects of Temperature and Reduction on N16 Fimbriae 77
Assessment of the Purity
of Fimbrial Samples by SDS-PAGE-Immunoblot 83
Antigenic Relatedness of Actinomyces Fimbriae 90
Ouchterlony Analysis 90
XIEP-A 92
Bacterial Agglutination 106
Immunoblot Analysis 115
SUMMARY AND CONCLUSIONS 119
REFERENCES 127
BIOGRAPHICAL SKETCH
137

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION OF FIMBRIAE OF ACTINOMYCES NAESLUNDII N16
USING MONOCLONAL AND POLYCLONAL ANTIBODIES
By
Sandra L. Bragg
August 1988
Chairman: Dr. Arnold S. Bleiweis
Major Department: Microbiology and Cell Science
Two populations of fimbriae, which differ both in antigenicity and
biological activity, have been identified on Actinomyces viscosus T14V
cells. Although A. naeslundii serotype 1 isolates possess only one of
these fimbrial populations (type 2 fimbriae), there was functional
evidence to suggest that A. naeslundii serotype 3 strain N16 had both
types of fimbriae. The purpose of this study was to characterize the
fimbriae of A. naeslundii N16 immunologically by using both monoclonal
and polyclonal antibodies.
Three monoclonal antibodies (MAbs) to N16 were produced; all three
bound to N16 fimbriae as determined by immunoelectron microscopy. In a
solid-phase radioimmunoassay MAb 3B5.A1 reacted with 100% of the
A. naeslundii serotype 3 isolates tested, but it did not react with any
heterologous isolates. Type 1 and type 2 fimbriae were detected in
Lancefield extracts of N16 cells by crossed immunoelectrophoresis (XIEP)
125
using rabbit antiserum against N16 whole cells. When I-MAb 3B5.A1
was also incorporated into the gel, autoradiography indicated that MAb
v

3B5.A1 was specific for type 2 fimbriae. The N16 type 2 fimbriae were
purified by gel filtration and immunoaffinity chromatography on a MAb
3B5.A1 column.
Polyclonal antisera specific for N16 type 1 or type 2 fimbriae were
produced by immunizing rabbits with fimbrial immunoprecipitins excised
from XIEP gels. These antisera were used to identify fimbrial bands on
immunoblots of SDS-PAGE resolved N16 fimbrial antigens. Although N16
fimbriae could not be completely dissociated, type 1 fimbrial subunits
at 65 kd, 57-60 kd, and a weaker doublet at 53-54 kd and type 2 subunits
at 63 kd and a doublet at 39-40 kd were identified in fimbrial extracts
reduced at 100°C.
Fimbriae-specific polyclonal and monoclonal antibodies were used in
various immunological assays to determine that (a) N16 type 1 fimbriae
are not related antigenically to type 2 fimbriae, (b) each type of
fimbriae has epitopes that are present on the corresponding fimbriae of
certain heterologous strains, and (c) MAb 3B5.A1 recognizes a
serotype-specific epitope residing on the type 2 fimbriae of
A. naeslundii serotype 3 strains.
vi

INTRODUCTION
Many structures found on the cell surfaces of microorganisms
exhibit properties that enable microorganisms to cause disease. These
properties include (a) adherence to host surfaces and other microbes,
(b) evasion of host defenses, and (c) destruction of host tissues. In
addition, surface molecules carry antigenic determinants that form the
basis of many serological and immunological identification
procedures. The fimbriae of certain species of Actinomyces are one
example of surface components that not only mediate adherence both to
host surfaces and to other microbes but also possess important
antigenic determinants.
Actinomyces are gram-positive, nonacidfast, nonsporeforming,
nonmotile bacteria that are highly variable in morphology, most
characteristically diphtheroidal, or filamentous and branched. They
ferment carbohydrate with production of acid but no gas, and except
for A. viscosus, all species are catalase-negative. Their cell walls
do not contain diaminopimelic acid, a characteristic that
differentiates them from Arachnia propionica, a morphologically
similar organism. Their natural habitat is the oral cavity of man and
other animals (96). All Actinomyces species, as well as Arachnia
propionica. are potential agents of actinomycosis (45).
Classical actinomycosis is a chronic granulomatous disease
characterized by the formation of abscesses and draining sinuses. It
is an endogenously acquired infection that can involve the soft tissue
1

2
and bone in any area of the body. Clinically, actinomycosis is
usually categorized as cervicofacial, thoracic, or abdominal. Of
these categories, cervicofacial actinomycosis is the most common, and
Actinomyces israelii is the most important etiological agent of
actinomycosis in humans. However, the extraoral infections of
classical actinomycosis are not as prevalent as intraoral periodontal
infections, and it has been suggested that the clinical classification
scheme be broadened to include a periodontal category (56).
During the last twenty-five years, most of the research efforts
dealing with Actinomyces species have been directed toward trying to
delineate the role that Actinomyces species play in the formation of
dental plaque and the development of periodontal disease. In the
early 1960s A. viscosus was found to be the etiologic agent of a
transmissible periodontal disease in Syrian hamsters (59,65). That
discovery initiated an explosion of research activity focused on the
pathogenicity of Actinomyces species in the oral cavity of man and
other animals because periodontal disease, unlike classical
actinomycosis, was and still is a major public health problem.
Virtually all humans and many animals accumulate dental plaque on
their teeth, and the formation of plaque may be followed by the
development of both caries and periodontal disease (49).
Periodontal disease is a collective term for a variety of chronic
inflammatory diseases of the structures that support the teeth.
Periodontal disease can range from mild inflammation of the marginal
gingiva (gingivitis) to severe forms of periodontitis, in which
extensive destruction of soft tissues and resorption of alveolar bone
can result in loss of the teeth. Although the microbial etiology and

3
pathology of the different types or stages of periodontal disease may
vary (98) , the formation of dental plaque is the common first step in
the development of periodontal disease (49). However, successful
colonization of the tooth surface or other surfaces of the oral cavity
depends upon the ability of oral microbes to anchor themselves, either
directly or indirectly, to these surfaces to avoid being swept away by
the flow of saliva (47,104). Thus, an understanding of the mechanisms
by which microbes attach to surfaces in the oral cavity and to other
microbes is crucial to understanding the sequence of events leading to
periodontal disease.
Studies with experimental animals indicated that both A. viscosus
and A. naeslundii could form dental plaque and initiate the
pathological changes associated with periodontal disease
(55,58,59,60,97). However, most of the attention has been focused on
A. viscosus, particularly in studies on mechanisms of attachment to
oral surfaces and other bacteria (6,13,14,16,19,21,22,27,48,72,81,82,
92,106,107) and mechanisms for tissue destruction (1,8,9,10,39,40,51,
53,74,78,94).
There were two factors that contributed to the placing of more
emphasis on A. viscosus than A. naeslundii as a subject for
periodontal research. One was the recognition that these two species
tended to be distributed differently in the human oral cavity in that
A. viscosus preferentially colonized the teeth, whereas A. naeslundii
was associated with mucosal epithelial surfaces (34,35). The second
was the existence of the T14V-T14AV model system for comparative
studies of the properties that enable T14V to be virulent (i.e., cause
periodontal disease) in experimental animals and T14AV to be avirulent

4
(6,51). Although it was soon determined that the virulence-
associated differences in the antigens of T14V and T14AV were
quantitative rather than qualitative (19,22,90), T14V became the focus
of intensive study because it was found to have two different
adherence-related properties. These were the ability to coaggregate
with Streptococcus sanguis via a lactose-inhibitable lectin (82) and
the ability to adsorb well to saliva-coated hydroxyapatite (24).
A. naeslundii, on the other hand, coaggregated streptococci (36) but
did not bind well to saliva-coated hydroxyapatite (24). Some of these
early studies established an association between the presence of
fibrils or fimbriae on the surface of A. viscosus cells and
adherence-related functions (19,22,82).
Fimbriae are proteinaceous surface appendages that are found on
many gram-negative and gram-positive bacteria and that often mediate
attachment to host surfaces. Fimbriae from five strains of
A. viscosus and two strains of A. naeslundii have been isolated and
characterized as to their amino acid compositions (21,76,77,106).
Polar uncharged and nonpolar amino acids together made up 62-74% of
the total amino acids, whereas basic amino acids accounted for only
10-17% of the total; aspartic and glutamic acid generally comprised
20-24% of the total amino acids (76). The minimum molecular weight of
a fimbrial subunit as calculated from amino acid data was determined
to be approximately 25 kd for T14V (type 1) fimbriae (106) or 30 kd
for A. viscosus WVU627 fimbriae (77). However, a method for
completely dissociating Actinomyces fimbriae into subunits has not
been discovered (12).

5
Over the last decade, the fimbriae of A. viscosus and
A. naeslundii have been the subject of intensive study. A great deal
of progress has been made in identifying the adherence-related
functions of the fimbriae and defining the molecular basis of the
interactions of certain Actinomyces cells with other microorganisms
and surfaces within the oral cavity. Nearly all of the studies have
focused on only two strains, A. viscosus serotype 2 strain T14V and
A. naeslundii serotype 1 strain WVU45, and spontaneously occurring
mutants derived from these two strains. Two populations of
Actinomyces fimbriae, which differ both in antigenicity and biological
activity, have been identified (20,84). Type 1 fimbriae mediate
adherence to saliva-coated hydroxyapatite in vitro and to the salivary
pellicle on teeth in vivo (23). Type 2 fimbriae via their
lactose-inhibitable lectin activity mediate adherence to other oral
bacteria and mammalian cells (11,12,15,44,68,80,93,103). The
functional and molecular properties of the fimbriae from these two
strains have been recently reviewed (84).
Both types of fimbriae are present on T14V and other serotype 2
isolates of A. viscosus, whereas WVU45 and other A. naeslundii
serotype 1 isolates possess only type 2 fimbriae (20). In 1974 Jordan
et al. (57) described a new serotype of A. naeslundii based on their
studies of strain N16 and 20 similar isolates. These strains had been
isolated from cervical plaque in a sampling of 59 Down's syndrome
patients who had moderate to severe periodontal disease (64). In
studies with gnotobiotic rats and hamsters, Jordan and co-workers have
shown that N16 can cause heavy plaque deposits and severe periodontal
pathology, including alveolar bone loss, root surface caries,and even

6
enamel caries in some animals (57,60). Strain N16 and isolates that
are biochemically and serologically identical to it have been
designated A. naeslundii serotype 3 (46).
There is both functional and immunological evidence to suggest
that A. naeslundii serotype 3 strain N16 has both types of fimbriae.
The N16 isolate exhibits lactose-reversible coaggregation with
Streptococcus sanguis 34 (18). It produces its own neuraminidase and
agglutinates human erythrocytes in a reaction that is reversed by
lactose (29). It possesses numerous surface fibrils or fimbriae and
adsorbs to saliva-treated hydroxyapatite (SHA) in vitro as avidly as
T14V (26). In fact, Clark et al. (25) demonstrated that on average
A. naeslundii serotype 3 adsorbed better to SHA than any other species
or serotype of Actinomyces tested. Strain N16 agglutinated with MAbs
against T14V type 2 fimbriae (14). It reacted with rabbit IgG against
T14V type 1 fimbriae in a dot enzyme immunoassay (28), and its
adsorption to SHA was partially inhibited by the antibody (23,28).
Although most of our knowledge of Actinomyces fimbriae has been
based on studies of A. viscosus serotype 2 T14V and A. naeslundii
serotype 1 WVU45, A. naeslundii serotype 3 was the Actinomyces species
most frequently isolated from subgingival dental plaque from sites
with moderate, severe, or juvenile periodontitis (87), and in another
study A. naeslundii serotype 3 was considered to be one of the most
likely etiological agents of gingivitis (86). Thus, A. naeslundii
serotype 3 strain N16 appears to be a logical choice for a study of
surface antigens of a periodontopathic actinomycete.

7
The goals of this study were to (a) produce monoclonal antibodies
against surface antigens of A. naeslundii serotype 3 strain N16, (b)
select at least one MAb that exhibited serotype specificity, and (c)
use the MAb as a tool to isolate and characterize the surface
component carrying the serotype-specific epitope.

MATERIALS AND METHODS
Antigen Preparation
Cultures
A. naeslundii N16 (WVU820), WVU1267, WVU1468, WVU1527, and
WVU1528 were obtained from M. A. Gerencser, West Virginia University.
W1629, W2273, and W2821 were obtained from the Centers for Disease
Control, Atlanta, GA. UF92 and UF524 were fresh clinical isolates
provided by J. E. Beem, University of Florida, Gainesville, FL. The
identity of each of these ten isolates was confirmed by biochemical
tests and by direct staining with serotype-specific fluorescent
antibody reagents; all ten isolates were designated A. naeslundii
serotype 3. Approximately 50 additional isolates, including
representatives of all the recognized serotypes of A. naeslundii,
A. viscosus, A. israelii, A. odontolyticus, Arachnia propionica,
Rothia dentocariosa, and Bacterionema matruchotii were obtained from
culture collections at the Centers for Disease Control for use as
heterologous organisms in evaluating the specificity of monoclonal
antibodies. Other strains used in this study were kindly provided by
P. E. Kolenbrander, National Institute of Dental Research, Bethesda,
MD, and by W. B. Clark and P. J. Crowley, University of Florida.
Stock cultures of all isolates used in this study were maintained by
. . . . o
lyophilization in skim milk and by storage at -20 C in Trypticase
soy broth (TSB; BBL Microbiology Systems, Cockeysville, MD) containing
20% (v/v) glycerol.
8

9
Culture Conditions
Cells for all procedures were obtained by culturing the isolates
in TSB supplemented with 2.5 g of K^HPO^ per liter in flasks that
could be chemically sealed to provide the atmospheric environment
appropriate for each isolate. After inoculation of a TSB-containing
flask with an actively growing TSB culture (2% v/v), the flask was
plugged with a rubber stopper through which an open-ended screw-capped
tube filled with cotton had been inserted (7). Prior to tightening of
the screwcap, the cotton was saturated with equal volumes of 10%
Na^CO^ and 1 M KH2P0^ to generate an aerobic 4- C02
environment or with 10% Na^O^ and pyrogallol solution to generate
an anaerobic + C02 environment (45). The cultures were incubated
without shaking at 37°C. Cells were harvested from cultures in mid
exponential to early stationary phase by centrifugation (10,000 x g,
10 min), washed twice, and stored in buffer at 4°C. The buffers
used for washing and making cell suspensions varied depending on the
procedure for which the cells were intended; they are noted in each
specific method.
Preparation of Crude Soluble Antigens
Several different methods were used to obtain crude soluble
antigens from whole cells.
Ammonium sulfate fractionation of French pressure cell
supernatants. N16 cells harvested from 8.5 1 of 36 h TSB cultures
were suspended in 0.05 M phosphate buffer, pH 7.2, to form a thick
paste; then the cell suspension was passed once through a French
pressure cell (Aminco Model J4-3337, American Instrument Co., Silver
Springs, MD) at 10,000 psi to remove the fimbriae (106). The

10
pressed cell suspension was centrifuged (48,300 x g, 20 min) to pellet
intact cells and cell walls; then the crude supernatant was
ultracentrifuged at 160,000 x g for 24 h to pellet the fimbriae. The
pellet was partially resuspended in 0.1 M Tris-HCl, pH 7.5, by
sonicating for 1 min at full power with a Kontes microultrasonic cell
disruptor (Kontes, Vineland, NJ). After centrifugation at 23,700 x g
for 10 min, the fimbriae-containing supernatant was processed by
ammonium sulfate fractionation to obtain precipitates at 10, 20, 30,
40, and 50% (w/v) (NH^)^SO^. The precipitates were collected by
centrifugation (30,900 x g, 15 min), dissolved in and dialyzed against
deionized water, and lyophilized. Lyophilized antigens were dissolved
in 0.5% Triton X-100 by adding 100 yl to 2 mg dry weight and
sonicating for 10 sec. The lyophilized material was highly resistant
to solubilization, so lyophilization was not used on subsequent
batches of fimbriae.
Continuous flow sonication. N16 cells (10% packed cells by
volume) in Tris-buffered saline (TBS; 0.025 M Tris-HCl, 0.15 M NaCl,
—4 —4
10 M CaCl2, 10 M MgCl2, and 0.02% NaN3, pH 7.6) were
passed at a flow rate of 5 ml/min three times through a continuous
flow sonifier cell disruptor (Heat Systems Ultrasonics, Inc.,
Farmingdale, NY) set at 100 W (14).
Virtis homogenization. A suspension of N16 cells in buffer was
agitated twice at 23,000 rpm for 5 min in a Virtis Model 45
homogenizer (The VirTis Company, Inc., Gardiner, NY) (77).
Extraction with 8 M lithium chloride. N16 cells were suspended
in 8 M LiCl, pH 7.0, (10% packed cells by volume) and stirred
o
continuously for 1 h at 25 C (82).

11
Lancefield extraction. N16 cells were suspended in 0.04 N HC1 in
saline (5 ml per g wet weight of cells); the suspension was heated for
15 min in a boiling water bath, cooled, and neutralized (90).
For the last four methods described above, the cells and
supernatants were separated by centrifugation (10,000 x g, 10 min),
and the supernatants were concentrated by ultrafiltration (PM10
membrane; Amicon Corp., Danvers, MA). The protein content of each
sample was determined by the Bio-Rad Protein Assay, a Coomassie blue
dye binding assay (3) (Bio-Rad Laboratories, Richmond, CA). Total
carbohydrate was estimated by the phenol-sulfuric acid procedure (33).
Preparation of Fimbriae for Purification
Sonication
Batch A. N16 cells were harvested from 12.5 1 of 36 h TSB
cultures, washed, and resuspended in TBS to a total volume of 240 ml
(10-207» v/v). Fimbriae were removed by sonicating 30 ml aliquots in
15 sec bursts at 100 W. After 0.5, 1, 2, 3, 4, 5, and 6 min of
sonication, cells and supernatants were separated by centrifugation
(10,000 x g, 10 min). The cells were resuspended in fresh buffer, and
a few drops of the suspension were set aside for subsequent
examination by hemagglutination (HA) and electron microscopy (EM)
before the next cycle of sonication was started. All the supernatants
were pooled and centrifuged (27,000 x g, 30 min) to remove
particulates. The crude 6 min sonicate was concentrated to 10 ml by
ultrafiltration (Amicon PM10 membrane) and by addition of dry Sephadex
G-25 prior to chromatography on Bio-Gel A-5m.
Batch B. N16 cells were harvested from 27.5 1 of 24-29 h TSB
cultures, washed, and resuspended in TBS to a total volume of 750 ml

12
(10-20% v/v) . Fimbriae were removed from 720 ml of cell suspension by
sonicating 30 ml aliquots in 30 sec bursts at 100 W. After sonication
for a total of 2 min, the cell suspensions were centrifuged at 10,000
x g for 10 min to separate intact cells from supernatants. The
sonicated cells were resuspended in fresh buffer for examination by
HA. The supernatant or crude 2 min sonicate was further clarified by
centrifugation (27,000 x g, 30 min), then concentrated by
ultrafiltration (Amicon PM10 membrane) to 180 ml. The Amicon
concentrated sonicate was mixed with an equal volume of saturated
. . o
(NH^)2^4 to give 50% saturation. After incubating at 4 C
overnight, the precipitate was collected by centrifugation (10,000 x g,
30 min), dissolved in water, and re-precipitated with (NH^J^SO^
at 35% final saturation. The precipitate from the 35% (NH^J^SO^
saturation was collected by centrifugation, resuspended in water, and
dialyzed against TBS. This was the starting material for gel
filtration chromatography.
French press shearing
N16 cells were harvested from 30 1 of 16-24 h TSB cultures,
washed once in TBS, and resuspended in TBS (5 ml of TBS per 1 of
culture harvested). The cell suspension was passed once through a
French pressure cell (Model J4-3337; American Instrument Company,
Silver Spring, MD) at 10,000 psi.
The amount of lysis caused by the French press was estimated to
be 1.2%, as determined by the following equation:
% lysis = (A260/A280 of untreated) - (A260/A280 of treated) X 100
A260/A280 of untreated

13
Intact cells were removed from the pressed cell suspension by
centrifugation at 10,000 x g for 10 min. The French press supernatant
was clarified further by centrifugation at 48,300 x g for 20 min; the
clear supernatant was stored at -80°C until processed.
Gel Filtration Chromatography
Columns were packed with Bio-Gel A-5m (exclusion limit 5 x 106
daltons for globular proteins; Bio-Rad Laboratories). The void and
total volumes were determined with blue dextran 2000 and potassium
ferricyanide, respectively. Crude sonicates were applied to the
columns and eluted with TBS. A Marriotte flask was used to maintain a
constant operating pressure. For all chromatographic techniques,
effluents and eluates were monitored by A with a Uvicord II UV
analyzer and recorder, and fractions were collected with an Ultrorac
fraction collector (LKB Instruments Inc., Rockville, MD). The Batch A
sonicate was run on a 12 x 750 mm column packed to a bed volume of
77.5 ml; the flow rate was 8 ml/h, and 3 ml fractions were collected.
The Batch B sonicate, a 35% saturated (NH^) SO^ fraction, was
applied in several aliquots (20 ml each at 3.9 mg protein per ml) to a
25 x 1000 mm column packed to a bed volume of 410 ml; the flow rate
was 25 ml/h, and 5 ml fractions were collected. Column fractions were
assayed for (a) total protein by the Bio-Rad Protein Assay with bovine
albumin as the standard, (b) total carbohydrate by the phenol-sulfuric
acid method with glucose as the standard, and (c) fimbriae reactive
with MAb 3B5.A1 by an enzyme immunodot assay. Some fractions were
also examined by SDS-PAGE.

14
Treatments of Fimbriae by Physical/Chemical Means
Acetone precipitation. Five volumes of cold acetone were added
to the sample. After having been mixed thoroughly, the sample was
. o
incubated at -20 C for at least 10 min. Precipitates were collected
by centrifugation (10,000 x g, 5 min) and dried by evaporation under
vacuum. Precipitates were reconstituted in an appropriate buffer.
Magnesium chloride precipitation. Four parts of 1 M MgCl^ were
mixed with one part sample to obtain a final concentration of 0.1 M
MgCl^• The sample was incubated at 4°C for 24 h, then examined
for evidence of precipitation.
Freon extraction. Equal volumes of sample and Freon 113
(trichlorotrifluoroethane) were mixed thoroughly by vortexing. After
centrifugation (2000 x g, 30 min), the upper aqueous phase was
collected.
Heat. Samples were placed in a waterbath at 37°C for 60 min,
at 65°C for 30 min, or at 100°C for 5 min or 60 min.
Sonication. Samples in capped microfuge tubes were placed in an
ice slurry in a cup horn sonicator (Heat Systems-Ultrasonics, Inc.,
Plainview, NY) and sonicated continuously at full power for 1, 5, or
10 minutes.
Urea. An acetone precipitate of the sample was dissolved in a
volume of 8 M urea in 0.05 M Tris-HCl, pH 8.0, equal to the original
volume of sample; then it was incubated at 37°C for 1 h.
Guanidine hydrochloride. An acetone precipitate of the sample
was dissolved in saturated (8.6 M) guanidine HC1 at a final
concentration of 1 ml of guanidine HCl per mg total protein. After
. . o ,
incubation at 37 C for 1 h, the sample was diluted with and dialyzed
against 10 mM EDTA, 0.05 M Tris-HCl, 0.15 M NaCl, 0.1% NaN^, pH 7.5.

15
Acid. One aliquot was treated by Lancefield extraction; it was
o
adjusted to 0.04 N HC1 in saline and heated at 100 C for 15 min.
o
Two others were adjusted to 0.1 N HC1 and heated at 100 C for 5 min
o
or at 37 C for 1 h. All were neutralized by addition of NaOH after
treatment.
Base. The sample was adjusted to 0.1 N NaOH and incubated at
37°C for 1 h; then it was neutralized with HCl.
Periodate oxidation. The sample was adjusted to 0.1 M sodium
metaperiodate in 0.05 M acetate buffer, pH 4.5, and incubated in the
dark at 4°C for 24 h; then ethylene glycol was added to 0.3 M final
concentration to consume excess periodate.
Enzymatic digestions. Solutions of lysozyme (Cat. No. 36-324,
Miles Laboratories, Naperville, IL), mutanolysin (M-3765, Sigma
Chemical Co., St. Louis, MO), papain (Sigma P-4762), and Pronase (Cat.
No. 537088, Calbiochem, San Diego, CA) were prepared in 0.1 M sodium
phosphate, pH 6.2. Solutions of a-chymotrypsin, trypsin (Sigma
T-8253, Type III), Staphylococcus aureus V8 protease (Sigma P-8400,
Type XVII), and proteinase K (Sigma P-0390, Type XI) were prepared in
0.05 M Tris-HCl, pH 8.0. Acetone precipitates of crude N16 French
press supernatants were dissolved in 0.1 M phosphate-buffered saline,
pH 6.2, or 0.05 M Tris-buffered saline, pH 8.0, depending on the
enzyme to be used.
Antigen samples containing 25 yg of total protein were
incubated (37°C, 30 min) with serial dilutions of lysozyme ranging
from 0.1 yg to an upper limit of 7 yg (175 units) of lysozyme per
sample. Likewise, samples were treated with mutanolysin in amounts
ranging from 0.3 Units to 17.5 Units. To ensure that the lysozyme and

16
mutanolysin were active, a suspension of A. naeslundii PK19 cells was
treated with each enzyme, and muramidase activity was detected as a
decrease in turbidity (A ) of the cell suspension.
6 00
Samples that were to be treated with proteases were heated at
o
100 C for 2 min in the presence of 0.5% SDS prior to the addition of
enzyme solutions in order to make the proteins more susceptible to
digestion and to inactivate any endogenous proteases that might be
present in the crude antigen sample. Samples containing 500 vig of
total protein were mixed with 50 pg of enzyme (chymotrypsin,
trypsin, papain, Pronase, proteinase K, or V8 protease) for a 10:1
ratio of protein:enzyme, and the appropriate buffer was added to bring
the final enzyme concentration to 100 yg per ml. The
. . o
protease-treated samples were incubated in a 37 C waterbath for 1 h.
At the end of the incubation period each sample was prepared
immediately for SDS-PAGE and stored at -80°C until it could be
evaluated.
Aliquots of bovine serum albumin were treated exactly like the
N16 samples as a positive control for activity of the proteases and
for detection of protease contamination in the muramidases. Samples
that received buffer in place of enzyme served as untreated controls.
Samples containing enzyme only were also used as controls.
Treated samples were subjected to SDS-PAGE; the SDS-PAGE resolved
proteins were transferred to nitrocellulose. These blots were reacted
with either R10 rabbit IgG against N16 type 1 fimbriae or R2P rabbit
IgG against N16 type 2 fimbriae in an indirect EIA to visualize
immunoreactive fimbrial bands.

17
Antibody Production
Immunization of Mice
The cell suspensions for immunizations were formalin-killed whole
cells suspended in phosphate-buffered saline (PBS; 0.01 M phosphate,
0.85% NaCl, pH 7.4) containing 0.3% formalin. Cell aggregates were
dispersed by sonication, and the turbidity of each cell suspension was
adjusted to a No. 8 McFarland Standard. To obtain antigen-primed
spleen cells for fusions, 10-week old female BALB/c mice were
immunized with A. naeslundii N16 cells. Two mice that received 0.1 ml
intravenously on days 1, 8, and 16 were sacrificed on day 20 for
fusion of their spleen cells with X63Ag8.653 myeloma cells. Two
additional mice received 0.1 ml mixed with an equal volume of Freund's
complete adjuvant intraperitoneally on day 1 and 0.1 ml without
adjuvant intravenously on day 19 and were sacrificed on day 22 for a
fusion with P3X63Ag8 cells.
Monoclonal Antibodies
Hybridomas were produced, using the method of Simrell and Klein
8 7
(95), by fusing 10 spleen cells from N16-immunized mice with 10
myeloma cells in the presence of 50% polyethylene glycol-1000. For
one fusion myeloma cell line P3X63Ag8, which secretes immunoglobulin
molecules with gamma-1 heavy chains and kappa light chains (66), was
used. For another fusion, the Kearney myeloma cell line X63Ag8.653,
which does not express any immunoglobulin chains (63), was used.
Hybrid cells were selected by growth in Dulbecco's modified Eagle's
medium (DMEM) containing hypoxanthine, aminopterin, and thymidine (HAT
medium). Culture supernatants were screened for specific antibody by
solid-phase radioimmunoassay (RIA). Hybridomas secreting antibody to

18
N16 were cloned by limiting dilution by seeding a 96-well tissue
culture plate at a density of one-half cell per well; each well
4
contained 5 x 10 mouse peritoneal exudate cells as a feeder layer.
Three different clones were selected for further evaluation. They
were propagated in tissue culture and as ascites tumors in female
BALB/c mice primed by intraperitoneal injection of 0.5 ml pristane
(2,6,10,14-tetramethylpentadecane; Aldrich Chemical Company, Inc.,
Milwaukee, WI). Culture supernatants and sera or ascites fluids from
hybridoma-bearing mice were stored at -20°C. Hybridoma cells in
DMEM containing 30% fetal calf serum and 10% dimethylsulfoxide were
stored in liquid nitrogen.
Isotyping
The class and subclass of immunoglobulin secreted by each
hybridoma were determined initially by immunodiffusion in which
subclass-specific anti-heavy chain antisera (Meloy Laboratories, Inc.
Springfield, VA) were reacted with the hybridoma culture
supernatants. These results were confirmed subsequently by testing
hybridoma culture supernatants or ascites fluids with an enzyme
immunoassay isotyping kit (Mouse Typer, Bio-Rad Laboratories).
Polyclonal Antibodies
Polyclonal antisera monospecific for type 1 and type 2 fimbriae
of A. naeslundii N16 were produced in female New Zealand white rabbits
by immunization with immunoprecipitins cut from crossed
immunoelectrophoresis (XIEP) gels (71). Type 1 and type 2 fimbriae
from partially purified samples of N16 fimbriae were separated and
precipitated by XIEP versus rabbit antiserum R29 raised against N16
whole cells. Sections of gel containing the immunoprecipitate were

19
cut from several XIEP patterns. The appropriate segments (a minimum
total of 10 cm per immunogen) were pooled, washed exhaustively with
saline to remove excess soluble reactants, and solubilized in 1 ml of
6 M KI, One-fourth of the immunogen was mixed with an equal volume of
complete Freund's adjuvant and injected subcutaneously into multiple
sites. The remainder of the immunogen was emulsified with an equal
volume of incomplete Freund's adjuvant and administered in 3
subcutaneous injections at approximately weeks 3, 5, and 7. Three
rabbits received type 1 fimbrial immunoarcs, and three were immunized
with type 2 fimbrial immunoarcs. Antibody responses were monitored by
microtiter plate EIA, and additional injections of immunogen were
given as needed to boost or maintain antibody titers. The rabbits
were bled periodically from the central artery of the ear throughout
the schedule and were exsanguinated by cardiac puncture 3-9 months
after the first injection.
RIO against N16 type 1 fimbriae and R2P against N16 type 2
fimbriae were the two rabbit antisera used most often in this study.
J. 0. Cisar, National Institute for Dental Research, kindly provided
the following samples of monospecific rabbit IgG: R59 against
A. viscosus T14V type 1 fimbriae, R55 against T14V type 2 fimbriae,
and R70 against A. naeslundii WVU45 type 2 fimbriae. Rabbit antiserum
raised against immunoarcs of Histoplasma capsulatum was kindly
provided by P. Standard, Division of Mycotic Diseases, Centers for
Disease Control, for use as a negative control.
DEAE Chromatography
IgG was purified from rabbit antisera or hybridoma culture
supernatants by chromatography on DEAE Bio-Gel A (Bio-Rad

20
Laboratories). Antisera and supernatants were precipitated with
(NH4)2S04 at 50% saturation. The precipitates were dissolved
and equilibrated with 0.01 M phosphate buffer, pH 7.8. Samples were
applied to DEAE Bio-Gel A columns, and IgG was eluted with
equilibrating buffer. Purified IgG was concentrated by
ultrafiltration and stored at 4°C with 0.1% NaN^ as preservative.
Protein A-Sepharose Chromatography
Protein A-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ)
was packed in a column with flow adaptors. Hybridoma culture
supernatants or rabbit antisera were precipitated with ammonium
sulfate at 50% saturation. The precipitates were dissolved in water
and equilibrated against starting buffer. Samples were applied and
the unbound fraction eluted in 0.05 M Tris-HCl, 0.25 M NaCl, 0.1%
NaN^, pH 8. IgG was eluted with 7 M urea in 0.05 M Tris-HCl, pH 8.
Radiolabelins
125
Purified immunoglobulins in PBS and Na I (5 pCi per pg
protein) were placed in glass tubes coated with IODO-GEN
(1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril; Pierce Chemical
Co., Rockford, IL). The samples were incubated for 10 min at 25°C,
and then removed from the reaction vessel to terminate the iodination.
125 125
I-labeled Igs were separated from free Na I by gel filtration
on PD-10 columns (Pharmacia) equilibrated with PBS containing 1%
125
bovine serum albumin and 0.1% NaN^. All I-labeled samples
contained 90-100% TCA-precipitable counts per minute.
Immunoaffinity Chromatography
MAb 3B5.A1, purified from hybridoma culture supernatants by
protein A-Sepharose chromatography, was coupled to Affi-Gel 10

21
(Bio-Rad) and packed in a K16/20 column with flow adaptors
(Pharmacia); bed volume was 9.5 ml. Samples were applied by
continuously cycling them through the column in an ascending direction
at a flow rate of 10 ml/h. Unbound and bound fractions were eluted at
a flow rate of 40 ml/h in an ascending or descending direction,
respectively. The unbound fraction was eluted with 0.05 M Tris-HCl,
0.25 M NaCl, 0.1% NaN^, pH 8. The bound fraction was eluted with 7
M urea in 0.05 M Tris-HCl, pH 8.
Rabbit IgG, 250 mg in 0.1 M HEPES, pH 7.5, purified by DEAE and
protein A-Sepharose chromatography from antisera monospecific for N16
type 1 or type 2 fimbriae, was coupled to 50 ml of Affi-Gel 10 and
packed in a K26/40 column with flow adaptors. The coupling efficiency
for each gel was >90%. Initially samples were eluted as described for
the MAb immunoaffinity column; however, on the basis of results of a
dissociation experiment, 6 M NaSCN was substituted for 7 M urea in the
buffer for elution of the bound fimbriae.
Dissociation Experiment
For protein A-Sepharose chromatography and initial experiments
with immunoaffinity chromatography, 7 M urea was used as the
dissociating buffer for release of bound molecules from the ligand.
However, to experimentally determine the most effective dissociating
buffer to use with each immunoaffinity column, various dissociating
buffers were evaluated in a microtiter plate EIA for their efficacy in
releasing antibody bound to antigen.
The wells of a 96-well Immulon 2 plate (Dynatech Laboratories,
Inc., Alexandria, VA) were coated with a crude fimbriae-containing
sonicate of N16 cells. Aliquots of the antibody samples used to

22
prepare the three imntunoaf f inity columns, i.e., MAb 3B5.A1, rabbit IgG
anti-N16 type 1 fimbriae, and rabbit IgG anti-N16 type 2 fimbriae,
were the antibody samples tested in the dissociating experiment; the
optimal dilution of each antibody had been determined by indirect EIA
versus the fimbriae-coated plates. An indirect EIA was performed as
described below, except that prior to the addition of the enzyme
conjugate, each well received 200 ul of a dissociating or control
buffer for 1 h at 25°C. Buffers were evaluated in triplicate, and
the average A^gQ of the triplicate samples was used in the following
formula for measuring the efficacy of each buffer in dissociating the
antigen-antibody bonds:
% release of Ab 1 - (A490 with dissociating buffer)
from fimbriae = (A490 with control buffer) X 100
The control buffer was the buffer normally used to elute the unbound
fraction during imntunoaffinity chromatography, i.e., 0.05 M Tris-HCl,
0.25 M NaCl, 0.1% NaN3, pH 7.5 at 25°C.
In order to rule out the possibility that the dissociating
buffers themselves were removing the fimbriae from the plate or
adversely affecting the fimbriae in some other manner rather than
merely dissociating the antibody, an indirect EIA was performed on a
fimbriae-coated plate that was first incubated with dissociating
buffers at maximum concentration overnight at ambient temperature. If
the dissociating buffers were releasing fimbriae from the plates or
irreversibly denaturing fimbrial epitopes, the A^gQ of wells that
were incubated with dissociating buffers would be lower than that of
controls.

23
Assays
Electron Microscopy
For electron microscopy (EM) of whole cells, the specimens were
prepared by the pseudoreplica technique as described by Martin et al.
(75), using 0.5% uranyl acetate for negative staining. For
immunoelectron microscopy (IEM), N16 cells were incubated with a 1:100
dilution of rabbit antiserum overnight at 4°C, washed with pH 7.2
PBS, and incubated (2 h, 25°C) with a 1:10 dilution of
gold-conjugated goat anti-rabbit IgG (Auroprobe EM GAR G10, Janssen
Life Sciences Products, Piscataway, NJ). Then the gold-labeled cells
were prepared for EM and negatively stained as described above.
For thin section IEM colloidal gold (diameter, approximately 15
nm) was prepared by citrate reduction of chlorauric acid (43) and
coupled to goat anti-mouse IgG by the method of Horisberger and Rosset
(54).
N16 cells were grown in brain heart infusion broth (48 h,
37°C), then harvested and washed with pH 7.2 PBS by vacuum
filtration on Nucleopore filters (45 p pore size). The cells were
reacted with monoclonal antibody by floating the filters on hybridoma
culture supernatant (immune sample) or on myeloma cell line P3X63Ag8
culture supernatant (negative control) for 1 h at 25°C. The filters
were washed extensively with PBS, and then floated on gold-conjugated
goat anti-mouse IgG for 1 h at 25°C. Filters were washed in PBS,
fixed for 1 h in 2.5% glutaraldehyde in PBS with 0.5% tannic acid,
post-fixed in 1% OsO^, dehydrated, embedded in Spurr's resin, and
sectioned. Thin sections were stained with uranyl acetate and lead
citrate and examined on a JEOL 100-CX electron microscope.

24
Hemagglutination
The assay described below, as performed with unstabilized
erythrocytes (RBC), is basically that of Costello et al. (29).
Preparation of neuraminidase-treated RBC. Human type 0 blood was
drawn in anticoagulant and diluted with four volumes of pH 7.2 PBS.
RBC were collected by centrifugation at 750 x g for 10 min and washed
twice more in the same manner. A 15% (v/v) suspension of RBC in
neuraminidase (30 pg per ml; Cat. No. N2876 type V neuraminidase
from Clostridium perfringens, Sigma Chemical Co.) in pH 5.0 PBS was
incubated in a 37°C waterbath for 2 h. Neuraminidase-treated RBC
(NTRBC) were washed three times and suspended in TBS containing 0.4%
BSA. NTRBC had to be used within a day or two, unless they were
stabilized by treatment with formaldehyde or glutaraldehyde.
Formaldehyde stabilization of NTRBC. After neuraminidase
treatment an aliquot of NTRBC was washed in pH 7.2 PBS to remove the
enzyme and acid buffer. Then a 10% (v/v) suspension of NTRBC in PBS,
pH 7.2, was mixed with an equal volume of 3.7% formaldehyde. The
suspension was incubated at 25°C with occasional stirring for 4-6 h,
then at 37°C with continuous stirring for 14-18 h. The
formaldehyde-stabilized NTRBC were washed 4X with 10 volumes of TBS
and 3X with TBS containing 0.47» BSA. They were stored at 4°C in
TBS-BSA. These cells were evaluated versus freshly prepared and aged
preparations of NTRBC that had not been treated with formaldehyde to
determine whether formaldehyde stabilization prolonged the "life" of
the NTRBC.
Glutaraldehyde stabilization of NTRBC. A 1-27» (v/v) suspension
of RBC in cold 17» glutaraldehyde in pH 8.2 PBS was incubated in an ice

25
bath for 30 min with intermittent mixing (2). The glutaraldehyde-
treated RBC (G-RBC) were collected by centrifugation (750 x g, 10 min)
and washed once in saline. To block potentially reactive free
aldehyde groups, a 10% (v/v) suspension of G-RBC in 0.1 M glycine was
incubated at 25°C for 1 h. Then G-RBC were washed 5-10 times in
TBS. They were stored in TBS with 0.4% BSA at 4°C. Glutaraldehyde
stabilization could be used either before or after neuraminidase
treatment.
Test procedure. Starting with a bacterial cell suspension
equivalent to A = 2.0, serial two-fold dilutions in TBS with
6 50
0.4% BSA were made in a U-bottom microtiter plate, leaving 25 pi per
well. Then 25 ul of a 1% (v/v) suspension of NTRBC in TBS-BSA were
added to each well and mixed for 1 min. The reactions were read
immediately and after overnight incubation at ambient temperature. To
test for lactose-reversibility, 50 pi of 0.04 M lactose (0.02 M
final concentration) were added. Alternatively, the effects of
various inhibitors were tested by adding 50 pi of inhibitor to the
bacterial cells prior to the addition of NTRBC.
CoaRRregation
The protocol described below is based on the methods of Mclntire
et al. (82) and Cisar et al. (18).
Tube assay. Bacterial cell suspensions in TBS were adjusted to
A650 = 2.0-2.1 (1-cm cuvette, Beckman Model 25 spectrophotometer).
In a 10 x 75 mm test tube 0.2 ml of an actinomycete cell suspension
was mixed with an equal volume of a streptococcal cell suspension.
For controls, 0.2 ml of buffer was added in place of one of the cell
suspensions. The suspensions were mixed by vortexing, then incubated

26
overnight at ambient temperature. They were mixed again before the
reactions were graded on a scale of 0 to 4+ : 0 = no visible
aggregates; 1+ = small uniform aggregates in suspension; 2+ = definite
aggregates that did not settle immediately; 3+ = large aggregates that
settled rapidly, leaving some turbidity in the supernatant; 4+ = large
aggregates that settled immediately, leaving a clear supernatant.
Microtiter plate assay. A microtiter plate coaggregation assay
was developed to replace the standard tube assay. The procedure was
similar to that described for the tube assay, except that 25 yl of
each cell suspension were mixed in a U-bottom microtiter plate.
Bacterial Agglutination
In a U-bottom microtiter plate serial dilutions of the antibody
samples were made in TBS containing BSA (4 mg/ml). Normal sera and
diluent were used as negative controls. To 25 ^1 of antibody were
added 25 yl of a suspension of bacterial cells in TBS adjusted to
A^^0 = 1.0 (1 cm cuvette, Beckman Model 25 spectrophotometer), and
the plate was shaken for 1-2 min. Reactions were read after overnight
. . o
incubation at 25 C (14).
Radioimmunoassay
A solid-phase radioimmunoassay (RIA) was developed as a rapid,
sensitive screening assay for detection of monoclonal antibodies to
surface antigens of N16 and other isolates. Polyvinyl chloride
microtiter plates (U-bottom, Dynatech Laboratories, Inc.) were coated
with formalin-killed whole cells by placing 25 y1 of a No. 4
McFarland cell suspension in each well for 1 h at 25°C. Excess
antigen was rinsed out, and free sites were blocked by the addition of
a few drops of 10% agamma horse serum to each well. The antigen-

27
coated wells were incubated (1 h, 25°C) with 25 yl of undiluted
hybridoma culture supernatant. The wells were washed three times with
o
PBS containing 1% fetal calf serum and incubated (1 h, 25 C) with 25
125
yl of I-labeled rabbit anti-mouse IgG (heavy and light chain
specific) containing 30,000-50,000 counts per minute (cpm). After the
unbound radiolabeled second antibody had been washed away, individual
wells were cut out, and the bound radioactivity was measured in a
125
gamma counter. Reactions in which I-cpm were at least twice that
of the negative control were considered to be positive. Culture
supernatants from the myeloma lines used for fusion served as negative
controls. Murine antisera were used as positive controls if positive
culture supernatants or ascites fluids were not available.
Indirect Enzyme Immunoassay
Indirect enzyme immunoassays (EIA) were performed in Immulon 2
96-well flat-bottomed polystyrene microtiter plates (Dynatech
Laboratories, Inc.). Wells were coated with antigens by placing 50
yl of whole cells or soluble antigens optimally diluted in TBS in
each well and drying at 37°C. Antigen-coated plates were stored at
ambient temperature until used. Plates were washed once with blocking
buffer (PBS with 1% BSA), then 3 times with PBS with 0.05%
polyoxyethylene sorbitan monolaurate (Tween 20) (PBST). Antibodies
diluted in PBST were added to the wells (25 yl per well) and
incubated at 25°C for 1 h. Plates were washed 3 times with PBST.
Then 100 yl of horseradish peroxidase-labeled goat anti-rabbit or
mouse IgG optimally diluted in PBST were added and incubated at 25°C
for 1 h. After the excess conjugate had been removed by washing, 200
yl of substrate solution (0.1 mg of ortho-phenylenediamine and

28
2 yl of 3% ^2°2 Per citrate-phosphate buffer, pH 5.0) were
added and incubated at 25°C for 15-30 min. Reactions were stopped
with 25 yl/well of 4 M H SO , and the absorbance at 490 nm was
2 4
read on a Dynatech MR 600 Microplate Reader (Dynatech Laboratories,
Inc.). Absorbance readings >0.2 above the negative control were
considered positive.
Enzyme Immunodot Assay
Antigens were affixed to strips of nitrocellulose by applying
2-yl drops at 5-mm intervals and allowing them to dry at 25°C
(79). The antigen-coated strips were processed by enzyme immunoassay
essentially as described by Tsang et al. (99). The strips were washed
4 times for 5 min each time with PBS with 0.37» Tween 20 (PBSTW) with a
quick rinse in deionized water after each PBSTW wash. The
antigen-coated strips were immersed in antibodies diluted in PBSTW and
incubated with gentle agitation for 1 h at 25°C. Then the strips
were washed as before to remove unbound antibodies. The strips were
covered with the appropriate peroxidase-conjugated anti-Ig and
incubated for 1 h at 25°C. Unbound conjugate was removed by washing
as described above, followed by a final wash with PBS. The strips
were incubated with substrate (50 mg of 3,3'-diaminobenzidine and 10
yl of 30% H202 per 100 ml of PBS) for 10 min or until the spots
had developed the desired degree of intensity. The reaction was
stopped by rinsing the strips thoroughly with water.
Immunodiffusion
Immunodiffusion was performed in 17. agarose in PBS with wells
formed by use of a microimmunodiffusion template (89).

29
Laurell Rocket Immunoelectrophoresis
Laurell rocket immunoelectrophoresis (LRI) was performed as
described by Powell et al. (90) in 0.75% agarose in 0.043 M sodium
barbital buffer, pH 8.3, at a constant current of 8 mA per 50 mm x 75
mm slide. Gels were dried and stained with 0.57» Coomassie brilliant
blue R-250 in ethanol:glacial acetic acidrwater (4.5:1.0:4.5).
Crossed Immunoelectrophoresis with Autoradiography
Glass slides (50 mm x 75 mm) were coated with 7 ml of 0.757»
agarose in barbital buffer (0.0375 M barbital, 2 mM calcium lactate,
0.057» NaN^, pH 8.6). After the antigen wells were cut and filled,
the gels were electrophoresed at 8 mA constant current for 1-3 h for
separation of antigens in the first dimension. Then the gel above the
antigen wells was replaced with 3 ml of agarose containing an
unlabeled rabbit antiserum raised against whole cells and an
125
I-labeled Ig (50). After electrophoresis in the second dimension
overnight at 4 mA constant current, gels were washed, dried, and
exposed to Kodak XAR-2 film with a Dupont Cronex Lightning Plus
intensifying screen at -70°C. Gels were stained with Coomassie
brilliant blue R-250 and photographed.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Socium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed as described by Tsang et al. (102). Samples
were prepared to obtain final concentrations of 17» SDS, 0.9 M urea,
0.057» bromophenol blue, 87» glycerol, and 0.25 mg total protein per
ml. Some samples also received dithiothreitol (DTT) or
2-mercaptoethanol for reduction of disulfide bonds. Samples were
heated at 37°C for 30 min, at 65°C for 15 min, or at 100°C for

30
5 min. Gels consisting of a 5-20% linear gradient resolving gel and a
3% stacking gel 0.75 mm thick were cast using the Pharmacia GSC-2 177
mm x 200 mm vertical slab system (Pharmacia). The gels were
electrophoresed in the Pharmacia GE 2/4 LS electrophoresis chamber
using a discontinuous buffer system with 0.424 M Tris-HCl, pH 9.18, in
the lower reservoir and 0.04 M boric acid, 0.04 M Tris, 0.1% SDS in
the upper reservoir. Generally 0.25 pg of protein per mm width of
sample lane was applied. Either high molecular weight markers
(Bio-Rad) and low molecular weight markers (Pharmacia) or prestained
protein molecular weight standards (Bethesda Research Laboratories,
Gaithersburg, MD) were included in each gel (101). After
electrophoresis protein bands were visualized by the silver stain as
described by Tsang et al. (100), which is a combination of the methods
of Merril et al. (85) and Morrissey (88).
Immunoblot
SDS-PAGE resolved proteins were transferred electrophoretically
to nitrocellulose (0.2 p, Schleicher and Schuell, Keene, NH); then
antigens were visualized by an indirect enzyme immunoassay, using
peroxidase conjugates (Bio-Rad Western blotting grade) and
diaminobenzidine/ ^02 as the substrate. These methods have been
described in detail by Tsang et al. (99,102). For detecting unstained
molecular weight markers or other proteins on blots, nitrocellulose
strips were stained with India ink (52) or Aurodye forte (Janssen Life
Sciences Products, Piscataway, NJ).

RESULTS AND DISCUSSION
Monoclonal Antibodies
Three different hybridomas secreting antibodies to A. naeslundii
serotype 3 strain N16 were produced. Hybridomas 3B5.A1 and 2A3.B3,
both obtained from the fusion with the non-Ig-producing Kearney
myeloma cell line, secreted IgG and IgG monoclonal antibodies,
2a 3
respectively. The third hybridoma, 2B5.B6, which was a product of the
fusion with an IgG^(k)-secreting myeloma cell line, secreted IgM
monoclonal antibodies along with the Ig chains of the myeloma parent.
Preliminary indications of differences in specificity and the
range of reactivity of each of the three monoclonal antibodies were
obtained by radioimmunoassay, using whole cells as antigens. When
tested against a panel of ten isolates of A. naeslundii serotype 3,
monoclonal antibodies 3B5.A1 and 2A3.B3 reacted with all 10 isolates,
whereas monoclonal antibody 2B5.B6 reacted with only A of the 10
isolates (Table 1). On the basis of these results, monoclonal
antibodies 3B5.A1 and 2A3.B3 appeared to have potential as serotyping
reagents, provided that they did not also react with heterologous
serotypes; monoclonal antibody 2B5.B6 had too narrow a range of
reactivity to be useful for serotyping, unless it could be used as an
epidemiological tool for fine typing below the serotype level. To
determine whether or not the monoclonal antibodies would cross-react
with other organisms, the panel of cells for the RIA was expanded to
include 51 heterologous isolates: 17 isolates of A. naeslundii that
31

32
TABLE 1. Summary of RIA results
for isolates within the N16 serogroup
Isolate
Monoclonal antibody
3B5.A1 2A3.B3 2B5.B6
N16
+a
+
+
WVU1267
+
+
-
WVU1468
+
+
-
WVU1527
+
+
+
WVU1528
+
+
+
W1629
+
+
-
W2273
+
+
-
W2821
+
+
+
UF92
+
+
-
UF524
+
+
—
a + = ^â– 25I_Cpm > 125j_Cpm 0f negative control x 2;
- = l^I-cpm < 125j_Cpm 0f negative control x 2 for the average
1^^I-cpm of samples run in duplicate. 12^I-cpm of negative
controls ranged from 53 to 78, whereas •L2^I-cpm of + reactions
ranged from 1919 to 6283.

33
did not belong to the N16 serogroup (serotype 3), 13 isolates of
A. viscosus, 8 isolates of A. israelii, and 13 isolates of less
closely related species. When assayed using polyclonal antisera,
A. naeslundii and A. viscosus are closely related antigenically, and
antisera raised to whole cells of N16 could be expected to cross-react
with A. viscosus serotype 2 in particular. However, monoclonal
antibody 3B5.A1 did not react with any heterologous species or
serotypes (Table 2). On the other hand, monoclonal antibody 2A3.B3
cross-reacted with 7 of 17 non-serotype 3 A. naeslundii isolates, as
well as 7 of 11 A. viscosus serotype 2 isolates. However, the
125
I-cpm for all but one of these cross-reactions were less than 34%
125
of the I-cpm of the least reactive A. naeslundii serotype 3
isolate. Monoclonal antibody 2B5.B6 cross-reacted with 4 of 17
isolates representing other serotypes of A. naeslundii, and these
cross-reactions were as strong as the homologous reactions. None of
the three monoclonal antibodies reacted with isolates of A. israelii.
A. odontolyticus, A. pyogenes, A. propionica, R. dentocariosa,
P. avidum, or Bacteroides gingivalis. These results suggested that
monoclonal antibody 3B5.A1 recognizes a serotype-specific antigenic
determinant but that monoclonal antibodies 2A3.B3 and 2B5.B6 see
epitopes common to other species or serotypes.
The location of the epitopes to which the monoclonal antibodies
were binding was presumed to be the cell surface because whole cells
were used as immunogens and as antigens in immunoassays to measure
monoclonal antibody activity. The most immunogenic and biologically
significant surface components of A. naeslundii and A. viscosus
isolates are their fimbriae. Electron microscopy of A. naeslundii N16

34
TABLE 2. Summary of the specificities
of three monoclonal antibodies to N16 as determined by RIA
Species/serotype
No. isolates
tested
Monoclonal antibody
3B5.A1 2A3.B3 2B5.B6
Actinomyces naeslundii/3
10
10a
10
4
A. naeslundii/1, 2, 4
17
0
7
4
A. viscosus/1
2
0
0
0
A. viscosus/2
11
0
7
0
A. israelii/1
6
0
0
0
A. israelii/2
2
0
0
0
A. odontolyticus
3
0
0
0
A. pyogenes
1
0
0
0
Arachnia propiónica
3
0
0
0
Rothia dentocariosa
2
0
0
0
Propionibacterium avidum
3
0
0
0
Bacteroides Rinfcivalis
1
0
0
0
a Number of isolates that gave positive reactions, as defined in
Table 1. The range of ^^I-cpm for positive reactions was as
follows: 2547-6283 for 3B5.A1 against homologous isolates; 1919-6025
for 2A3.B3 against homologous isolates; 2097-2628 for 2B5.B6 against
homologous and heterologous isolates. Except for 3207 125I-cpm
against A. naeslundii W1250, 2A3.B3 gave 161-649 l^I-cpm against
heterologous serotypes of A. naeslundii and 180-328 12^I-cpm against
A. viscosus serotype 2 isolates.

35
cells revealed that they possess abundant fimbriae (Figure 1). The
location and density of the epitopes recognized by each monoclonal
antibody, as determined by EM of N16 cells immunolabeled with colloidal
gold, are shown in Figure 2. All three monoclonal antibodies bound to
epitopes residing on the fimbriae, but based on the far greater number
of gold particles seen on cells immunolabeled with MAb 3B5.A1, the
3B5.A1 epitope appeared to be more abundant than those recognized by
MAbs 2A3.B3 and 2B5.B6.
All three of the MAbs against N16 fimbriae were used on occasion
in later experiments. However, most of this study focused on the use
of MAb 3B5.A1 because preliminary evaluations indicated that (a) it
reacted with all 10 A. naeslundii serotype 3 strains by RIA, (b) it
appeared to recognize a unique, serotype-specific epitope, and (c) on
the basis of the density of immunogold labeling, it appeared to
recognize an epitope that was more abundant than those of the other
two MAbs.
Evidence for the Presence of Both Types of Fimbriae
on A. naeslundii Serotype 3 Strains
Evidence for the Presence of Type 1 Fimbriae
Although this study did not include any functional assays to
demonstrate the presence of type 1 fimbriae on N16 or other
A. naeslundii serotype 3 isolates, Clark et al. (23,25,26,28) have
presented evidence that 3 of the 10 strains listed in Table 1 possess
type 1 fimbriae. In one study they showed by EM that N16 and UF92
cells had fimbriae; both strains adsorbed very well to saliva-coated
hydroxyapatite (SHA) relative to the adsorption of T14V, 100% for N16
and 84% for UF92; and their adherence to SHA could be blocked by
purified T14V fimbriae (26). In another study N16 and G1468 (WVU1468)

36
Figure 1. Electron micrograph of A. naeslundii N16 cells negatively
stained with 0.5% uranyl acetate. Note abundant fimbriae
on the cell surface. Bar, 1 pm.

Figure 2. Electron micrographs of thin sections of immunogold labeled
A. naeslundii N16 cells demonstrating the binding of the
monoclonal antibodies to fimbrial epitopes. The cells were
incubated with MAb-containing culture supernatants of
myeloma or hybridoma cell lines and then with goat anti¬
mouse IgG conjugated to colloidal gold (diameter, 15 nm).
Bar, 0.25 ym.
A) P3X63Ag8, negative control; B) MAb 2B5.B6; arrows
indicate gold particles; C) MAb 2A3.B3; D) MAb 3B5.A1;
E) MAb 3B5.A1; F) MAb 3B5.A1

38

Figure 2.
—continued

40

Figure 2.
—continued

42

43
adsorbed to SHA better than T14V, despite the fact that each had a
lower hydrophobic index than T14V (25). N16 and WVU1468 reacted in an
immunodot EIA with rabbit IgG specific for the T14V type 1 fimbriae
(28), and rabbit IgG anti-T14V type 1 fimbriae was able to partially
inhibit adsorption of N16 cells to SHA and give 917» inhibition for
WVU1468 (23,28). The ability of N16, WVU1468, and UF92 to adsorb well
to SHA and the fact that T14V fimbriae or antibodies to T14V type 1
fimbriae can inhibit the adsorption suggests that these A. naeslundii
serotype 3 strains possess type 1 fimbriae. Immunological assays
performed in this study (data to be presented later) showed that all
of the A. naeslundii serotype 3 strains listed in Table 1 have type 1
fimbriae.
Evidence for the Presence of Type 2 Fimbriae
Hemagglutination. Many human strains of A. viscosus and
A. naeslundii can agglutinate human types A, B, 0, and AB erythrocytes
(29,37). With untreated RBC, hemagglutination occurs immediately at
4°C, but the reaction takes longer at room temperature or 37°C.
With neuraminidase-treated RBC, hemagglutination occurs immediately at
all three temperatures (29). Hemagglutination by A. viscosus and
A. naeslundii cells can be completely reversed by lactose. The
mechanism proposed for this reaction involves two steps: 1) the
unmasking of G-galactoside-containing receptors on the RBC when
terminal sialic acid residues are removed by neuraminidase released
from Actinomyces cells, and 2) the multivalent binding of these
receptors by multiple low affinity, lactose-reversible lectin sites on
the surface of the actinomycete (29,37). Since the lactose-reversible
lectin activity is a function of type 2 fimbriae (20), HA can be used

44
to determine whether or not Actinomyces cells have type 2 fimbriae.
Costello et al. (29) reported that N16 and WVU820 (N16) produced
neuraminidase and agglutinated human RBCs as described above. On the
other hand, Ellen et al. (37) reported that strain 820, which is
presumably WVU820 (N16), did not agglutinate non-sialidase-treated
horse, sheep, guinea pig, or human RBCs. Since Ellen et al. measured
HA after only a 5 min incubation, the inability of WVU820 to
agglutinate RBCs was probably a result of not allowing sufficient time
for the WVU820 neuraminidase to remove the terminal sugar blocking the
receptor.
In this study the 10 A. naeslundii serotype 3 strains listed in
Table 1 were tested for HA activity. Preliminary experiments were
performed with unfixed NTRBC, but unfixed NTRBC were found to be so
unstable that they generally had to be used within 24 h of
preparation. Even then they often gave weak HA reactions or lysed
during assays. In an effort to improve their shelf-life, NTRBC were
fixed with formaldehyde or glutaraldehyde and compared to unfixed
NTRBC. HA reactions with fixed NTRBC were stronger than with unfixed,
and glutaraldehyde-fixed RBC were superior to formaldehyde-fixed
cells. Not only did glutaraldehyde-fixed NTRBC agglutinate better
than unfixed cells, but they also had a shelf-life at 4°C that could
be measured in years instead of hours. The characteristics of the
agglutination reactions between N16 cells and human RBC (in terms of
the effects of temperature, incubation time, etc. as reported by
Costello et al.) were the same for glutaraldehyde-fixed RBC or NTRBC
as for unfixed RBC or NTRBC, except that HA with fixed cells was
stronger and, therefore, more difficult to reverse. All 10

45
A. naeslundii serotype 3 isolates agglutinated G-NTRBC, and all of the
HA reactions were reversed by lactose. Twelve sugars and EDTA were
tested for their ability to inhibit HA by N16 cells. Glucose,
fructose, mannose, sucrose, xylose and a-methyl galactoside
exhibited no inhibition of HA at concentrations of inhibitor < 25 mM.
Of the substances that did inhibit at concentrations < 25 mM, the
relative potency of the inhibitors was as follows: EDTA > lactose >
fi-methyl galactoside > talóse > fucose > galactose > N-acetyl
galactosamine. These results on the relative potency of inhibitors of
the N16 HA reaction are similar to the results of Ellen et al. (37)
for the inhibition of HA by A. naeslundii serotype 2 W752 cells and
also the results of Mclntire et al. (81,82) for inhibition of
coaggregation between T14V and Streptococcus sanguis strain 34 cells.
N16 cells also agglutinated sheep and guinea pig RBC, and HA was
completely inhibited with EDTA or lactose at a final concentration of
25 mM.
Coaggregation. Many human strains of A. viscosus and
A. naeslundii can agglutinate certain strains of oral streptococci
(18.19.36.69.81.82). These coaggregation reactions involve different
mechanisms for interaction, one of which is the binding of a
carbohydrate moiety on S. sanguis 34 cells by a lectin on Actinomyces
cells in a reaction that can be completely reversed by lactose
(18.69.81.82). Coaggregation, like hemagglutination, is a function of
type 2 fimbriae (20,92), so it also can be used to determine whether
or not Actinomyces cells have type 2 fimbriae.
Cisar et al. (18) showed that WVU820 (N16) and W1527 (WVU1527)
exhibited lactose-reversible coaggregation with S. sanguis 34 cells.

46
Their observations were confirmed in this study, using both the tube
and microtiter plate coaggregation assays (data not shown).
In summary, there was ample evidence from our preliminary studies
to suggest that N16 and other A. naeslundii serotype 3 strains had
both type 1 and type 2 fimbriae. The next step was to find a good way
to remove the fimbriae from the cells and obtain crude soluble
fimbrial extracts suitable for purification of fimbriae and for use as
antigens in immunological assays.
Antigen Preparation: Results of Preliminary Experiments
In the early stages of this research several different methods
for preparing soluble fimbrial extracts of N16 cells were tried.
First, the method of Wheeler and Clark (106) for purifying VA-1
fibrils (type 1 fimbriae) from A. viscosus T14V cells by ammonium
sulfate fractionation of French pressure cell supernatants was
attempted. Laurell rocket immunoelectrophoresis of the N16 fractions
prepared in this manner, i.e. 10, 20, 30, 40, and 50% (w/v) ammonium
sulfate saturated samples, revealed multiple antigens in each fraction
when antiserum to N16 whole cells was employed. When these fractions
125
were used to coat wells in the RIA, MAb 3B5.A1 gave I-cpm with
the 20% and 30% ammonium sulfate fractions that were 5-8 times higher
than those obtained with the 10%, 40%, or 50% fractions; thus the 20%
and 30% fractions appeared to be enriched for the antigen that 3B5.A1
recognizes. Each fraction was examined by transmission electron
microscopy for the presence of fimbriae, but the 20% fraction was the
only one in which fimbriae were readily observed.
Although the French pressure cell was an effective means for
removing fimbriae from cells with minimal cell lysis, access to this

47
equipment was not readily available. So, several other methods for
extracting fimbrial antigens were investigated: Lancefield extraction,
sonication, Virtis homogenization, and extraction with 8 M lithium
chloride. N16 cells, before and after being treated by these various
procedures, were tested for lectin activity by hemagglutination.
Since type 2 fimbriae of Actinomyces exhibit lectin activity, the
efficacy of each treatment of the cells in removing type 2 fimbriae
could be compared in this manner; presumably, type 1 fimbriae would be
removed also. Sonication and Lancefield extraction reduced the lectin
activity of N16 cells from 2+ to 0, whereas Virtis homogenization and
extraction with 8 M lithium chloride had little, if any, effect on the
lectin activity. Electron microscopy confirmed that sonication and
Lancefield extraction were much more effective than Virtis
homogenization or lithium chloride in removing fimbriae from N16
cells. The observation that treatment of N16 cells with 8 M LiCl had
no detectable effect on the fimbriae is contrary to the observation of
Mclntire et al. (82) that A. viscosus T14V cells treated in such a
manner lost their fimbriae and, consequently, their ability to
coaggregate with S. sanguis 34.
The total protein and total carbohydrate extracted per gram wet
weight of cells by these procedures are compared in Table 3.
Sonication released greater than 15 times more protein than either
Virtis homogenization or lithium chloride extraction, and the protein
to carbohydrate ratio was greater than 1.0 in each case. For
Lancefield extraction the protein to carbohydrate ratio was only
0.14. Lancefield extraction was eliminated from consideration as a
method for extracting fimbriae for subsequent purification because it

48
released 7 times more carbohydrate than protein and because of the
likelihood of protein denaturation resulting from acid hydrolysis.
However, it was the preferred method for preparing soluble antigens
for immunoelectrophoresis because (a) it was the most convenient way
to extract antigens from a large number of strains, and (b) it
invariably gave strong, symmetrical type 1 and type 2 fimbrial rockets
in LRI and XIEP.
TABLE 3. Total protein and total carbohydrate
released from N16 cells by several methods for extracting fimbriae
Method of extraction
CHOa
Protein*3
Continuous flow sonication
5.0
11.2
Virtis homogenization
0.5
0.6
8 M LiCl
0.3
0.4
Lancefield extraction0
22.8
3.1
mg total carbohydrate released per g wet weight of cells.
b
mg total protein released per g wet weight of cells.
c A different method was used to determine the wet weight of
cells subjected to Lancefield extraction, so the quantity of CHO and
protein extracted by that method cannot be compared directly to the
quantities extracted by the other methods.
Although French pressure cell shearing and continuous flow
sonication were my first and second choices, respectively, for
obtaining crude extracts for purification of N16 fimbriae, lack of
access to the appropriate equipment at the proper time ultimately
dictated that the initial crude fimbrial extracts be obtained by a
batch method of sonication. Later in the study, a French pressure
cell extract was also processed.

49
Identification of N16 Type 1 and Type 2 Fimbriae in
Crude Antigen Extracts by XIEP-A
Immunoelectron microscopy had been used to demonstrate that the
3B5.A1 epitope was located on N16 fimbriae, but whether those fimbriae
were type 1 or type 2 fimbriae had not yet been established. Analysis
of N16 crude sonicates by XIEP against rabbit antiserum to whole cells
revealed the presence of two major antigens that were thought to
correspond to type 1 and type 2 fimbriae (Figure 3, upper panels).
Cisar et al. (14) had shown that in XIEP Ag 1 (type 1 fimbriae) of
T14V migrated closer to the anode than Ag 2 (type 2 fimbriae).
Whether the type 1 and type 2 fimbriae of all Actinomyces strains
maintained the same relative electrophoretic mobilities was not
known. In fact, since crude sonicates of N16 cells contained other
antigens besides fimbrial antigens, and the antiserum used to
precipitate the antigens was not specific for fimbriae, the two N16
rockets did not necessarily have to be fimbrial antigens.
In order to establish the identity of the two N16 rockets and to
determine which type of fimbriae MAb 3B5.A1 recognized,
125
fimbriae-specific I-Ab was incorporated into the Ab-containing
portion of the gel along with the non-radiolabeled polyspecific
125
antiserum. When I-Ab specific for T14V type 1 fimbriae was used,
autoradiography of the gel demonstrated that the N16 rocket closer to
the anode corresponded to type 1 fimbriae (Figure 3A). When the gel
125
contained I-MAb 3B5.A1, autoradiography revealed that MAb 3B5.A1
bound to the other rocket (Figure 3B). Since (a) MAb 3B5.A1 binds to
fimbriae, (b) it did not bind to type 1 fimbriae, and (c) only two
types of Actinomyces fimbriae have been identified, by a process of
elimination it was determined that the antigen that migrated more

50
Figure 3. Identification of A. naeslundii N16 type I and type 2
fimbriae by XIEP-A. N16 cell surface antigens extracted by
sonication were separated by electrophoresis in the first
dimension with the anode to the left. Then they were
precipitated by electrophoresis (at a right angle to the
first dimension) into gel containing R29 rabbit antiserum
against N16 whole cells and ^25i-labeled IgG specific for
fimbriae. Two rockets precipitated by R29 are seen in the
Coomassie-stained gels (upper panel); the corresponding
autoradiographs (lower panel) indicate that the rocket
closer to the anode represents type 1 fimbriae, whereas the
other rocket represents type 2 fimbriae. In this and all
subsequent figures of XIEP patterns, the anode is on the
left for electrophoresis in the first dimension and at the
top for electrophoresis in the second dimension. The
type 1 fimbrial rocket is always on the left and the type 2
fimbrial rocket on the right.
A) •1-25i-R59 specific for the type 1 fimbriae of
A. yiscosus T14V binding to the type 1 fimbriae of N16;
B) -*-25I-MAb 3B5.A1 binding to the type 2 fimbriae of N16.

51
slowly toward the anode was type 2 fimbriae and that MAb 3B5.A1 was
specific for type 2 fimbriae.
Immunological detection of type 2 fimbriae throughout various
purification procedures and purification of type 2 fimbriae by
immunoaffinity chromatography were made possible by the availability
of MAb 3B5.A1. Also, XIEP-A proved to be a useful technique for
monitoring antigen fractions for the presence of both types of
fimbriae, as well as non-fimbrial antigens.
Purification of N16 Type 2 Fimbriae From a Crude Sonicate
Batch A
Aliquots of the untreated control cells and the sonicated cells
collected at different time intervals were assayed for lectin activity
by HA to monitor the removal of type 2 fimbriae as measured by the
decline in HA activity. Cells sonicated for only 30 sec had the same
HA titer as the untreated control cells. With cells sonicated from 1
to 6 min total, the HA titers of the cells declined as the total
sonication time increased. Most of the decrease in lectin activity
had occurred after 2 min of sonication, but even after sonication for
a total of 6 min, the cells still retained weak HA-positive activity.
Electron microscopy on the cells sonicated for 6 min revealed that
nearly all the cells had lysed and the cell walls appeared to be
devoid of fimbriae.
When the crude 6 min sonicate was applied to the Bio-Gel A-5m
column and eluted with TBS, two major protein-rich peaks were eluted,
one just after the void volume and the other near the total volume
(Figure 4). When the column fractions were dotted on nitrocellulose
and reacted with MAb 3B5.A1 in an indirect EIA, fractions 11-24 gave

Figure 4. Elution profile for chromatography of A. naeslundii N16 crude
sonicate on Bio-Gel A-5m. The hatched bar represents fractions
strongly reactive with MAb 3B5.A1 as determined by immunodot EIA.

2.2
Ln
LO

54
3-4+ reactions, but the intensity of the immunodots decreased sharply
beyond fraction 24. Although fractions 18-24 also contained type 2
fimbriae, only fractions 11-17 (designated A-5m peak 1) were pooled,
because fractions beyond the first major protein peak contained
increasing levels of carbohydrate. The relatively high carbohydrate
content of all the fractions in this batch probably was a reflection
of the substantial amount of cell breakage that occurred when the
cells were sonicated. When A-5m peak 1 was examined by XIEP against
rabbit antiserum to N16 whole cells, both types of fimbriae and some
nonfimbrial antigens were detected. A-5m peak 1 from Batch A was not
purified further because another batch, less contaminated with
cytoplasmic components, was prepared.
Batch B
Two minutes of sonication caused a 16-fold decrease in the 2+ HA
endpoint relative to the untreated cell suspension, suggesting
significant removal of type 2 fimbriae from the cells. Electron
microscopy of negatively stained cells confirmed that sonication
removed some but not all fimbriae from the cells. When the crude
sonicate was precipitated with (NH^) SO^ at 50% saturation and
again at 35% saturation, the supernatant fractions after dialysis and
concentration were examined for the presence of fimbriae by XIEP. The
results indicated that some of both types of fimbriae were lost to the
supernatant fraction with each precipitation; however, no attempt was
made to quantitate the amounts of fimbriae or total protein not
precipitated.
The precipitate from 35% (MH^) SO^ saturation of the crude
2 min sonicate contained 390 mg total protein. This sample was

55
chromatographed in 5 separate aliquots on a Bio-Gel A-5m column; a
representative elution profile is shown in Figure 5. This elution
profile is similar to that obtained with Batch A (Figure 4), except
that the Batch B fractions contained much lower levels of total
carbohydrate. The first of two major protein-rich peaks that were
eluted from the column contained most of the MAb 3B5.A1 reactive
(type 2) fimbriae as determined by immunodot EIA on the column
fractions. All of the fractions representing the first peak (peak 1)
from each run, i.e., fractions 14-32 in Figure 5, were pooled; the
A-5m peak 1 pool contained 159 mg total protein.
A 5 mg aliquot of the peak 1 pool was chromatographed on the MAb
3B5.A1 immunoaffinity column. Analysis of the column fractions by
XIEP-A indicated that the unbound fraction resembled the starting
material, which suggested overloading of the column, whereas the bound
fraction appeared to contain only type 2 fimbriae.
The unbound fraction from the first immunoaffinity run was
rechromatographed on the MAb column. This resulted in a reduction in
type 2 fimbriae relative to type 1 in the unbound fraction and
recovery of type 2 fimbriae in the bound fraction with only minor
contamination with type 1 fimbriae (Figure 6). Thus, MAb 3B5.A1,
which is specific for the type 2 fimbriae of N16, is an effective tool
for the immunochromatographic purification of this structural entity.
The fimbrial immunoprecipitin arcs that were used to immunize
rabbits for the production of polyclonal antibodies specific for N16
type 1 or type 2 fimbriae were obtained from these fractions, as shown
in Figure 7 (refer also to Materials and Methods).

Figure 5. A representative elution profile for Bio-Gel A-5m chromatography of
aliquots of the fraction obtained by precipitating A. naeslundii
N16 crude sonicate with (NH^^SC^ at 357» saturation. The
hatched bar represents fractions strongly reactive with MAb 3B5.A1
as determined by immunodot EIA.

mg/ml

58
Figure 6. Results of immunoaffinity chromatography of A-5m peak 1 on
the MAb 3B5.Al-Affi-Gel 10 column as demonstrated by
XIEP-A. The antibody-containing portion of each gel
included R29 antiserum against N16 whole cells and
125I-labeled MAb 3B5.A1 specific for type 2 fimbriae.
The Coomassie-stained gels (upper panel) show all the
antigens precipitated by the polyspecific rabbit antiserum,
whereas their corresponding autoradiographs (lower panel)
identify the type 2 fimbriae.
A) A-5m peak 1, the starting material for immunoaffinity
chromatography, contained both types of fimbriae; B) The
unbound fraction also contained both types of fimbriae, but
there was a reduction in type 2 fimbriae relative to type 1;
C) The bound fraction contained mostly type 2 fimbriae with
a barely detectable quantity of type 1 fimbriae (arrow).

59
Figure 7. Source of immunogens for production of rabbit antisera
monospecific for N16 type 1 or type 2 fimbriae. Segments
of fimbrial immunoprecipitates were excised from the areas
indicated on the XIEP gels. R29 against N16 ceils was the
precipitating antiserum in both gels.
A) Type 1 fimbrial immunoarcs for production of RIO
antiserum;
B) Type 2 fimbrial immunoarcs for production of R2P
antiserum.

60
When RIO antiserum against N16 type 1 fimbrial immunoprecitins or
R2P against N16 type 2 fimbriae were examined by immunoelectron
microscopy against N16 cells, both antisera reacted with epitopes
located on the fimbriae (Figure 8; RIO reaction not shown). These
antisera were used to make polyclonal rabbit IgG immunoaffinity
columns for the purification of both types of fimbriae from the N16
French press supernatant.
Purification of N16 Fimbriae from the French Press Supernatant
In addition to the purification of N16 type 2 fimbriae from a
crude sonicate by sequential chromatography first on Bio-Gel A-5m and
then on the MAb immunoaffinity column, an attempt was made to purify
N16 type 1 fimbriae and type 2 fimbriae directly from a crude French
press supernatant by immunoaffinity chromatography on rabbit IgG
(RIgG) anti-N16 type 1 fimbriae and rabbit IgG anti-N16 type 2
fimbriae columns, respectively. When a 90 ml aliquot (252 mg protein)
was chromatographed on the polyclonal anti-N16 type 2 fimbriae
immunoaffinity column, the unbound fraction contained both types of
fimbriae, and the bound fraction, desorbed with 7 M urea buffer,
contained type 2 fimbriae with perhaps a trace of type 1 fimbriae, as
determined by immunodot EIA. Since the unbound fraction still
contained plenty of type 2 fimbriae, approximately half of it was
rechromatographed and eluted as before. The quantity of bound protein
desorbed on each run was so low that it was virtually undetectable by
A^ggj also, the recovery from the second run appeared to be lower
than the first, suggesting that the bound antigen was not being
desorbed completely with 7 M urea.

61
Figure 8. Electron micrograph of A. naeslundii N16 cells showing
indirect immunogold labeling of type 2 fimbriae. The cells
were incubated with R2P antiserum (rabbit anti-N16 type 2
fimbrial immunoarcs) and then with goat anti-rabbit IgG
conjugated to colloidal gold (diameter, 10 nm). The
gold-labeled cells were prepared for EM by the
pseudoreplica technique and were negatively stained with
0.5% uranyl acetate. Bar, 1 pm.

62
A dissociation experiment was undertaken to assess the efficacy
of various buffers in dissociating the antigen-antibody complexes
formed between N16 fimbriae and the anti-fimbrial antibodies used to
prepare the immunoaffinity columns; the results are summarized in
Table 4. Although 8 M urea buffer released 85% of Ag-bound MAb
3B5.A1, it released only 28.8% of rabbit IgG anti-N16 type 1 fimbriae
and 18.5% of rabbit IgG anti-N16 type 2 fimbriae. Thus, the 7 M urea
buffer normally used as the dissociating buffer for immunoaffinity
chromatography would be ineffective at desorbing bound fimbriae from
either of the polyclonal rabbit IgG anti-N16 fimbriae immunoaffinity
columns. The most effective dissociating buffer for the polyclonal
immunoaffinity columns was 6 M NaSCN, which gave 95.4% release of
rabbit IgG anti-N16 type 1 fimbriae and 91.0% release for rabbit IgG
anti-N16 type 2 fimbriae. There was no evidence that any of the
dissociating buffers irreversibly denatured fimbrial epitopes or
released fimbriae from the plate during these assays.
Based on the results of the dissociation experiment, the
desorbing buffer was changed to 6 M NaSCN, 0.05 M Tris-HCl, pH 7.5,
and the rabbit IgG anti-N16 type 2 fimbriae immunoaffinity column was
desorbed again to release the fimbriae that were not released by 7 M
urea. The bound fractions recovered from the ’type 2’ immunoaffinity
column were pooled, concentrated, and dialyzed versus TBS by
ultrafiltration (Amicon YM10). Crystalline (NH^^SO^ was added
to 100% saturation, and the precipitated sample was stored at 4°C
until further processing.
The unbound material recovered from the rabbit IgG anti-N16 type
2 fimbriae immunoaffinity column was chromatographed on the anti-N16

63
TABLE 4. Summary of the efficacy of various dissociation buffers in
disrupting binding between antibodies to N16 fimbriae and N16 fimbriae
coated on Immulon 2 microtiter plates
Antibody samples
Dissociation buffer RIgG a-1 RIgG a-2 MAb 3B5.A1
NaSCN, 6 M
NaSCN, 3 M
NaSCN, 1 M
GuHCl, 6 M
GuHCl, 3 M
GuHCl, 1 M
Urea, 8 M
Urea, A M
Urea, 2 M
Urea, 1 M
Glycine-HCl, pH 2.5
Glycine-HCl/10% C2H602 b, pH 2.5
NH4OH, pH 11.5
NH40H/10% C2H602, pH 11.5
95.4a
91.0
85.6
47.8
23.4
83.6
16.9
6.4
78.0
93.0
84.5
ND
46.5
30.2
82.2
17.1
8.6
76.6
28.8
18.5
85.0
5.3
3.9
73.9
3.1
2.1
33.2
12.1
0.0
7.7
81.7
64.9
86.7
72.3
54.0
85.6
16.8
7.4
82.8
26.8
10.8
73.6
Percent release of antibody from fimbriae-coated plates.
Ethylene glycol.

64
type 1 fimbriae immunoaffinity column. Since the column did not bind
all the type 1 fimbriae on the first pass, the unbound fraction was
rechromatographed for a total of three runs. The bound fractions were
eluted with 6 M NaSCN, pooled, concentrated, and dialyzed versus
saline by ultrafiltration. Examination of the unbound and bound
fractions by immunodot EIA revealed that rabbit Ig was leaching from
the column during desorption of the bound fimbriae with 6 M NaSCN.
Crystalline (NH^) SO4 was added to the unbound and bound
fractions, and they were stored as 75% and 100% saturated solutions at
4°C.
Fimbrial samples from the French press supernatant, as well as
samples obtained from the Batch A and Batch B sonicates, were examined
by SDS-PAGE and immunoblot. However, before one can understand the
results of those experiments, an explanation of the nature of
Actinomyces fimbriae and the patterns they exhibit on immunoblots is
essential.
Effects of Various Physical and Chemical Treatments
on N16 Fimbriae
The fimbriae of E. coli and other gram-negative bacteria are
polymers of smaller subunits, and under the appropriate conditions,
they can be dissociated into their constituent monomers. The
following experiments were undertaken to (a) determine whether or not
N16 fimbriae had a subunit architecture or exhibited other properties
similar to those reported for the fimbriae of E. coli, (b) provide
additional evidence that N16 type 1 fimbriae differed from type 2
fimbriae, (c) see whether certain procedures caused changes in the
molecular weight or antigenicity of fimbrial bands.

65
Aliquots of the N16 crude French press sample were subjected to a
variety of physical and chemical treatments. The first was addition
of MgCl^• Unlike E. coli fimbriae (83), N16 fimbriae did not
precipitate with a final concentration of 0.1 M MgCl^.
Since the N16 crude French press supernatant contained both types
of fimbriae, as well as non-fimbrial components, the effects of the
other treatments on each type of fimbriae were demonstrated by
SDS-PAGE immunoblot analysis, using antibodies monospecific for type 1
or type 2 fimbriae. For some of these experiments a convenient method
for exchanging buffers and concentrating samples was needed. Acetone
precipitation appeared to be the ideal choice, but first it had to be
established that acetone precipitation would give total recovery of
fimbriae without adversely affecting their immunological reactivity.
When acetone precipitates of the crude fimbrial sample were compared
to the original sample, they gave identical patterns on immunoblots.
Acetone precipitation was thus employed whenever necessary to
accomplish the objectives cited above.
The effects of some of the physical and chemical treatments on
N16 type 1 and type 2 fimbriae are shown in Figures 9 and 10,
respectively. Exposure to Freon appeared to have no effect on N16
fimbriae; therefore, if extensive lysis were to occur during
sonication or French press shearing of cells to remove fimbriae,
lipids could be extracted from the crude fimbrial sample with Freon
before applying it to gel filtration or immunoaffinity columns.
Treatment with mutanolysin or lysozyme (lysozyme not shown) did
not change the fimbrial patterns observed on immunoblots. This
suggested that the ladderlike series of bands >100 kd represented

Figure 9. Immunoblot analysis of the effects of various physical and
chemical treatments on N16 type 1 fimbriae. Aliquots of
the N16 antigen sample were treated as indicated, then
prepared for SDS-PAGE by heating at 100 C for 5 min without
reduction. Each lane was loaded with 1 pg of total
protein, based on the protein concentration prior to
treatment. Prestained molecular weight standards were
mixed with two of the samples; their positions on the blot
were marked with a ballpoint pen, and approximate molecular
weights are expressed in kilodaltons. The blot was
developed with RIO IgG (anti-N16 type 1 fimbriae) at 1 yg
per ml as the primary antibody.

Periodate-oxidized
Untreated
Lancefield-extracted
0.1 N HC1, 37 C, 1 h
0.1 N NaOH, 37 C, 1 h
8 M urea
Mutanolysin
Untreated
0.1 N HC1, 100 C, 5 min
0.1 N HC1, 100 C, 5 min
Untreated
Freon-extracted
Sonicated 10 min
Sonicated 5 min
Sonicated 1 min
100 C, 60 min
100 C, 5 min
65 C, 30 min
37 C, 60 min

Figure 10. Immunoblot analysis of the effects of various physical and
chemical treatments on N16 type 2 fimbriae. This
experiment was identical to that described in Figure 9,
except that the blot was developed with R2P IgG (anti-N16
type 2 fimbriae) as the primary antibody, and the lane
order of the samples is reversed.

I
I
I
I
l
I
I
t
MMi «»IMMb
37 C, 60 min
65 C, 30 min
100 C, 5 min
100 C, 60 min
Sonicated 1 min
Sonicated 5 min
Sonicated 10 min
Freon-extracted
Untreated
0.1 N HC1, 100 C, 5 min
0.1 N HC1, 100 C, 5 min
Untreated
Mutanolysin
8 M urea
0.1 N NaOH, 37 C, 1 h
0.1 N HC1, 37 C, 1 h
Lancefield-extracted
Untreated
Periodate-oxidized
O'
to

70
fimbriae of different lengths rather than a fimbrial protein attached
to different lengths of peptidoglycan.
Sonication generated some fragments that were not evident in the
untreated control. The longer the sample was sonicated, the more
fragments were generated and the greater the intensity of the
immunological reaction. However, the generation of different
molecular weight bands by sonication did not appear to be completely
random. The highest molecular weight material was broken down into
smaller fragments, but the breakdown products generally were the same
molecular weight as fragments already present in the untreated
control. The fimbriae were fairly resistant to breakage into
fragments smaller than 100 kd; after 10 min of continuous sonication,
most of the fimbrial bands were still greater than 100 kd, even though
the most immunodominant bands were in the 35-65 kd range.
N16 fimbriae were not affected by 8 M urea or saturated guanidine
hydrochloride, treatments that would dissociate some types of E. coli
fimbriae into subunit monomers (61,62).
Another method that has been reported to disaggregate E. coli
fimbriae, i.e. 0.1 N HCl at 100°C for 5 min (31), did not have a
similar effect on N16 fimbriae. Instead of causing dissociation into
monomers, hot acid caused loss of band resolution, probably as a
result of acid hydrolysis. This also occurred with Lancefield
extraction (0.04 N HCl at 100°C for 15 min). However, 0.1 N HCl at
37°C for 1 h had little, if any, effect on N16 fimbriae; and heating
o
at 100 C in the absence of a pH<2 did not cause loss of band
resolution. So, it was the combination of a very acidic pH and
o
100 C that was destructive to the N16 fimbriae.

71
Base was even more destructive than acid, as judged by the total
loss of band resolution and substantially reduced antigenicity when
N16 fimbriae were incubated with 0.1 N NaOH at 37°C for 1 h.
Sodium periodate oxidation also caused some loss of band
resolution but not to the extent seen with acid or base hydrolysis.
There was some loss of antigenicity with increasing temperature
and, as might be expected, heating at 100°C for 1 h was the most
destructive.
N16 type 1 fimbriae could be distinguished from type 2 fimbriae
by their different patterns on immunoblots, i.e. the different
molecular weight distribution of the unreduced immunodominant bands.
Disregarding their intrinsically different patterns, the two fimbrial
types generally behaved similarly in response to the various
treatments. For example, neither was affected by high concentrations
of urea or guanidine HC1, but both were most adversely affected by
incubation with 0.1 N NaOH. The decrease in antigenicity of N16
fimbriae when incubated with NaOH or heated at 100°C for 60 min is
consistent with the findings of Masuda et al. (77) for A. viscosus
WVU627 fimbriae. Although they reported that WVU627 fimbriae were
also labile when incubated with 0.1 N HC1 at 37°C for 60 min, a
similar effect was not observed with N16 fimbriae. However,
immunoblot analysis of SDS-PAGE-resolved proteins may not have been
the best way to examine the effects of the various physical and
chemical treatments because denaturation of the proteins by heating in
SDS-PAGE sample buffer may mask the true effects of the other
treatments. It might have been better to use an assay that could
quantitate the amount of immunoreactive fimbriae remaining after

72
treatment, perhaps by using LRI, XIEP, EIA, or radioimmuno-
precipitation. On the other hand, SDS-PAGE immunoblots were probably
the best way to see the effects of sonication.
A. naeslundii N16 fimbriae were not disaggregated by any of the
methods that would dissociate E. coli fimbriae, pili, or flagella
(31,61,62,70,83). These results are consistent with the observation
that A. viscosus T14V fimbriae cannot be completely dissociated either
(12,84).
Immunoblots were also used to assess the effects of various
proteases on the molecular weight distribution and immunological
reactivity of N16 type 1 and type 2 fimbrial bands. All the proteases
eliminated the immunoreactive bands >200 kd for both types of
fimbriae. Samples treated with papain, Pronase, or proteinase K
exhibited a total loss of immunoreactive type 1 and type 2 fimbrial
bands. However, since papain caused only limited digestion of the BSA
control as determined from the presence of multiple bands in the range
below 35 kd on a silver-stained SDS-PAGE gel, it is possible that
papain and perhaps the other enzymes destroyed the fimbrial epitopes
without completely digesting the fimbriae.
The effects of chymotrypsin, trypsin, and V8 protease on N16 type
1 and type 2 fimbriae are shown in Figure 11 and Figure 12,
respectively. Each of these enzymes caused limited digestion of both
types of fimbriae, but the resulting immunoblot patterns were
different for each type of fimbriae. Trypsin caused a total loss of
band resolution for type 1 fimbriae; except for two discrete bands at
<14 kd, trypsin-treated type 1 fimbriae gave an immunoreactive smear
from 40-200 kd on immunoblot. On the other hand, the immunoreactive

Figure 11. Immunoblot analysis of the effects of digestions with
different proteases on N16 type 1 fimbriae. The lanes
containing untreated or enzyme-treated N16 antigens were
loaded with 2 pg of N16 protein, based on the
concentration of the N16 sample prior to treatment,
whereas the lanes containing enzyme only (lanes 3, 5, and
8 from left to right) were loaded with quantities of
enzyme equivalent to those in the enzyme-treated samples.
All samples were unreduced, except for the one marked R.
Prestained molecular weight standards were mixed with the
untreated control in lane 1. The blot was developed with
RIO IgG (anti-N16 type 1 fimbriae) at 2.5 pg per ml as
the primary antibody.

Untreated
Trypsin-treated
Trypsin
Chymotrypsin-treated
Chymotrypsin
V8 protease-treated, R
V8 protease-treated
V8 protease

Figure 12. Immunoblot analysis of the effects of digestions with
different proteases on N16 type 2 fimbriae. This
experiment was identical to that described in Figure 11,
except that the blot was developed with R2P IgG (anti-N16
type 2 fimbriae) as the primary antibody, and the lane
order reads right to left.

11 m mm
III i"
li l III
4>* 00
hO
on
â– p'
co
ON
00
vO
200
V8 protease
V8 protease-treated
V8 protease-treated, R
Chymotrypsin
Chymo t ryp sin-treated
Trypsin
Trypsin-treated
Untreated
ON

77
type 2 fimbrial bands after trypsin treatment were mostly between the
14 and 43 kd markers, and for the most part, the bands were discrete
rather than unfocused. The effect of chymotrypsin on type 1 fimbriae
was very similar to that of trypsin, whereas for type 2 fimbriae, the
pattern obtained by digestion with chymotrypsin was quite different
from that obtained with trypsin. V8 protease-treated samples were
less immunoreactive than the trypsin- or chymotrypsin-treated samples,
even though V8 protease appeared to be less efficient than the other
two enzymes at digesting the higher molecular weight fragments.
Effects of Temperature and Reduction on N16 Fimbriae
On immunoblots the most immunoreactive N16 type 1 and type 2
fimbrial bands undergo a shift in apparent molecular weight in
response to increases in the temperature for SDS-PAGE sample
preparation, as demonstrated in Figure 13. In unreduced samples
heated at 37°C for 30 min there are a series of type 1
immunoreactive fimbrial bands at or slightly below the 43 kd molecular
. o
weight marker, whereas after heating at 100 C for 5 min, the
immunodominant bands are in the 57-65 kd range. Both sets of bands
are present in the sample heated at 65°C for 15 min, so the
immunoblot pattern at this intermediate temperature shows the
"transitional state” of proteins switching from their lower molecular
weights at 37°C to higher molecular weights at 100°C. Reduction
with 0.01 M DTT (or 1% 2-mercaptoethanol) further simplifies the
pattern of bands so that with samples reduced at 100°C the primary
type 1 fimbrial bands on immunoblots are as follows: the uppermost
band has an apparent molecular weight of about 65 kd; there is a broad
area of immunoreactivity in the 57-60 kd range, which represents at

Figure 13. Immunoblot analysis of the effects of temperature and
reduction on the type 1 and type 2 fimbriae of
A. naeslundii N16. Aliquots of the N16 antigen sample
were treated with SDS-PAGE sample buffer, with or without
0.1 M dithiothreitol (DTT), by heating at the temperature
and times indicated. Each lane of the SDS-PAGE gel was
loaded with 1 pg of protein. Approximate molecular
weights are expressed in kilodaltons. The left half of
the blot was developed with RIO IgG at 5 pg/ml, and the
right half was developed with R2P IgG at 5 pg/ml.

co
ON
00
37
c,
30
min,
-
DTT
37
c,
30
min,
+
DTT
65
c.
15
min,
-
DTT
65
c,
15
min,
+
DTT
100
c,
5
min,
-
DTT
100
c,
5
min,
+
DTT
100
c,
5
min,
+
DTT
100
c,
5
min,
-
DTT
65
c,
15
min,
+
DTT
65
c,
15
min,
-
DTT
37
c,
30
min,
+
DTT
37
c,
30
min,
-
DTT
£
r»*
H
3
(D
H
(D
ro
K3
o
o
-'sj
v£>

80
least two bands, including a component at 57 kd that reacts with
normal RIgG; then there is a weaker doublet at about 53-54 kd.
In unreduced samples heated at 37°C, the immunoblot pattern for
type 2 fimbrial bands shows a series of bands near the 43 kd marker,
o
whereas unreduced samples heated at 100 C show a very reactive
doublet at about 62 and 63 kd and several closely spaced bands at
o
about 39-40 kd. As with the type 1 pattern, the 65 C type 2 pattern
o o
showed the transition from the 37 C to the 100 C pattern. Type 2
fimbriae were also affected by reduction in that the prominent doublet
at 62-63 kd in the nonreduced sample is replaced by a single 63 kd
band, and a minor doublet is still evident at 39-40 kd. On type 2
blots there was a very weakly immunoreactive band at about 23 kd that
was present in reduced and nonreduced samples prepared at any
temperature. A similar band at 18 kd (not visible in Figure 13) was
sometimes visible on type 1 blots. These low molecular weight bands
might represent fimbrial subunits (E. coli fimbrial subunits are
approximately this size); however, they may just be products of
degradation.
The observation that A. naeslundii N16 fimbrial proteins are heat
modifiable is consistent with the report of Yeung et al. (108) that
A. viscosus T14V type 1 fimbriae have a heat modifiable subunit with
an apparent molecular weight of about 50 kd at 37°C or 65 kd at
100 C. Similarly, Yeung et al. reported that the fimbrial subunit of
A. naeslundii WVU45 type 2 fimbriae has an apparent molecular weight
of about 48 kd at 37°C or 60 kd at 100°C (109, M. K. Yeung,
personal communication).
It is likely that the observed shifts in molecular weights of
fimbrial proteins in response to heat are the result of changes in

81
conformation. Strong intramolecular interactions at the lower
temperature may prevent complete unfolding of the protein and
saturation with SDS, thereby altering the mobility in gels. The
results obtained with the cloned T14V type 1 fimbrial subunit (108)
tend to support such an explanation. Intermolecular interactions of a
noncovalent or covalent nature could also prevent complete
denaturation of fimbrial complexes and account for some of the
observed differences in the 37°C and 100°C immunoblot patterns.
The observation that N16 fimbriae were susceptible to reducing
agents differs from all previous reports on fimbriae from other
strains of Actinomyces (76,77,108). However, Masuda et al. (76) were
unable to get many of their fimbrial samples to migrate into 5 or 7%
SDS-PAGE gels, and their single percentage gels would not have the
resolving power of the 5-20% gradient gels used in this study. Thus,
solubility problems and inadequate resolution of proteins with very
similar molecular weights might have prevented them from detecting the
effects of reduction on fimbrial bands. This is especially likely in
light of the finding of Yeung et al. (108) that the cloned T14V type 1
fimbrial subunit migrated as a single band in 10 or 12% gels but as a
doublet in 5-12% gradient gels.
If one were examining the effect of reduction on a purified
protein complex that migrated as a single band on SDS-PAGE gels, one
would expect that a reduction would cause a single higher molecular
weight band to dissociate into one or more lower molecular weight
bands, depending on whether the complex was composed of identical or
dissimilar polypeptide chains held together by disulfide bonds.
However, fimbrial immunoblot patterns are too complex to allow such a

82
simple interpretation. A comparison of the differences between the
patterns of type 2 fimbriae prepared with or without reduction at
100°C indicates that reduction caused changes in the visible bands
at virtually every molecular weight level. There are too many bands
present to speculate on whether any particular band was derived from
another, but the fact that the reduced samples appear to have fewer
and better focused bands than nonreduced samples suggests that the
reduction of intra- or intermolecular disulfide bonds allows complete
denaturation and SDS saturation of the proteins, thereby overcoming
conformational variations that affect the mobilities and apparent
molecular weights of fimbrial fragments.
Another possible explanation for the presence of fewer reactive
bands in reduced samples is that reduction may destroy the fimbrial
epitopes on some bands so that they are no longer visible on
immunoblots. This explanation would be more plausible if the blots
were developed with monoclonal antibody; but since they were developed
with polyclonal antibodies, it is not likely that destruction of
epitopes could explain these results.
In summary, since complete dissociation of N16 fimbriae was not
possible, immunoblot patterns exhibited a multitude of fimbrial
bands. The simplest pattern of bands was obtained with fimbrial
samples heated at 100°C in the presence of DTT or
2-mercaptoethanol. In addition to the ladderlike series of bands in
the high molecular weight range, there were several strongly
immunoreactive bands (referred to as fimbrial subunits) in the 35-65
kd range. A. viscosus T14V type 1 and type 2 fimbriae have been
reported to give immunoblot patterns (32,108) similar to those

83
obtained in this study with N16 fimbriae. The fimbrial subunit
patterns for unreduced and reduced A. naeslundii N16 type 1 and type 2
fimbriae are summarized in Figure 14.
Assessment of the Purity of Fimbrial Samples by SDS-PAGE-Immunoblot
When aliquots of the type 1 and type 2 fimbriae purified from the
polyclonal antibody immunoaffinity columns were examined by SDS-PAGE
and immunoblot, it was apparent from the presence of immunoglobulin
heavy and light chains on the silver-stained SDS-PAGE gel (not shown)
that both samples were contaminated with RIgG that had leached off the
immunoaffinity columns. Consequently, immunoblot analysis of these
two fractions was complicated by the reaction of the peroxidase
conjugate with RIgG present in the fimbrial samples. The immunoblot
patterns of these two samples as well as those for the crude French
press supernatant, Batch A A-5m peak 1, Batch B 35% saturated
(NH^)^SO^ precipitate of the crude sonicate, and two samples of
the MAb-purified type 2 fimbriae, are shown in Figure 15.
The fimbrial subunits for both type 1 and type 2 fimbriae appear
to be present in all the samples except the two MAb-purified
fractions, although the weak type 1 bands in the type 2 fimbriae
purified from the RIgG column may actually be a reaction of conjugate
with immunoglobulin heavy chains contaminating the preparation. The
65 kd type 1 fimbrial subunit is readily apparent only in the type 1
fimbriae recovered from the RIgG column, indicating that this fraction
was enriched for type 1 fimbriae. That band is also present, but not
as obvious, in the French press supernatant, sonicate, and A-5m peak 1
samples, all of which should contain both types of fimbriae. The 63
kd type 2 fimbrial subunit is present in the type 2 fimbrial samples,

84
Type 1 Type 2
R NR R NR
43
26
Figure 14. Line drawing summarizing the "fimbrial subunit" patterns
obtained on immunoblots for A. naeslundii N16 type 1 and
type 2 fimbriae for samples heated at 100 C for 5 min.
R = reduced with 0.01 M DTT; NR = unreduced. Approximate
molecular weights are expressed in kilodaltons.

Figure 15. Immunoblot analysis of several preparations of N16
fimbriae at different stages of purification. Crude
French press (FP) supernatant was the starting material
for immunoaffinity purification of type 1 and type 2
fimbriae from the RIgG anti-type 1 and anti-type 2
columns, respectively. The middle lane contains the
Batch B 35% precipitate of the crude 2 min
sonicate. Also shown are A-5m peak 1 from Batch A and two
samples of type 2 fimbriae purified on the MAb 3B5.A1
column. All samples were reduced with DTT (0.01 M final
concentration). The blot was developed with a combination
of RIO IgG at 5 pg/ml and R2P IgG at 2.5 yg/ml, so
both types of fimbriae are evident.

I I
4N 00
ro
O
4^
U>
On
00
FP supernatant
Type 1 fimbriae/RIgG
Type 2 fimbriae/RIgG
Sonicate/35% ppt
A-5m peak 1
Type 2 fimbriae/MAb-1
Type 2 fimbriae/MAb-2

87
but the 39-40 kd doublet is not apparent. In fact, the 39-40 kd
doublet was present only in the crude French press supernatant, so
there is some question as to whether or not it is a type 2 fimbrial
subunit.
An attempt was made to remove the RIgG contaminating the
immunoaffinity purified type 1 and type 2 samples by adsorption with
protein A-Sepharose, but SDS-PAGE analysis of the samples after
protein A adsorption indicated that immunoglobulin chains were still
present in the fimbrial samples.
The protein A-adsorbed fimbrial samples and several of the other
samples were examined again by immunoblot analysis as shown in Figure
16. Duplicate blots were developed with either RIO against N16 type 1
fimbriae or R2P against N16 type 2 fimbriae. Also included for
comparison to N16 fimbriae were samples of purified T14V type 1 and
type 2 fimbriae. The T14V fimbrial samples appeared to be pure since
type 2 fimbriae did not react with RIgG against N16 type 1 fimbriae or
vice versa. On the other hand, the N16 type 2 fimbriae recovered from
the MAb immunoaffinity column exhibited minor contamination with type
1 fimbriae, as determined previously by XIEP (Figure 6C). The subunit
pattern for T14V type 1 fimbriae on the immunoblot developed with RIgG
against N16 type 1 fimbriae differs from the N16 type 1 fimbrial
subunit pattern, whereas the type 2 patterns for each appear to be
more similar. Another interesting observation is that the 63 kd N16
type 2 subunit (in the French press supernatant reacted with anti-type
2) appears as a doublet. On all the other immunoblots it appeared to
be a single band, but this sample was electrophoresed longer than
usual, and better resolution was obtained.

Figure 16. Iiranunoblot analysis of A. naeslundii N16 and A. viscosus
T14V fimbrial samples. All samples were reduced with DTT
at 100 C. The left half of the blot was developed with
RIO IgG anti-N16 type 1 fimbriae, and the right half was
developed with R2P IgG anti-N16 type 2 fimbriae.

N)
I
l
N>
O- O' vo o
U) 00 O
III I
FP supernatant
T14V type 1 fimbriae
Type 1 fimbriae/RIgG
RI&G
8
Type 2 fimbriae/RIgG
Type 2 fimbriae/MAb
T14V type 2 fimbriae
A-5m peak 1
I
K
t T * !fl1
t X. a - FP supernatant
T14V type 1 fimbriae
Type 1 fimbriae/RIgG
RI&G
Type 2 fimbriae/RIgG
Type 2 fimbriae/MAb
T14V type 2 fimbriae
A-5m peak 1
Is»
oo
vO
Anti-Type 1 Anti-Type

90
In summary, the N16 fimbrial samples obtained by immunoaffinity
chromatography were enriched for one of the fimbrial types, but none
of the samples were completely pure. The MAb immunoaffinity column
appears to offer the most promise for purification of type 2 fimbriae,
but it might be necessary to incorporate a detergent into the starting
buffer to prevent nonspecific adsorption of type 1 fimbriae. As for
the RIgG columns, it was probably too much to expect that they could
produce pure fimbriae from totally crude starting material. However,
since they required such harsh conditions to elute bound fimbriae that
some RIgG also was stripped from the columns, it is unlikely that
these immunoaffinity columns would be as useful as MAb columns.
Antigenic Relatedness of Actinomyces Fimbriae
Ouchterlony Analysis
Like the type 1 and type 2 fimbriae of A. viscosus T14V (20), the
type 1 and type 2 fimbriae of A. naeslundii N16 were found to be
unrelated antigenically, as demonstrated by the reactions of
non-identity between type 1 and type 2 fimbrial precipitins in
immunodiffusion (Figure 17A). A reaction of partial identity between
the type 2 fimbrial bands precipitated by MAb 3B5.A1 and the
polyclonal reference antiserum (Figure 17B) provided additional
evidence that MAb 3B5.A1 is specific for an epitope on the type 2
fimbriae; however, the presence of a spur suggests that there are
other epitopes on N16 type 2 fimbriae that elicit the formation of Abs
in a polyclonal response to the antigen. MAbs 2A3.B3 and 2B5.B6 did
not precipitate either type of fimbriae, perhaps because (a) the
proportions of antigen and antibody were not at equivalence or (b) the

91
Figure 17. Immunodiffusion reactions of antibodies to N16 fimbriae.
The center wells contained N16 fimbriae partially purified
from a French press supernatant by precipitation at 20%
(w/v) (NH4)2SO4. Rabbit antiserum R29 against N16
whole cells precipitated both type 1 and type 2 fimbriae.
A) A reaction of non-identity between type 1 and type 2
fimbriae. Antisera were RIO against N16 type 1 fimbriae
and R2P against N16 type 2 fimbriae.
B) A reaction of partial identity between the type 2
fimbrial bands precipitated by MAb 3B5.A1 and the
polyclonal reference antiserum R29. MAb 2A3.B3 and
MAb 2B5.B6 did not precipitate the fimbriae.

92
density of fimbrial epitopes was too low, as suggested previously by
the sparse distribution of gold particles in immunoelectronmicroscopy.
XIEP-A
The antigenic relatedness among fimbriae from different isolates
of A. naeslundii and A. viscosus was examined by XIEP-A. Unlabeled
homologous antisera raised against whole cell antigens were
incorporated into gels to precipitate fimbrial and sometimes non-
125
fimbrial antigens; in addition, an I-labeled antibody specific
for one type of fimbriae was incorporated into the gel so that
autoradiography would reveal which type of fimbriae reacted with the
monospecific antibody. Six fimbriae-specific antibodies were examined,
and the results of reactions with A. naeslundii antigens are
summarized in Table 5.
RIO rabbit IgG anti-N16 type 1 fimbriae reacted strongly with the
type 1 fimbriae of all 10 A. naeslundii serotype 3 isolates but not
with the type 2 fimbriae (Figure 18). It cross-reacted with the type
1 fimbriae of A. naeslundii serotype 2 W1544, but it did not react
with A. naeslundii serotype 1 W826 (WVU45), a serotype that has been
reported to lack type 1 fimbriae (17).
R2P rabbit IgG anti-N16 type 2 fimbriae reacted strongly with the
type 2 fimbriae of the 10 A. naeslundii serotype 3 isolates, but it
was essentially negative for type 1 fimbriae and the fimbriae of other
serotypes of A. naeslundii. Heterologous fimbriae were sometimes
visible on autoradiographs, but the intensity was so weak that it was
difficult to determine whether the reactions were the result of
nonspecific binding or very low levels of cross-reactivity; normal
rabbit IgG gave similar results.

Table 5. Summary of the reactions of fimbriae-specific 125I-labeled antibodies
with Lancefield-extracted type 1 and type 2 fimbriae of A. naeslundii isolates
Antigen
RIO
R2P
125i-Antibodya
MAb R59
R55
R70
Type
1
Type
2
Type
1
Type
2
Type
1
Type
2
Type
1
Type
2
Type
1
Type
2
Type
1
Type
2
N16
4b
0
0
4
0
4
4
0
1
1
1
1
WVU1267
4
0
0
4
0
4
4
0
1
4
1
2
WVU1468
4
0
0
4
0
4
4
0
1
4
1
2
WVU1527
4
0
0
4
0
4
4
0
1
1
1
1
WVU1528
4
0
0
4
0
4
4
0
1
1
1
1
W1629
4
0
0
4
0
4
3
1-2
1
1
1
2
W2273
4
0
0
4
0
4
4
0
1
1
1
1
W2821
4
0
0
4
0
4
4
0
1
1
1
1
UF92
4
0
0
4
0
4
4
0
1
4
1
1
UF524
2
0
0
4
0
4
4
1
1
1
1
1
W826 (WVU45)
_c
0
-
0
-
0
-
0
-
0
-
4
W1544
2-3
0
0
0
0
0
3
1
0
4
0
0
a RIO =
rabbit IgG anti-N16
type 1
fimbriae
R2P =
rabbit IgG anti-N16
type 2
fimbriae
MAb =
MAb 3B5.A1 anti-N16
type 2
fimbriae
R59 =
rabbit IgG anti-T14V
type 1
. fimbriae
R55 =
rabbit IgG anti-T14V
type i
! fimbriae
R70 = rabbit IgG anti-WVU45 type 2 fimbriae
b Reactions of l^I-antibodies with fimbrial rockets in XIEP-A were graded on a scale of
0 to 4 relative to the intensity of reaction with the homologous control. The homologous
reaction for R59 was 4,1 (type 1, type 2 fimbriae, respectively), and the homologous reaction for
R55 was 1,4.
c
W826 (WVU45) does not have type 1 fimbriae (17).
\o
u>

Figure 18. Reactions of 10 isolates of A. naeslundii serotype 3 with RIO
anti-N16 type 1 fimbriae by XIEP-A. All antigens were Lancefield
extracts of whole cells. The gel for electrophoresis in the
second dimension contained 0.5-0.75% R29 antiserum against N16
cells plus 125x-iabeled RIO RIgG.
A) Stained gels demonstrating the presence of type 1 and type 2
fimbriae.
B) Autoradiographs (23.5 h) demonstrating that RIO binds to the
type 1 fimbriae of each isolate.

vO
Ln

A
A.
W 1 629
A\
W2273
4
W2821
A
UF92
//\
UF524
B
A
A
A
A
l\
Figure 18. —continued

97
MAb 3B5.A1 gave the same pattern of reactivity as the polyclonal
anti-N16 type 2 fimbriae: it reacted only with the type 2 fimbriae of
the A. naeslundii serotype 3 isolates (Figure 19), thereby providing
confirmation for the RIA data, which indicated that MAb 3B5.A1
recognizes a serotype-specific epitope.
R59 rabbit IgG anti-T14V type 1 fimbriae cross-reacted with the
type 1 fimbriae of all 10 A. naeslundii serotype 3 isolates, as well
as the isolate representing A. naeslundii serotype 2, W1544.
R55 rabbit IgG anti-T14V type 2 fimbriae exhibited limited
reactivity with fimbriae of A. naeslundii isolates. It reacted
strongly with the type 2 fimbriae of only 3 of 10 A. naeslundii
serotype 3 isolates (WVU1267, WVU1468, and UF92) and also with
A. naeslundii serotype 2 W1544 type 2 fimbriae. With the other 7
A. naeslundii serotype 3 strains both fimbrial rockets were visible on
the autoradiographs, but the intensity of reaction would be graded no
more than 1+ on a 0 to 4+ scale. This antibody did not react with
A. naeslundii W826 fimbriae.
R70 rabbit IgG anti-WVU45 type 2 fimbriae gave a very strong
reaction only with the type 2 fimbriae of the homologous strain. Both
fimbrial rockets were clearly visible on autoradiographs of
heterologous isolates, but most could be rated 1+ relative to the 4+
homologous reaction. The type 2 fimbrial rockets of WVU1267, WVU1468,
UF524, and W1629 might be graded slightly higher.
Since R70 was produced by immunizing a rabbit with affinity
purified type 2 fimbriae from a strain that does not have any type 1
fimbriae (17), the presence of any reaction with type 1 fimbriae,
especially one equal in intensity to the reaction with type 2

Figure 19. Reactions of 10 isolates of A. naeslundii serotype 3 with MAb
3B5.A1 anti-N16 type 2 fimbriae by XIEP-A. All antigens were
Lancefield extracts of whole cells. The gel for electrophoresis
in the second dimension contained 0.5-0.75% R29 antiserum against
N16 cells plus 125x_iabeled MAb 3B5.A1 IgG.
A) Stained gels demonstrating the presence of type 1 and type 2
fimbriae.
B) Autoradiographs (20 h) demonstrating that MAb 3B5.A1 binds to
the type 2 fimbriae of each isolate.

vO
vO

Figure 19.
continued

101
fimbriae, was somewhat surprising. However, it points out the
problems associated with trying to interpret weak reactions on
autoradiographs. In this instance the weak reactions may be a result
of the presence of preimmune Abs to fimbriae of other strains; or
perhaps the fimbriae used as immunogen were attached to cell wall
components that elicited Abs to non-fimbrial determinants present also
in Lancefield extracts of cells. When immunoprecipitates of RIgG and
fimbriae are used as immunogens, as they were in this study and others
(17,92), there is the added possibility of eliciting anti-allotype or
125
anti-idiotype Abs such that I-RIgG might bind to the RIgG
precipitating the fimbrial Ags. Thus, weak reactions that result from
binding of specific Abs to fimbrial epitopes present in low numbers
are virtually impossible to distinguish from reactions that would be
considered "false positives".
The ability of the six anti-fimbriae antibodies, as well as a
normal rabbit IgG control, to react with isolates of A. viscosus
serotype 2 was assessed with Lancefield extracts of M100, W1053, and
T14V. The Lancefield-extracted antigens of these three strains gave
patterns of reactivity in XIEP-A similar to those shown for purified
(non-Lancefield extracted) T14V fimbriae (Figure 20). On a scale of 0
to 4 + , the reactions with T14V fimbriae were graded as shown in
Table 6. The grades were assigned according to the relative
intensities of the reactions on the original autoradiographs because
125
the reactions with heterologous I-Abs (RIO and R2P) appear to be
more intense in the photographs than they were in the original
autoradiographs.

Figure 20. Reactions of fimbriae-specific antibodies with A. viscosus T14V
fimbriae by XIEP-A. The antigen for each panel was 6.4 pg of
purified T14V fimbriae (kindly provided by J.O. Cisar). The
stained gels (upper panels) show both types of fimbriae
precipitated by UF-1 rabbit antiserum against T14V cells. The
24 h autoradiographs (lower panels) demonstrate the binding of
the following ^5i_iabeled antibodies:
A) R59 anti-T14V type 1 fimbriae;
B) R55 anti-T14V type 2 fimbriae;
C) RIO anti-N16 type 1 fimbriae;
D) R2P anti-N16 type 2 fimbriae.

103

104
TABLE 6. Reactions of fimbriae-specific 125I_]^at,e^e(j antibodies
with A. viscosus T14V
fimbriae
T14V fimbrial
antigens3
125I-Antibody
Type 1
Type 2
R59
rabbit
IgG anti-T14V type 1 fimbriae
4b
0
R55
rabbit
IgG anti-T14V type 2 fimbriae
0-1
4
R70
rabbit
IgG anti-WVU45 type 2 fimbriae
0-1
0-1
RIO
rabbit
IgG anti-N16 type 1 fimbriae
1-2
0
R2P
rabbit
IgG anti-N16 type 2 fimbriae
0
1-2
MAb
3B5.A1
anti-N16 type 2 fimbriae
0
0
a Sample of purified T14V fimbriae (types 1 and 2) obtained
from J.O. Cisar.
b Reactions of l^I-antibodies w¿th fimbrial rockets in
XIEP-A graded on a scale of 0 - 4+ relative to the intensity of the
reactions of normal rabbit IgG and homologous antibodies, respectively.

105
Polyclonal Abs to N16 type 1 or type 2 fimbriae cross-reacted
with the corresponding type of A. viscosus serotype 2 fimbriae, but
the reactions were much weaker than the homologous reactions. MAb
3B5.A1 did not react with A. viscosus serotype 2 strains in XIEP-A.
Thus, when examined by XIEP-A, all 10 strains of A. naeslundii
serotype 3, one strain of A. naeslundii serotype 2 (W1544), and 3
strains of A. viscosus serotype 2 (T14V, W1053, and M100) had both
types of fimbriae as determined by reactions with fimbriae-specific
Abs. In every case, the Coomassie-stained patterns revealed that type
1 fimbriae migrated slightly more toward the anode than type 2,
although in a few instances, the rockets were nearly superimposed (see
UF92 and UF524 in Figure 18). Also, the type 1 and type 2 precipitin
bands of each strain showed a reaction of non-identity, thereby
extending to A. viscosus and A. naeslundii strains in general the
observation, initially made on the basis of studies of T14V fimbriae
(14,92), that type 1 and type 2 fimbriae are immunologically distinct
entities.
However, there are shared determinants among fimbrial populations
of the same type as demonstrated by the reaction of RIO or R59 with
the type 1 fimbriae of A. naeslundii serotypes 2 and 3 and A. viscosus
serotype 2. Likewise, R2P and R55 reacted with the type 2 fimbriae of
heterologous species. The fact that R59 against T14V type 1 fimbriae
reacted strongly with the type 1 fimbriae of all 10 strains of
A. naeslundii serotype 3, whereas R55 and R70 against the type 2
fimbriae of T14V and WVU45, respectively, reacted with only a few
strains suggests that the type 1 fimbriae of different strains are
more closely related antigenically than are the type 2 fimbriae.

106
These observations will be discussed more fully after presentation of
the results of the bacterial agglutination assays.
Bacterial Agglutination
The monoclonal and polyclonal antibodies specific for N16
fimbriae were evaluated by agglutination versus cells of different
species and serotypes to assess the incidence of shared fimbrial
epitopes among heterologous isolates. Although this had been done to
a very limited extent by XIEP-A using fimbrial antigens that had been
extracted with hot acid, bacterial agglutination assays permitted a
more extensive survey and also enabled the evaluation of antigenic
relatedness among fimbriae in their native configuration.
Rabbit anti-N16 type 1 fimbriae, rabbit anti-N16 type 2 fimbriae,
and two MAbs reactive with N16 fimbriae were tested by bacterial
agglutination against a total of 106 isolates of various oral
bacteria. The panel of isolates included representatives of the
following species: Actinomyces bovis, A. israelii, A. naeslundii,
A. odontolyticus, A. pyogenes, A. viscosus, A. meyeri, Arachnia
propionica, Bacteroides loescheii, Rothia dentocariosa, Streptococcus
mutans, S. salivarius, S. cricetus, S. sobrinus, S. mitis, S. rattus,
and S. sanguis. Only isolates of A. naeslundii and A. viscosus were
agglutinated by any of these anti-fimbrial antibodies. Since fimbriae
are present on many of the other species, for example A. bovis (73),
A. israelii (26,73), B. loescheii (105), and S. sanguis (41), the
inability of antibodies against N16 fimbriae to agglutinate them
suggests that their fimbriae are not antigenically related to N16
fimbriae.

107
The bacterial agglutination patterns of selected isolates
representative of various serotypes of A. naeslundii and A. viscosus
are shown in Table 7.
RIO against N16 type 1 fimbriae agglutinated A. naeslundii
serotypes 2 and 3, Fillery cluster 3 (42), and A. viscosus serotype 2
strains, but it did not agglutinate strains of A. naeslundii serotypes
1 and 4. Clark et al. (28) obtained the same reaction pattern using
RIgG against T14V type 1 fimbriae in an immunodot EIA. R2P against
N16 type 2 fimbriae agglutinated both serotypes of A. viscosus and all
A. naeslundii isolates except serotypes 2 and 4. MAb 3B5.A1
agglutinated only the A. naeslundii serotype 3 isolates and one of the
Fillery cluster 3 isolates, whereas MAb 2B5.B6 agglutinated less than
50% of the A. naeslundii serotype 3 strains plus several strains that
are difficult to serotype. The agglutination results, for the most
part, were consistent with the results obtained by RIA and XIEP-A.
Although N16 type 1 fimbriae are unrelated antigenically to type
2 fimbriae, as demonstrated by immunodiffusion and XIEP-A, the results
of both XIEP-A and bacterial agglutination indicate that within a
fimbrial population (type 1 or type 2), there are antigenic
determinants that are common to other serotypes and species, as well
as determinants that are unique to certain serotypes or other subsets
of a species, as first suggested by the agglutination reaction
patterns of MAbs against T14V type 2 fimbriae (14).
Other investigators have used many of the strains listed in Table
7 to study the functional and antigenic relationships among
Actinomyces fimbriae. Some of their findings, which have been based
almost exclusively on the use of Abs specific for T14V or WVU45

108
TABLE 7. Bacterial agglutination patterns of selected isolates
Antibodies3 specific for N16 fimbriae
Antigens
RIO
R2P
3B5.A1
2B5.B6
A.
naeslundii serotype 1
W826 (WVU45)
0b
320
0
0
X569, W734, W869 (WVU398A)
0
10
0
0
A.
naeslundii serotype 2
W752
10
0
0
0
W1544
20
0
0
0
A.
naeslundii serotype 3
N16 (WVU820)
10
160
>20480
640
WVU1267
40
5120
>20480
0
WVU1468
20
640
>20480
0
WVU1527
40
640
>20480
2560
WVU1528
20
320
>20480
1280
W1629
10
160
>20480
0
W2273
80
5120
>20480
0
W2821
80
5120
>20480
10240
UF92
80
>20480
>20480
0
UF524
160
2560
>20480
0
A.
naeslundii Fillery cluster 3
B74
20
80
0
0
B120, RIK21
40
160
0
0
H3-3-8
40
5120
>20480
0
RIK149
20
320
0
0
A-
naeslundii serotype 4
WVU924, WVU963
0
0
0
0
A.
naeslundii untypables
X600
10
1280
0
640
W735
0
1280
0
640
WVU911
0
640
0
640
UF76
0
2560
0
1280
PK29
40
1280
0
2560
WVU903
160
2560
0
10240
WVU825
0
80
0
0
W1911
160
320
0
0
E38W3
20
80
0
0
W1716
0
640
0
0
A.
viscosus serotype 1
M482(T6), M484, X602, A755
oc
10
0
0
A757, A758, A759
0
20
0
0
A.
viscosus serotype 2
T14V
40
320
0
0
T14AV
640
1280
0
0
W859
160
320
0
0
W863, W1628
0
80
0
0
W872
40
10
0
0
M100
10
320
0
0
W1053
10
40
0
0

109
Table 7—continued
a RIO = rabbit antiserum against N16 type 1 fimbriae
R2P = rabbit antiserum against N16 type 2 fimbriae
3B5.A1 = MAb 3B5.A1 ascites against N16 type 2 fimbriae
2B5.B6 = MAb 2B5.B6 ascites against N16 type 2 fimbriae
b If the cells did not agglutinate at a 1:10 dilution of
antibody, the reaction was recorded as 0. Positive reactions were
expressed as the reciprocal of the highest dilution of antibody that
gave complete agglutination.
c Although all A. viscosus serotype 1 isolates were negative at
a 1:10 dilution of RIO antiserum, all were positive at dilutions
ranging from 1:2 to 1:32 when tested against protein A - purified RIgG
at 1 mg/ml.

110
fimbriae, will be discussed in relation to the results obtained in
this study with Abs specific for the fimbriae of N16.
In the original classification scheme of Fillery et al. (42),
based on numerical taxonomy and serology, all human isolates of
A. viscosus and A. naeslundii were assigned to one of six clusters.
Since the A. viscosus clusters (1, 2, 4, and 6) could not be easily
differentiated even with antifibril antisera (76), all A. viscosus
isolates now are generally assigned to cluster 1 and A. naeslundii
isolates to cluster 3 or cluster 5 (38), a separation that has been
supported by DNA hybridizations (30). The relationships between
cluster assignment and serotype (46) will be explained in the
following discussion.
A. naeslundii serotype 1, often referred to as cluster 5 or
"typical" A. naeslundii, has only one type of fimbriae, type 2
fimbriae. In a study of the type strain (WVU45 = W826 = ATCC 12104)
and a spontaneously occurring mutant, WVU45M, Cisar et al. (17)
observed that WVU45 was agglutinated by RIgG specific for T14V type 2
fimbriae but not with RIgG specific for T14V type 1 fimbriae and that
R70 RIgG specific for WVU45 fimbriae agglutinated T14V but not PK455,
a mutant of T14V that lacks type 2 fimbriae. Also, the mutant
(WVU45M) did not agglutinate with R70 or with RIgG specific for T14V
type 1 or type 2 fimbriae, and it did not have fimbriae when examined
by immunoelectron microscopy. The agglutination of A. naeslundii
serotype 1 strains by R2P against N16 type 2 fimbriae but not by RIO
against N16 type 1 fimbriae is consistent with their conclusion that
these strains possess only type 2 fimbriae.

Ill
In the same study Cisar et al. reported that strain W1544
agglutinated with RIgG specific for either type of T14V fimbriae,
which suggested that W1544 had both types of fimbriae. The existence
of a second serotype of A. naeslundii was first reported in 1972 (4),
and W1544 was designated as a typical representative of A. naeslundii
serotype 2 (5). These strains appeared to be catalase-negative
variants of A. viscosus serotype 2 (personal observation). Evidence
in support of this hypothesis was provided by numerical taxonomy
(38,42) and indirect immunofluorescence with antifibril antisera (38)
using strain W752; also, Coykendall and Munzenmaier (30) showed that
by nuclease DNA hybridization W752 had 98% homology with WVU627,
a cluster 1 or "typical" A. viscosus serotype 2 isolate, and only 46%
and 33% homology with cluster 3 and cluster 5, respectively. On the
basis of these studies, A. naeslundii serotype 2 strain W752 was
assigned to cluster 1 with all the A. viscosus serotype 2 isolates.
Clark et al. (25) showed that W752 and W1544 adsorbed as well to SHA
as most A. viscosus serotype 2 strains, which suggests that these
strains have type 1 fimbriae and, therefore, are more similar to
typical A. viscosus than to typical A. naeslundii in that regard.
W1544 and W752 also have been shown to exhibit the lectin activity
associated with type 2 fimbriae (29,37). Thus, on the basis of both
functional and antigenic characteristics it is clear that these
strains have both types of fimbriae. In view of the extremely close
similarity between A. naeslundii serotype 2 and A. viscosus serotype 2
strains, it is interesting to note that W752 and W1544 did not react
with R2P antiserum, even though R2P agglutinated all of the
A. viscosus serotype 2 strains tested.

112
However, the most interesting results were those obtained with
the A. naeslundii serotype 3 and Fillery cluster 3 isolates. These
two groups are generally considered to be equivalent (25,38). B74 and
B120 were among the original isolates assigned to cluster 3 by Fillery
et al. (42). In that study these "atypical” A. naeslundii strains
fell in between the typical A. viscosus isolates (cluster 1) and the
typical A. naeslundii isolates (cluster 5) on a similarity matrix.
Like typical A. naeslundii, cluster 3 strains were catalase-negative;
like typical A. viscosus, they had galactose rather than galactosamine
in their cell wells, and they completely hydrolyzed aesculin. Like
W752 (A. naeslundii serotype 2) but unlike any of the other cluster 1
isolates or cluster 5 isolates, cluster 3 isolates had electro-
phoretically fast moving 6-phosphogluconate dehydrogenase. It is
unfortunate that their study did not include N16 or any of the other
A. naeslundii serotype 3 strains (46,57) for a direct comparison to
cluster 3 isolates. Even in the DNA hybridization study of Coykendall
and Munzenmaier (30), which did include WVU820 (N16) and B74, the two
strains were evaluated separately by different methods.
Clark et al. (23,25,28) have demonstrated both similarities and
differences between A. naeslundii serotype 3 and cluster 3 strains.
The relative hydrophobicity of N16 or WVU1468 was lower than that of
each of 5 cluster 3 isolates (B74, B120, RI-K47, RI-K21, and H3-3-8),
but all the strains adsorbed equally well to SHA (25). Even though
all the isolates were positive by immunodot EIA versus RIgG to T14V
type 1 fimbriae (28), they were not affected to the same degree when
RIgG against T14V type 1 fimbriae was used to inhibit adsorption of

113
the cells to SHA; for example, WVU1468 was inhibited 91% but H3-3-8
was not inhibited at all (23).
The agglutination of all 5 of the cluster 3 isolates by the
polyclonal antiserums against N16 type 1 and type 2 fimbriae, as shown
in Table 7, is evidence of antigenic relatedness between cluster 3 and
serotype 3 strains. On the other hand, since the 3B5.A1 epitope
appears to be an important antigenic determinant for the A. naeslundii
serotype 3 isolates, its absence on 4 of the 5 cluster 3 strains,
including B74, suggests that there are at least two serogroups in
cluster 3, one with the 3B5.A1 epitope and the other without it. The
absence of the 3B5.A1 epitope on B74 fimbriae might explain why
antiserum to B74 fibrils, after adsorption with A. viscosus cells,
reacted with slightly less than 50% of cluster 3 isolates by indirect
immunofluorescence (38).
The strains originally identified by Gerencser and Slack (46) as
A. naeslundii serotype 4 do not appear to belong to this species. In
this study the two strains examined were not antigenically related to
N16 fimbriae as demonstrated by bacterial agglutination, and WVU924
did not react in XIEP-A with any of the Abs against fimbriae of N16,
WVU45, or T14V. Also, Clark et al. have shown that WVU963 did not
absorb to SHA (25) or react with RIgG against T14V type 1 fimbriae
(28). However, these strains make up a serologically identifiable
group (46), and similar strains are often isolated from the oral
cavity, although now they are usually referred to as just Actinomyces
serotype 963 without any species designation (87).
The group of isolates designated A. naeslundii untypables
includes strains that resemble A. naeslundii physiologically and

114
biochemically but whose reactions with the fluorescent antibody
reagents used for serotyping do not allow unequivocable placement into
one serotype or another. The agglutination of many of the untypables
with MAb 2B5.B6, a MAb that reacts with less than 50% of the isolates
of the homologous serotype, points out the potential difficulties of
serotyping with polyclonal reagents. Also, the agglutination of W735,
WVU911, and UF76 with MAb 2B5.B6 and R2P but not with RIO suggests
that the 2B5.B6 epitope is on the type 2 fimbriae, an observation that
was confirmed by its reactivity with type 2 fimbrial subunits on
immunoblots. Gerencser and Slack (46) had suggested that WVU903 and
WVU825 represented different serotypes within serotype 1. Although it
is possible that WVU825 belongs in that serotype because it appears to
lack type 1 fimbriae like other isolates of that group, clearly WVU903
cannot be considered to be a typical serotype 1 isolate. Its
agglutination with RIO suggests that it has type 1 fimbriae, and Clark
et al. have shown that it adsorbs well to SKA (25).
In summary, the agglutination of A. viscosus isolates by
polyclonal antiserums RIO and R2P against A. naeslundii N16 fimbriae
suggests that the fimbriae of these two species possess common
epitopes, but the reactions of MAbs 3B5.A1 and 2B5.B6 indicate that
certain fimbrial epitopes are unique to a species or serotype or other
subgroup within a species. The presence of both common and unique
epitopes on the same structure or molecule provides an explanation for
the difficulty in obtaining serotype-specific or even species-specific
polyclonal reagents and suggests that the use of a MAb or a panel of
MAbs with the desired specificities would be a better approach.

115
Immunoblot Analysis
Blot strips of SDS-PAGE resolved proteins from N16 crude French
press supernatant were developed with various monoclonal and
polyclonal antibodies specific for fimbriae of N16 , T14V, or WVU45;
the results for reactions with DTT-reduced antigen are shown in
Figure 21. All antibodies specific for fimbriae reacted with the
ladderlike series of high molecular weight bands, whereas normal sera
did not react with these bands. As for the reactions with fimbrial
subunits, all 3 MAbs against N16 fimbriae reacted with the type 2 band
at 63 kd but not with the 39-40 kd doublet. When the MAbs were tested
against immunoblots of unreduced N16 antigens (data not shown), they
all reacted with the high molecular weight bands and with the 62-63 kd
doublet seen in unreduced type 2 fimbriae but not with any lower
molecular weight bands. The failure of the MAbs to react with the
39-40 kd doublet may indicate that those are not fimbrial bands or
that those bands lack the appropriate epitopes. Evidence in support
of the latter explanation comes from the observation that polyclonal
R2P and R70 against N16 and WVU45 type 2 fimbriae, respectively,
reacted with the 39-40 kd doublet, whereas R55 against T14V type 2
fimbriae did not.
R59 against T14V type 1 fimbriae gave virtually the same pattern
of reactivity as RIO, confirming the antigenic relatedness between the
T14V and N16 type 1 fimbriae as determined by XIEP-A. R55 against
T14V type 2 fimbriae and R70 against WVU45 type 2 fimbriae reacted
with N16 type 2 fimbrial bands by immunoblot even though XIEP-A had
indicated little or no relationship.

Figure 21. Demonstration of the specificities of various monoclonal
and polyclonal antibodies for A. naeslundii N16 crude
French press supernatant antigens by immunoblot analysis.
The antigen was reduced with DTT at 100 C. The
specificities of these antibodies have been defined: RIO
and R2P = RIgG at 5 pg/ml; NR-5 and NR-10 = normal RIgG
at 5 and 10 yg/ml, respectively; 3B5-G and 3B5-A = MAb
3B5.A1 IgG at 10 yg/ml and ascites at 1:100,
respectively; 2A3 = MAb 2A3.B3 culture supernatant; 2B5 =
MAb 2B5.B6 ascites at 1:100; NM = normal mouse ascites at
1:100; AF = Aurodye forte total protein stain; R29 =
antiserum at 1:1500; R59, R55, and R70 = RIgG at
10 yg/ml.


118
In summary, the results of immunoblot analysis of the reactivity
of various fimbriae-specific antibodies with N16 antigens confirms the
reported specificities of these antibodies for type 1 or type 2
fimbriae and supports the proposed patterns for N16 fimbrial subunits
as seen in Figure 14.

SUMMARY AND CONCLUSIONS
The original goals of this study were to (a) produce monoclonal
antibodies against surface antigens of A. naeslundii serotype 3 strain
N16, (b) select at least one MAb that exhibited serotype specificity,
and (c) use the MAb as a tool to isolate and characterize the surface
component carrying the serotype-specific epitope. After it was
determined that the serotype-specific epitope was located on the
fimbriae, the following additional goals were set: (d) determine which
type of fimbriae carried the epitope, (e) produce polyclonal
antibodies against that fimbrial type or both fimbrial types, and (f)
use the polyclonal and monoclonal antibodies in a variety of
immunological assays to examine the antigenic relatedness between the
fimbriae of A. naeslundii N16 and the fimbriae of heterologous strains.
Three MAbs that bound to N16 cells in a solid-phase RIA were
produced; these were MAb 3B5.A1 (IgG„ ), MAb 2A3.B3 (IgG„), and
2a 3
MAb 2B5.B6 (IgM). When they were tested by RIA against a panel of
cells that included 10 A. naeslundii serotype 3 strains and 51
heterologous strains, MAb 3B5.A1 was the only one that reacted with
all of the homologous and none of the heterologous isolates. Thus, it
was the only MAb that appeared to be serotype-specific. MAb 2A3.B3
cross-reacted with other serotypes of A. naeslundii and with
A. viscosus serotype 2, whereas MAb 2B5.B6 reacted with only 40% of
the homologous isolates plus a few other A. naeslundii strains.
Electron microscopy of N16 cells indirectly immunolabeled with
119

120
colloidal gold indicated that all three MAbs bound to epitopes located
on fimbriae.
There was both immunological and functional evidence from the
work of other investigators to suggest that N16 had both type 1 and
type 2 fimbriae. In the present study, all of the A. naeslundii
serotype 3 strains were shown to have type 1 fimbriae by their
cross-reactions with rabbit IgG specific for A. viscosus T14V type 1
fimbriae. In this respect, serotype 3 strains differ from
A. naeslundii serotype 1 strains, which do not have type 1 fimbriae
(17). All of the serotype 3 strains were shown also to have type 2
fimbriae by their ability to cause lactose-inhibitable
hemagglutination. Since the type 2 fimbriae mediate hemagglutination
by Actinomyces cells, the removal of type 2 fimbriae from the cells
was monitored by measuring the decline in the HA titer of the treated
cells relative to untreated cells.
In preliminary experiments fimbriae were extracted from N16 cells
by several different methods. French press shearing and sonication
were found to remove fimbriae from cells much more effectively than
Virtis homogenization or lithium chloride extraction. Lancefield
extraction was a convenient way to prepare soluble antigens for
Laurell or crossed rocket immunoelectrophoresis, but it extracted too
much carbohydrate to be considered as a means for obtaining fimbriae
for purification.
Type 1 and type 2 fimbriae were detected in Lancefield extracts
and in crude sonicates or French press supernatants by crossed
immunoelectrophoresis using rabbit antiserum against N16 whole cells.
125
When I-MAb 3B5.A1 was also incorporated into the gel,

121
autoradiography indicated that MAb 3B5.A1 was specific for the type 2
fimbriae. MAb 3B5.A1 proved to be a valuable tool for purifying the
type 2 fimbriae of A. naeslundii N16. When crude sonicates of N16
cells were chromatographed on Bio-Gel A-5m columns, the fractions
containing type 2 fimbriae were identified by their positive reactions
with MAb 3B5.A1 in an immunodot EIA. The type 2 fimbriae were then
purified from these fractions by chromatography on a MAb 3B5.A1
immunoaffinity column. The type 2 fimbriae recovered from the MAb
column were slightly contaminated with type 1 fimbriae, probably as a
result of noncovalent interactions between the two types of fimbriae
or nonspecific adsorption to the column; this might have been avoided
by inclusion of a detergent, such as 0.05% Tween 20, in the buffer for
eluting the unbound fraction, as suggested by Cisar et al. (17).
Polyclonal antisera monospecific for N16 type 1 or type 2
fimbriae were produced by immunizing rabbits with fimbrial
immunoprecipitins excised from XIEP gels. Immunoaffinity columns
prepared from these antisera required much harsher elution conditions
than the MAb column in order to desorb bound antigens; consequently,
rabbit IgG leached from the columns, contaminating the fimbrial
samples.
The effects of various physical and chemical treatments on N16
fimbriae were assessed by immunoblot analysis of SDS-PAGE resolved
antigens. Fimbrial bands were not detectable in samples treated with
Pronase, proteinase K, or papain. Base and hot acid also altered the
fimbrial immunoblot patterns significantly. N16 fimbriae could not be
completely dissociated, but there was evidence for partial
dissociation into subunits. Type 1 and type 2 fimbriae could be

122
distinguished by their different subunit patterns on immunoblots, and
both were affected by increased temperature and by reduction. N16
fimbriae appear to have heat modifiable subunits like those reported
for T14V type 1 fimbriae and WVU45 type 2 fimbriae, but that would
have to be verified with purified or cloned subunit proteins as Yeung
et al. (108) have done. The fimbrial patterns on immunoblots of crude
N16 fimbriae are so complex that it is not possible to tell precisely
what shifts in molecular weight are occurring. When samples were
reduced at 100°C, N16 type 1 fimbrial subunits were observed at
65 kd, 57-60 kd, and a weaker doublet at 53-54 kd, and type 2 subunits
occurred at 63 kd with a doublet also at 39-40 kd in some samples.
Antibodies specific for the fimbriae of N16 (A. naeslundii
serotype 3), T14V (A. viscosus serotype 2), and WVU45 (A. naeslundii
serotype 1) were used in several different immunological assays,
including Ouchterlony analysis, crossed immunoelectrophoresis with
autoradiography, bacterial agglutination, and SDS-PAGE immunoblot, to
examine the antigenic relationships among Actinomyces fimbriae. The
results of these assays indicated that (a) N16 type 1 fimbriae are not
related antigenically to the type 2 fimbriae, (b) the type 1 fimbriae
of A. naeslundii serotypes 2 and 3 and A. viscosus serotype 2 have
common antigenic determinants, (c) type 2 fimbriae of certain
A. naeslundii and A. viscosus strains also have common antigenic
determinants, and (d) MAb 3B5.A1 recognizes a serotype-specific
determinant located on the type 2 fimbriae of A. naeslundii serotype 3
strains.
One of the more interesting findings was that MAb 3B5.A1 did not
agglutinate 4 of the 5 strains belonging to Fillery cluster 3. Since

123
A. naeslundii serotype 3 and Fillery cluster 3 are generally
considered to be equivalent (25,38), this data suggests that there are
at least two serogroups in cluster 3, one with the 3B5.A1 epitope and
the other without it. Also, the absence of the 3B5.A1 epitope on B74
could explain why antiserum to B74 fibrils, after adsorption with
A. viscosus cells, reacted with less than 50% of the cluster 3
isolates by indirect immunofluorescence (38). If a strain possessing
the 3B5.A1 epitope, for example cluster 3 strain H3-3-8 or any of the
A. naeslundii serotype 3 strains, were used to make cluster 3 specific
antifibril antisera, it would probably react with a higher percentage
of the cluster 3 isolates than B74 antiserum.
This study was primarily an immunological characterization of the
fimbriae of A. naeslundii N16 and was somewhat weighted toward the
type 2 fimbriae because all the MAbs reacted with type 2 fimbriae, in
particular MAb 3B5.A1, which recognized a serotype-specific epitope.
Consequently, there is plenty of room for additional studies,
particularly in regard to the functional and immunological properties
of the type 1 fimbriae and functional properties of type 2 fimbriae.
The difficulties of purifying one type of fimbriae when both are
present and the difficulties of preparing polyclonal antisera
monospecific for one fimbrial type have been discussed previously
(20). Although the production of MAbs can overcome the latter
problem, polyclonal antibodies are more useful in some instances. The
availablility of mutants lacking one or both fimbriae can facilitate
purification of fimbriae, production of monospecific antisera, as well
as attribution of functional properties to one type or the other
(17,67,68,84); Cisar et al. (16) have already shown that loss of one

124
fimbrial type did not alter expression of the other. Thus, any
additional studies with N16 should probably start with the isolation
of l+2 , 1 2+, and 1 2 mutants. Since fimbriae-specific
antibodies are already available, mutants could be isolated by the
colony blotting technique of Reis et al. (91); mutants of other
strains have been isolated successfully by enrichment techniques
(17,67). Since one of the N16 cell lines was observed to be a mix of
about 80% l+2 and 20% l+2+ (data not presented), isolation of
a coaggregation-defective (l+2 ) mutant of N16 should be
relatively easy.
It would also be helpful to have large panels of MAbs specific
for different type 1 and type 2 fimbrial epitopes. These could be
useful typing reagents, but more importantly, the availability of many
different MAbs would increase the likelihood of finding some that
could inhibit the functional activities of the fimbriae. Even some
polyclonal antisera that bind to a fimbrial type cannot inhibit its
functional activity (28), so the chances of finding a MAb that would
do so would be even slimmer.
Fab fragments of both polyclonal and monoclonal antibodies should
be tested for their ability to inhibit functional activities; for
example, MAb 3B5.A1 should be tested for its ability to inhibit
coaggregation, whereas antibodies to type 1 fimbriae could be assayed
for their ability to inhibit adsorption to SKA (23) or to proline-rich
protein (PRP)-coated hydroxyapatite beads (48). These antibodies
could be used to screen recombinants for expression of N16 fimbrial
genes encoding for functional domains; MAb 3B5.A1 could be used to
detect recombinants expressing the N16 type 2 fimbrial gene encoding

125
for the serotype-specific epitope. After subcloning to obtain the
smallest DNA fragment encoding for the desired fimbrial domain, the
cloned protein could be purified, using MAbs as a tool to monitor
purification or for immunoaffinity chromatography.
The nature of the cloned protein would determine the next step.
If it was the serotype-specific epitope, the protein could be tested
for its ability to inhibit binding of MAb 3B5.A1 to N16 cells. If it
was the binding domain of the lectin or the domain for adsorption to
SHA, the cloned protein would be tested as an inhibitor of
coaggregation or adsorption to SHA, respectively. Fimbrial subunits
could be cloned and the structural basis for heat modifiability
investigated, as Yeung et al. (108) have done for the 65 kd T14V type
1 subunit.
The amino acid sequence of the cloned protein and the DNA
sequence of the corresponding gene could be determined. The cloned
protein or synthetic peptides could be tested as inhibitors of the
appropriate binding activity or used as immunogens to elicit
antibodies that could inhibit binding. Intact fimbriae of T14V have
been tried as vaccines in an experimental animal model, and there was
a reduction in colonization of immunized animals relative to control
animals (23). However, even though Actinomyces fimbriae appear to
have potential as vaccines, it is unlikely that a vaccine containing a
single strain would be effective against all strains because the
antigenic determinants of one strain may not be near the site that
mediates adsorption to SHA in other strains (23). It is even more
unlikely that a synthetic peptide or cloned protein subunit would be

126
an effective, broad-spectrum vaccine. However, cloning the fimbrial
genes will give a clearer picture of the molecular basis for adherence
in Actinomyces and perhaps suggest other strategies for interfering
with the pathogenic mechanisms.

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D.C.

BIOGRAPHICAL SKETCH
Sandra L. Bragg was bom June 16, 1947 in DeLand, Florida. She
attended elementary school in Kirksville, Missouri, while her father
completed his medical education. In 1955 her family moved to Daytona
Beach, Florida, where she participated in competitive tennis. After
graduating from Seabreeze Senior High School in 1965, Sandra moved to
Atlanta, Georgia. In 1969, she received her Bachelor of Arts degree
with a major in religion from Emory University. She then completed
her Master of Science degree in biology at Georgia State University in
1972 and has since been employed as a microbiologist in the Division
of Mycotic Diseases at the Centers for Disease Control (CDC). She was
awarded a CDC scholarship to pursue the Doctor of Philosophy degree
and moved to Gainesville for two years in 1978 to complete her course
work. She then returned to CDC in 1980 and has worked on both her
dissertation research and CDC research projects in mycology since that
time.
137

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Professor of Microbiology and Cell
Science and of Oral Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
/as a
William B. Clark
Professor of Oral Biology and of
Immunology and Medical Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Edward M. Hoffmann
Professor of Microbi
Science and of Immunology and
Medical Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a__d_issertation for the degree of
Doctor of Philosophy.
Paul A. Klein
Associate Professor of Pathology and
Laboratory Medicine
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Associate Professor of
Microbiology and Cell Science

This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy. / / /J
August, 1988 U le Dean, Graduate School

UNIVERSITY OF FLORIDA
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