<%BANNER%>

Characterization of the Zps1p Cell Wall Protein from Saccharomyces cerevisiae


PAGE 1

CHARACTERIZATION OF THE Z PS1P CELL WALL PROTEIN FROM Saccharomyces cerevisiae By STEPHANIE L. DROBIAK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

PAGE 2

Copyright 2004 by Stephanie L. Drobiak

PAGE 3

This document is dedicated to my fianc a nd my family for all their help and support during the last few years.

PAGE 4

ACKNOWLEDGMENTS I would like to thank my future husband and my family for their moral support, my lab mates for their continuous help and friendship, and my mentor, Dr. Thomas Lyons, for his endless guidance and patience. iv

PAGE 5

TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES...........................................................................................................vi ABSTRACT......................................................................................................................vii 1 INTRODUCTION...................................................................................................1 Zps1p-like Proteins from Candida albicans and Aspergillus spp..........................2 Zps1p from Saccharomyces cerevisiae.................................................................10 Zinc-dependent Metalloproteases of the M35 Clan..............................................12 Comparison of the Zps1p-like Proteins and the M35 Metalloproteases...............14 2 RESULTS AND DISCUSSION............................................................................16 Regulation of ZPS1 Gene Expression...................................................................16 Partial Purification of Zps1p from Inclusion Bodies............................................17 3 CONCLUSIONS....................................................................................................26 4 MATERIALS AND METHODS...........................................................................27 Growth Media.......................................................................................................27 Solutions and Buffers for Yeast Transformations and -Galactosidase Assays...28 Bacterial and Yeast Strains...................................................................................29 Yeast Transformations..........................................................................................30 -Galactosidase Assays.........................................................................................31 Cloning of ZPS1 and Construction of an E. coli Expression Plasmid..................32 Expression of Zps1p in E. coli..............................................................................33 Estimation of Protein Purity by SDS-PAGE........................................................34 LIST OF REFERENCES...................................................................................................35 BIOGRAPHICAL SKETCH.............................................................................................39 v

PAGE 6

LIST OF FIGURES Figure page 1-1. Multiple sequence alignment of fungal cell wall proteins with related metalloproteases.........................................................................................................3 1-2. Active-site residues of deuterolysin.43.......................................................................13 1-3. Basic structural features of the Zps1p-like proteins and the metalloproteases in the M35 clan...................................................................................................................14 1-4. Active site structures. On the right is the known active site of the aspzincins, deduced from the crystal structures of deuterolysin43 and GfMEP.49 On the left is a possible structure of an active site within the Zps1p-like proteins..........................15 2-1. Zinc and iron responsiveness of the ZPS1-lacZ reporter. -Galactosidase activity in wild-type cells and zap1 mutant cells grown in CSD..............................................16 2-2. Zinc and iron responsiveness of the ZPS1-lacZ reporter. -Galactosidase activity in wild-type cells and rim101 mutant cells grown in CSD..........................................18 2-3. SDS-PAGE analysis of E. coli transformants containing the pET-22b(+)-ZPS1 expression vector......................................................................................................19 2-4. SDS-PAGE analysis of soluble and insoluble components of the cell lysate obtained from breakage of E. coli expressing Zps1p..............................................................20 2-5. SDS-PAGE analysis of the soluble and insoluble products obtained after solubilization and refolding of the inclusion body pellet.........................................20 2-6. SDS-PAGE analysis of the major protein peak collected after SEC (combined fractions 9 12)........................................................................................................21 2-7. SDS-PAGE analysis of the washed inclusion body pellet, solubilized in Buffer A containing 8 M Urea.................................................................................................23 2-8. SDS-PAGE analysis of the soluble and insoluble products obtained after solubilization and refolding of the inclusion body pellet.........................................24 vi

PAGE 7

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CHARACTERIZATION OF THE ZPS1P CELL WALL PROTEIN FROM Saccharomyces cerevisiae By Stephanie L. Drobiak December 2004 Chair: Thomas Lyons Major Department: Chemistry Fungal cell wall proteins are involved in establishing infection through interaction with host ligands and by mediating morphological changes that enhance pathogenicity. In recent years, research has focused on a family of fungal cell wall proteins that are structurally related to zinc-dependent metalloproteases of the M35 clan. Members of this protein family include Zps1p from Saccharomyces cerevisiae, Pra1 from Candida albicans, CpAspf2 from Coccidioides posadassii, Aspnd1 from Aspergillus nidulans, and Aspf2 from Aspergillus fumigatus. The proteins from C. albicans and Aspergillus spp. are known cell-surface antigens during fungal infections, and both Pra1 and Aspf2 bind specific ligands within mammalian hosts. Although expression of these proteins during fungal infection is well documented, their biological function remains unknown. In this thesis, we report preliminary work toward characterization of Zps1p from S. cerevisiae. Results indicate expression of ZPS1 to be regulated in response to zincand iron-limitation, as well as extracellular pH. In addition, we present the partial purification of vii

PAGE 8

recombinant Zps1p from bacterial inclusion bodies. Analysis of Zps1p is intended to provide the framework for future expression, purification, and characterization of the Zps1p-like proteins from the medically important fungi C. albicans and Aspergillus spp. viii

PAGE 9

CHAPTER 1 INTRODUCTION Many fungi are responsible for both superficial and systemic infections in man. Immunocompromised individuals are susceptible to fungal infections caused by a variety of pathogens, including Candida albicans and several Aspergillus species. Relevant diseases caused by these species include candidiasis, aspergilloma, invasive aspergillosis, and allergic bronchopulmonary aspergillosis (ABPA). Although these mycoses are well documented, many factors contributing to fungal pathogenesis are still not well understood. Efforts to better understand virulence factors often focus on components of the fungal cell wall. The fungal cell wall is a complex mixture of carbohydrates (80 to 90%), proteins (6 to 25%), and minor amounts of lipid (1 to 7%).1 As the outermost part of the cell, the wall initiates physical interaction between the microorganism and the environment, including the host. The host-parasite interaction, resulting in adhesion, is the first critical step in establishing infection and modulation of the host immune response. In addition, the cell wall mediates fungal cell-cell adhesion (flocculation), a first step in the morphological change from a unicellular yeast to growth as multicellular filaments (mycelia or hypha). Formation of mycelia enhances pathogenicity, allowing the invasion of host tissues, and is influenced by environmental variables including extracellular pH2;3 and nutritional status.4 For these reasons, fungal cell wall proteins (CWPs) have been of heightened interest. Not only are CWPs involved in intercellular binding, many possess enzymatic activity involved in cell wall biosynthesis and maintenance, and acquisition of 1

PAGE 10

2 extracellular nutrients.1 When the actions of CWPs negatively impact the viability of the host, the proteins are considered virulence factors that advance the establishment of infection. Due to their accessibility at the cell surface, and their critical role in intercellular interactions, CWPs are ideal targets for the development of antifungal drugs. A family of cell wall proteins from various fungi has been the focus of much research in recent years. Members of this family include Zps1p from Saccharomyces cerevisiae, Pra1 from Candida albicans, CpAspf2 from Coccidioides posadasii, and Aspnd1 and Aspf2 from Aspergillus nidulans and Aspergillus fumigatus, respectively. These CWPs share a number of key structural features, have high sequence homology, and exhibit significant similarity to a family of zinc-dependent metalloproteases of the M35 clan, known as the aspzincins5 (Figure 1-1). At present, the biochemical function of these proteins remains unknown. The focus of this research is to characterize the Zps1p cell wall protein from the yeast Saccharomyces cerevisiae. This document entails the preliminary work toward characterization of Zps1p through study of its structure, function, and gene regulation in S. cerevisiae. Analysis of Zps1p is intended to provide the framework for future expression, purification, and characterization of the Zps1p-like proteins from the medically important fungi C. albicans (Pra1) and Aspergillus spp. (Aspnd1 and Aspf2). The relevance of the Zps1p-like proteins is discussed below. Zps1p-like Proteins from Candida albicans and Aspergillus spp. The homologues from A. fumigatus and A. nidulans are known as Aspf26 and Aspnd1,7 respectively. In C. albicans, the homologue is known by many names: Pra1 (pH regulated antigen),8 FBF (fibrinogen binding factor),9 FBP1 (fibrinogen binding protein),10 and mp58 (58-kDa fibrinogen-binding mannoprotein).11

PAGE 11

3 Figure 1-1. Multiple sequence alignment of fungal cell wall proteins with related metalloproteases. Aspf2, Aspnd1, and Pra1 are all secreted proteins with four N-glycosylation sites (Asn-X-Ser/Thr) and eight cysteine residues perfectly conserved, suggesting a similar function.6;7;11 Both Pra1 and Aspf2 contain a serineand threonine-rich region of potential O-glycosylation close to the C-terminus, a purported cell wall binding domain (CWBD).12 Studies have shown that Pra1 does indeed contain O-linked sugar moieties.11 Aspnd1 lacks this Serand Thrrich region. However, the C-terminal region of Aspnd1 is glutamateand glutamine-rich, the purpose of which is unclear. Although their biochemical function remains unknown, these three proteins from Candida and Aspergillus are immunodominant antigens in fungal infections.

PAGE 12

4 Purification and characterization of various Aspergillus spp. cell wall proteins for use in immunodiagnosis of ABPA and other diseases led to the identification of Aspf2 and Aspnd1 as proteins that elicit a strong immune response.6;7;13;14 These discoveries were made by immunoblotting water-soluble extracts of Aspergillus spp. with sera from patients with ABPA. Sera from those infected with different forms of aspergillosis contain elevated levels of immunoglobulin G (IgG) and immunoglobulin E (IgE) antibodies specific for Aspergillus antigens, including Aspnd1 and Aspf2. These antigens are consistently recognized by serum samples from aspergilloma patients, but not with sera from control or healthy individuals.7;14 Deglycosylated forms of the purified proteins remain reactive to the antibodies, suggesting the N-glycosidic groups are not required for recognition by the aspergillosis serum samples tested. These data indicate that the epitopes recognized are located mainly in the polypeptide region.7;13 This hypothesis is further supported by antibody reactivity with the recombinant forms of Aspnd1 and Aspf2 which, when over-expressed in the prokaryote E. coli, lack the glycosidic moieties.15;16 Furthermore, all reactivity was abolished following protease treatment.7;13 During characterization of the Aspnd1 antigen from A. nidulans, Calera et al. observed sera that reacted with Aspnd1 also consistently reacted with antigens from A. fumigatus. Therefore, they tested the reactivity of purified anti-Aspnd1 specific IgG with several different A. fumigatus antigens including Aspf2 (also known as gp55). The various antigens reacted with the anti-Aspnd1, suggesting a close relationship between the immunodominat antigens from the two Aspergillus species, possibly through the existence of common peptide epitopes.7 Likewise, Banerjee et al. detected binding of

PAGE 13

5 anti-Aspf2 antibodies by Aspnd1. The relatedness of Aspnd1 and Aspf2 is supported through analysis of their primary structure. The similarity between the two proteins suggests they share several epitopes and should therefore elicit the formation of IgG and IgE antibodies able to recognize both antigens. To answer this question, Banerjee et al. compared the reactivity of purified Aspnd1 and Aspf2 toward IgE antibodies from ABPA serum. The mean IgE binding of purified Aspf2 was almost three fold higher than binding of Aspnd1. Differences in Aspnd1 and Aspf2 binding to IgE may result from differences in posttranslational modifications or the tertiary structures of these proteins.6 Comparison of IgE binding by both native and recombinant forms of these antigens indicates that the recombinant forms most likely have structures functionally comparable to the native proteins, since IgE binding is dependent on proper three-dimensional structure.15;16 Similarly, Pra1 from C. albicans elicits a strong immune response with sera from patients infected with candidasis. Antibodies against Pra1 are present in sera from patients with systemic candidasis,17;18 and Pra1 itself has been detected in the cell wall of clinical isolates of C. albicans.17 Deglycoslyation of Pra1 did not affect reactivity with anti-Pra1 antibodies, again suggesting the epitopes recognized are located mainly in the polypeptide region. An extensive epitope-scanning study, employing a complete set of overlapping dodecapeptides deduced from the Pra1 sequence, identified several immunoreactive continuous B-cell epitopes within the protein sequence. Six regions of elevated reactivity were identified, including four internal regions and both the amino and carboxy termini of the mature polypeptide. Of these regions, the C-terminal domain was

PAGE 14

6 highly reactive towards the anti-Pra1 antibodies and therefore subjected to further epitope mapping.18 Analysis of the epitopic region at the C-terminal domain of the Pra1 polypeptide identified the nonapeptide 290HTHADGEVH298 as the minimal region required to retain antibody-binding activity. Researchers further probed the significance of this epitopic region by synthesizing a synthetic peptide corresponding to the last ten amino acid residues at the C-terminus. The synthetic peptide, coupled to keyhole limpet hemocyanin (KLH), was used to immunize two mice. The serum samples obtained from the two immunized mice were able to recognize Pra1 from cell wall extracts of C. albicans with high specificity. Interestingly, this C-terminal sequence (CHTHxxGxxHC) is conserved in both Aspnd1 and Aspf2, as are the four internal epitope regions (Table 1-1). The conservation of linear epitopes within this family of cell wall proteins from various fungal pathogens provides additional support suggesting their role in the host-parasite interaction.18 Table 1-1. Sequence homology of five of the identified IgG epitopes.18 Pra1 Antigen Sequence epitope sequence Aspnd1 Aspf2 LRFGSK LRWGNE LRWGNE RKYF RKYF RKYF NDGWAGYW LEGWGGHW LEGWGGHW DVYA EVYA EAYA HTHADGEVH HTHEGGELH HTHEGGQLH Not only do these fungal antigens interact with antibodies within the host, but Aspf2 and Pra1 also bind specific host ligands. Fungal adhesion to host cells and tissues initiates establishment of infection and is considered a potential virulence factor. Aspf2

PAGE 15

7 binds the extracellular matrix protein laminin,6;19 and Pra1 binds the serum protein fibrinogen.9;11 Proteins in the extracellular matrix (ECM) are known to bind to A. fumigatus conidia (an infectious airborne form of the fungus). Binding of conidia to ECM ligands is believed to be a crucial first step initiating aspergillosis, and specific recognition of these ligands may greatly influence pathogenicity.20;21 One component of the ECM is laminin, a multidomain glycoprotein and major component of the basement membrane. Interaction of laminin with cell surface ligands facilitates cell-cell adhesion, cell migration and cell differentiation.1 As reported by Banerjee et al., laminin shows a dose-dependent interaction with Aspf2. Both native and recombinant Aspf2 demonstrate high binding affinity to laminin, with greater affinity observed by the native protein. With specific binding to laminin, and significant homology to Pra1 (which binds fibrinogen), the involvement of Aspf2 in fungal adherence to the ECM may play an important role in establishing pathogenicity.6 Blood serum proteins (e.g., serum albumin, transferrin, fibrinogen, complement fragments C3d and iC3b) are additional targets for fungal binding. Interactions of C. albicans with fibrinogen have been well characterized.1 In 1987, Bouali et al. identified a fibrinogen binding factor (FBF) on the surface of C. albicans germ-tubes and mycelium, the fungal forms most often found in infected tissues.9 Five years later, in 1992, Casanova et al. identified this FBF as a 58-kDa fibrinogen-binding mannoprotein (mp58), which is now known to be Pra1. Binding of Pra1 to fibrinogen is apparently specific, since binding to other mammalian proteins tested (laminin, fibronectin, C3d, type IV collagen) was not observed. O-deglycosylated Pra1 was unable to interact with

PAGE 16

8 fibrinogen, implying this carbohydrate domain may play a role in binding. The in vivo production of Pra1 during candidasis and its ability to bind fibrinogen suggest a role in infection.1;11 Another factor supporting an active role of Pra1 in candidasis is its differential expression in response to pH.8 The ability of C. albicans to grow and differentiate over a broad pH range is critical for its survival in a variety of environments and host tissues (e.g., blood, pH ~ 7.2; vaginal tract, pH ~ 4.5).3 Extracellular pH is an environmental signal that regulates the yeast-to-mycelia transition in vivo, a morphological change that greatly enhances invasion of host tissues.2 In C. albicans, gene expression is regulated by the Rim101p transcription factor in response to alkaline pH.22 Studies have shown the C. albicans Rim101p pH response pathway to be required for several host-pathogen interactions, and therefore essential for pathogenesis.23 Pra1 is a Rim101p target gene maximally expressed at neutral pH, with no detectable expression below pH 6.0. However, ambient pH is not the sole factor influencing expression. When cultured in rich medium (YPD) buffered at pH 7.0, no Pra1 production was detected. This result implies partial regulation by nutritional status, a hypothesis that remains to be tested.8 Although the effect of nutritional status on Pra1 expression has not been assessed, the nutrient regulation of the Apsergillus spp. antigens has been investigated. Researchers recognized that production of Aspnd1 and Aspf2 only occurred when the fungi are grown in certain conditions, especially in Czapek-Dox (CD) medium (3g NaNO3, 0.5g MgSO4.7H2O, 0.5g KCl, 55mg FeSO4, 1g KH2PO4, and 30g sucrose per liter). Therefore, the various components of CD medium were tested to determine which is influencing antigen production. Elucidation of the regulatory elements responsible for

PAGE 17

9 Aspnd1 and Aspf2 expression may provide clues to their function and potential roles in virulence.24 Variations of CD medium were quantitatively and qualitatively tested against a control medium, AMM (1% glucose, 0.6% NaNO3, 0.052% MgSO4, 0.052% KCl, 0.15% KH2PO4, and traces of FeSO4 and ZnSO4), known not to stimulate Aspnd1 or Aspf2 production under normal conditions. The type and amount of carbon or nitrogen source did not affect antigen production, nor did addition of iron to the CD medium. However, addition of -molar concentrations of zinc eliminated antigen synthesis in CD, while removal of zinc from AMM medium induced antigen production. Addition of other divalent metals (Co2+, Ni2+, Cu2+, Ca2+) had no inhibitory effects, with the exception of Cd2+ and Mn2+ (only slight inhibition). Currently, researchers are attempting to identify and characterize potential zinc response elements (ZREs) in the promoter regions of ASPND1 and ASPF2.24 Detected in the promoter region of ASPND1 are at least five potential PacC binding sites.7 PacC is a pH responsive transcription factor in Aspergillus spp. and is homologous to C. albicans Rim101p.25 The presence of putative PacC sites suggests possible regulation of Aspnd1 expression in response to ambient pH. Regulation of Aspnd1 and Aspf2 expression by zinc deficiency may play an important role in pathogenesis. During infection, the host environment is one of nutritional limitation. In efforts to starve invading pathogens, part of the acute-phase response of the human immune system is to redistribute micronutrients like iron and zinc to the liver.26 Therefore, this regulation may illustrate the role of zincs nutritional status as a signal for fungal pathogens of a host environment, initiating transcription of genes involved in zinc acquisition and transport or commencement of pathogenesis. Bacterial

PAGE 18

10 hemolysins offer precedent for the metalloregulation of virulence factors.27 The partial regulation of Pra1 expression by nutritional status may also have important implications in pathogenesis. The effect of zinc limitation on Pra1 expression may be worth investigation, since zinc deficiency has been shown to induce mycelium formation in several dimorphic yeasts.4;28 Zps1p from Saccharomyces cerevisiae Within the family of cell wall proteins, the homologue from Saccharomyces cerevisiae is Zps1p (Yol154w). Although S. cerevisiae is typically nonpathogenic, it is able to infect immunocompromised individuals29 and colonize complement factor five-deficient mice.30 Zps1p is a secreted cell wall protein,31 with two putative N-glycosylation sites and six cysteine residues conserved with respect to Pra1, Aspnd1, and Aspf2. Unlike the homologues from Candida and Aspergillus spp., Zps1p has a truncated C-terminal region lacking the potential cell wall binding domain and the conserved antigenicity determinant. Like its related fungal antigens, the function of Zps1p is also unknown. Disruption of the ZPS1 gene failed to reveal any strong phenotype and resulted in a viable strain, indicating that Zps1p is non-essential.32 Although the function of Zps1p is unknown, many research groups have provided information about its regulation. Interestingly, Zps1p expression is regulated by some of the same factors as the other fungal antigens, including zinc limitation and extracellular pH. In S. cerevisiae, the transcription factor Zap1p is activated by zinc deficiency.33 DNA microarray data has shown Zap1p regulates expression of 46 genes in S. cerevisiae under zinc deficient conditions. Of these genes, one of the most heavily induced is ZPS1. This result was confirmed by measuring zinc-regulation of a ZPS1-lacZ reporter

PAGE 19

11 construct, resulting from fusion of the ZPS1 promoter region (-1000 bp to ATG) to the lacZ reporter gene. Zap1p activates gene transcription during zinc deficiency by binding to a zinc response element (ZRE) upstream of the target genes start codon. The consensus ZRE recognized by Zap1p is ACCTTNAAGGT. Within the ZPS1 promoter region are two putative ZREs between -300 and -340 bp upstream of the start codon: ACCTTCAGGGT (-328 to -318) and ACCCTGAAGGT (-313 to -303). DNA microarray data indicated that ZPS1 was induced 14 fold by zinc deficiency, while ZPS1-lacZ fusion constructs were 10 times more inducible.34 Expression of Zps1p is also affected by alkaline pH. This regulation is dependant on the S. cerevisiae Rim101p transcription factor, which is homologous to the Rim101p and PacC transcription factors of Candida and Aspergillus, respectively. Using the ZPS1-lacZ construct, researchers observed a 100-fold increase in expression at pH 8 compared to pH 4, while alkaline induction did not occur in a rim101 strain. In addition, ZPS1 is more highly expressed in yeast strains harboring a hyperactive allele of Rim101p at pH 4.35 This direct regulation of ZPS1 expression by Rim101p is intriguing, for not only is Rim101p structurally similar to Zap1p, but these two proteins also interact in vivo,36 suggesting they may co-regulate ZPS1 expression. Potential regulation of ZPS1 by environmental iron status has also been implied in work studying iron-regulatory systems in yeast.37 In S. cerevisiae, iron homeostasis is regulated by the Aft1p transcription factor in response to low-iron conditions.38 In addition, S. cerevisiae contains a homologue of Aft1p, known as Aft2p, which regulates transcription of many of the same genes as Aft1p during iron deficiency.37 In strains harboring a hyperactive allele of Aft2p, activation of ZPS1 was increased over 8 fold

PAGE 20

12 when compared to wild type strains. This effect is dependent on Zap1p, suggesting Aft2p activity affects zinc metabolism.37 The data obtained through study of ZPS1 regulation further support its similarity to the fungal antigens from C. albicans and Aspergillus spp. Zinc-dependent Metalloproteases of the M35 Clan Based on sequence comparison and structural predictions, the Zps1p-like proteins show similarity to zinc-dependent metalloproteases of the M35 clan5 (known as the aspzincins). Included in this subfamily of secreted metalloendopeptidases (MEPs) are deuterolysin (neutral proteinase, NPII, aspzincin) from Aspergillus oryzae,39 penicilloysin (PlnC) from Penicillium citrinum,40 mep20 from both Aspergillus fumigatus and Aspergillus flavus,41 and the AVR Pi-ta avirulence determinant from Magnaporthe grisea.42 Many of these species are known pathogens. This protein family is characterized by a leader sequence directing the protein into the secretory pathway, a long pro-peptide that is cleaved during secretion, a mature polypeptide that contains three disulfide bonds, and two highly conserved motifs: HExxH and GTxDDxxYG.43 A crystal structure of deuterolysin,43 supported by site directed mutagenesis studies,39 indicated the two histidine residues of the HExxH motif, the second aspartate residue of the GTxDDxxYG motif, and two water molecules were the zinc binding ligands. The conserved glutamate is a catalytic residue, promoting the nucleophilic attack of a water molecule on the carbonyl moiety of the substrate. The conserved tyrosine residue interacts with the second zinc bound water molecule, possibly stabilizing the transition state by hydrogen bonding interactions.43 Figure 1-2 shows the crystal structure of the active site residues.

PAGE 21

13 Figure 1-2. Active-site residues of deuterolysin.43 More distantly related members of the M35 clan include GfMEP from Grifola frondosa, PoMEP from Pleurotus ostreatus,44 AmMEP from Armillariella mella,45 eprA1 from Aeromonas hydrophila,46 asaP1 from Aeromonas salmonicida,47 XAC2763 from Xanthomonas axonopodis, and XCC2062 from Xanthomonas campestris.48 Many of these species are also pathogenic. Furthermore, AmMEP from the edible mushroom A. mella is known to hydrolyze fibrinogen.45 The crystal structure of GfMEP has been solved. Despite one less disulfide bond, GfMEP possesses a near identical fold and active site as deuterolysin, suggesting a conserved mechanism.49 The substrate specificities of deuterolysin, PlnC, and mep20 are toward basic polypeptides. Both deuterolysin and PlnC show high activities on the basic nuclear proteins histone, protamine, and salmine, but very low activities on milk casein, hemoglobin, albumin, and gelatin.39 Further analysis of deuterolysins substrate specificity indicates high proteolytic activity toward the peptide bonds next to pairs of basic residues.50 GfMEP and PoMEP have strict specificity toward acyl-lysine bonds, also basic in nature.44 Analysis of the GfMEP structure reveals and electrostatically negative region that attracts a positively charged lysine side chain of a substrate.49

PAGE 22

14 Comparison of the Zps1p-like Proteins and the M35 Metalloproteases Although Zps1p and the related fungal antigens possess similarities to the M35 clan (i.e., secretory signal, conserved cysteine residues), they differ in their most highly conserved motifs. Figure 1-3 illustrates the basic structural features of the Zps1p-like proteins and the M35 proteases. Figure 1-3. Basic structural features of the Zps1p-like proteins and the metalloproteases in the M35 clan. The Zps1p-like proteins lack the HExxH and GTxDDxxYG motifs found in the metallo-proteases. However, they contain highly conserved HRxxH and D/ExxD/E motifs, which may serve as functional replacements enabling metal binding and potentially proteolytic activity. Figure 1-4 compares the known active site structure of the metalloproteases in the M35 clan with a possible structure in the Zps1p-like proteins.

PAGE 23

15 Figure 1-4. Active site structures. On the right is the known active site of the aspzincins, deduced from the crystal structures of deuterolysin43 and GfMEP.49 On the left is a possible structure of an active site within the Zps1p-like proteins. Despite their similarity to the M35 metalloproteases, it is quite possible that the Zps1p-like proteins do not act as metal-binding proteins or possess proteolytic activity. However, the HRxxH and D/ExxD/E motifs highly conserved within the Zps1p family of cell wall proteins may act as peptide binding ligands, enhancing potential virulence within a host. The significance of these motifs can be thoroughly probed through study of purified Zps1p structure and function.

PAGE 24

CHAPTER 2 RESULTS AND DISCUSSION Regulation of ZPS1 Gene Expression ZPS1 regulation in S. cerevisiae was studied using the ZPS1-lacZ reporter construct. -Galactosidase activity, reported in Miller units, was measured as a function of growth condition. To confirm the dependence of Zap1p on ZPS1 regulation, we monitored the responsiveness of the ZPS1-lacZ reporter to zinc deficiency. Simultaneously, we further probed the apparent regulation by iron status by measuring activity of the ZPS1-lacZ reporter in response to growth under iron deficient conditions and combined zincand iron-limitation (Figure 2-1). 050010001500200025003000350040004500Fe-/Zn-Fe+/Zn-Fe-/Zn+Fe+/Zn+Growth Condition-Galactosidase Activity(Miller Units) WT zap1 Figure 2-1. Zinc and iron responsiveness of the ZPS1-lacZ reporter. -Galactosidase activity in wild-type cells and zap1 mutant cells grown in CSD with or without 10M iron and/or zinc added. The ZPS1-lacZ reporter construct was indeed regulated by zinc in a Zap1p dependent manner, with no detectable expression in a zap1 knockout strain. Induction was only observed when the yeast were grown under zinc-deficient conditions, with no measurable increase in ZPS1-lacZ activity when the yeast were grown under solely iron 16

PAGE 25

17 deficient conditions. When the yeast were grown under both zincand iron-limitation, a significant increase in ZPS1-lacZ activity was observed. As the literature previously suggests, the increased ZPS1 expression by iron-deficiency may be due to Aft2p.37 To further investigate this hypothesis, future work may involve monitoring ZPS1 expression in strains lacking Aft2p, Aft1p, or both. Previously, reports have described ZPS1 regulation by Rim101p in response to alkaline pH.35 Therefore, we attempted to study ZPS1-lacZ activity in response to iron and/or zinc deficiency at both acidic and alkaline pH. Under standard growth conditions, the Chelex-treated synthetic defined medium (CSD) used to limit zinc and iron availability is at pH 4 (optimal for yeast growth). When the CSD medium was buffered to pH 8.0, the metals in the medium became insoluble and precipitated out of solution. Therefore we were limited to monitoring the effects of Rim101p on ZPS1-lacZ expression at acidic pH (Figure 2-2). When compared to wild type yeast, strains lacking Rim101p exhibit a significant decrease in ZPS1-lacZ activity, which remained a function of zinc-deficiency. This result suggests that Rim101p affects ZPS1 expression even at acidic pH, possibly by enhancing Zap1p regulation of this gene. These observations further support the hypothesis that Zap1p and Rim101p co-regulate ZPS1. Partial Purification of Zps1p from Inclusion Bodies We are currently attempting to purify recombinant Zps1p from Escherichia coli for use in characterizing Zps1p structure and function. Although Zps1p is not native in E. coli, expression of yeast proteins in bacterial systems has several advantages, including high yield and lack of glycosylation moieties, which may complicate protein purification.

PAGE 26

18 050010001500200025003000350040004500Fe-/Zn-Fe+/Zn-Fe-/Zn+Fe+/Zn+Growth Condition-Galactosidase Activity(Miller Units) WT rim101 Figure 2-2. Zinc and iron responsiveness of the ZPS1-lacZ reporter. -Galactosidase activity in wild-type cells and rim101 mutant cells grown in CSD (pH 4.0) with or without 10M iron and/or zinc added. As described in the Materials and Methods section, ZPS1 (lacking the leader peptide sequence) has been cloned by the polymerase chain reaction (PCR) and inserted into the pET-22b(+) expression vector for isopropyl--D-thioglactopyranoside (IPTG) inducible expression by bacteriophage T7 RNA polymerase in BL21(DE3) E. coli. When expressed, the mature form of Zps1p should have an apparent molecular weight of ~ 25.5 kDa. Approximately eight hours of induction by IPTG is required for optimum Zps1p yield (Figure 2-3). Following large scale induction (as described in Methods section), the resulting pellet was thawed and resuspended in 20 mL of cold 50 mM Tris(hydroxymethyl) aminomethane (Tris), buffered at pH 7.4, containing 1 mM phenylmethanesulfonly fluoride (PMSF), a protease inhibitor used to prevent degradation of Zps1p. The cells were lysed using several cycles of French press at 4oC. Multiple rounds of French press were required to adequately break the large cell pellet resulting from 8 h of growth. The resulting lysate was centrifuged at 19,000 rpm for 20 min at 4oC and the supernatant was decanted and saved.

PAGE 27

19 Figure 2-3. SDS-PAGE analysis of E. coli transformants containing the pET-22b(+)-ZPS1 expression vector not induced (lane C) or induced with IPTG for the number of hours indicated (lanes 2 8 h). For comparison, products from non-induced cells after 8 h growth are also shown (lane C2). In lane M is a molecular weight marker. At this time, the soluble (supernatant) and insoluble (pellet) components of the lysate were analyzed by SDS-PAGE to determine the location of Zps1p (Figure 2-4). Zps1p was present in the insoluble fraction, indicating the protein accumulates as inclusion bodies (dense aggregates of misfolded polypeptide). Formation of recombinant Zps1p inclusion bodies is not unexpected, given that expression of recombinant Aspnd1p in E. coli also results in inclusion body formation.15 To solubilize the inclusion bodies, 5 mL of 50 mM Tris (pH 7.4) containing 8 M Urea was used to denature the Zps1p aggregates by gentle mixing overnight. Next, we attempted to refold the denatured protein by single-step dilution. This entailed slowly (> 24 h) dripping 50 mL of buffer into the sample so to gradually decrease the concentration of Urea to ~ 0.7 M. Dilution was followed by dialysis to remove all traces of the denaturant and the sample was centrifuged at 8,000 rpm for 20 min at 4oC to collect any insoluble material. The soluble and insoluble components were analyzed for Zps1p

PAGE 28

20 content by SDS-PAGE (Figure 2-5). The results indicated Zps1p was successfully solubilized, with only trace amounts in the insoluble fraction. Figure 2-4. SDS-PAGE analysis of soluble and insoluble components of the cell lysate obtained from breakage of E. coli expressing Zps1p. Lane M, molecular weight marker; Lane C, non-induced E. coli (8 h growth); Lane I, IPTG induced E. coli (8 h growth); Lane S, soluble fraction; Lane P, insoluble fraction. In attempts to load the maximum sample volumes to each lane, runoff into neighboring lanes occurred (Lanes X). Figure 2-5. SDS-PAGE analysis of the soluble and insoluble products obtained after solubilization and refolding of the inclusion body pellet. Lane M, molecular weight marker.

PAGE 29

21 Next, we attempted to purify the soluble Zps1p by size-exclusion chromatography (SEC). The protein sample was concentrated, applied to a column containing Sephadex G-75 (Sigma) size-exclusion resin (molecular weight cutoff ca. 80 kDa), with a bed volume of approximately 310 mL. The protein was eluted using 50 mM Tris (pH 7.4). After elution of the void volume, 2 mL fractions were collected and analyzed for protein using the method of Bradford.51 The Bradford protein assay indicated that the protein eluted as one major peak (fractions 9 12) shortly after collection of the void volume. Fractions 9 12 were combined and the content was analyzed by SDS-PAGE (Figure 2-6). Figure 2-6. SDS-PAGE analysis of the major protein peak collected after SEC (combined fractions 9 12). Lane C, non-induced E. coli (8 h growth); Lane I, IPTG induced E. coli (8 h growth); Lane E, protein eluate from SEC column. The results indicated poor separation of Zps1p from contaminating proteins. The lack of separation may result from aggregation of Zps1p with other peptides, possibly due to unfavorable disulfide bridging involving one of Zps1ps six cysteine residues. Therefore, use of a reducing agent such as dithiothreitol (DTT) during the refolding,

PAGE 30

22 concentrating, and chromatographic steps may prove effective in decreasing unfavorable disulfide bond formation. To reduce the concentration of contaminating proteins that could unfavorably interact with Zps1p forming aggregates, a purification strategy was adapted from a published method.52 This method involves washing the inclusion body pellet with the detergent sodium deoxycholate (DOC) to remove impurities. First, the frozen cell pellet obtained after IPTG induction was thawed and resuspended in 20 mL of cold Buffer A (5% Glycerol, 50 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl) containing 1 mM PMSF and 0.1 mM DTT. The cells were lysed using several cycles of French press at 4oC. Next, DOC was added to the lysate to give a concentration of 0.2% (approximately 240 L of a 20% DOC stock), which is used to help liberate slightly insoluble proteins. The solution was mixed well, allowed to stand for 10 min at room temperature, and centrifuged at 13,000 rpm for 10 min at 4oC. The supernatant was decanted and saved for future analysis (Supernatant 1). Following collection by centrifugation, the inclusion body pellet appears as a white bulls-eye, which is the inclusion body protein, surrounded by a brownish layer. The brownish layer consists of contaminating cellular debris that can be effectively solubilized by washing the pellet with 2% DOC. Therefore, the inclusion body protein was washed by resuspending the pellet in 18 mL of Buffer A (containing 1 mM PMSF and 0.1 mM DTT) and 2 mL of 20% DOC. The solution was allowed to stand for at least 10 min at room temperature before being centrifuged at 13,000 rpm for 10 min at 4oC. The supernatant was decanted and saved for future analysis (Supernatant 2). The remaining pellet was washed one additional time and, after centrifugation, the

PAGE 31

23 supernatant was decanted and saved for future analysis (Supernatant 3). At this time, the washed inclusion body pellet was solubilized by resuspending in 5 mL Buffer A containing 8 M Urea and gently agitated overnight at 4oC. Prior to refolding by single-step dilution, the protein purity was assessed by SDS-PAGE to determine the effectiveness of the DOC wash (Figure 2-7). The gel showed few major bands, one being Zps1p, thus demonstrating the value of the DOC wash in purifying the inclusion body protein. Figure 2-7. SDS-PAGE analysis of the washed inclusion body pellet, solubilized in Buffer A containing 8 M Urea (Lanes P). Lane M, molecular weight marker; Lane C, non-induced E. coli (8 h growth); Lane I, IPTG induced E. coli (8 h growth). Next, we attempted to refold the solubilized inclusion body protein by single-step dilution using Buffer A. As before, this procedure involved slowly decreasing the concentration of Urea by dilution followed by dialysis. In an attempt to discourage unfavorable disulfide bridging, 0.1 mM DTT was added to the buffer during the refolding process. After dialysis, the sample was centrifuged at 8,000 rpm for 20 min at 4oC to pellet insoluble materials, and the supernatant was decanted and concentrated. The supernatant and pellet collected after refolding were analyzed by SDS-PAGE for protein content, as were the soluble fractions (Supernatant 1 3) collected after each treatment

PAGE 32

24 with DOC (Figure 2-8). Unfortunately, refolding was unsuccessful and Zps1p was present in the insoluble fraction. Figure 2-8. SDS-PAGE analysis of the soluble and insoluble products obtained after solubilization and refolding of the inclusion body pellet. Also shown are the supernatant fractions collected after each purification step. Lane M, molecular weight marker; Lane C, non-induced E. coli (8 h growth); Lane I, IPTG induced E. coli (8 h growth); Lane 1, Supernatant 1 (post-lysis); Lane 2, Supernatant 2 (after first DOC wash); Lane 3, Supernatant 3 (after second DOC wash); Lane S, soluble fraction (after refolding); Lane P, insoluble fraction (after refolding). It is unclear why the solubilized inclusion body protein failed to refold despite its improved purity and the addition of DTT (to prevent unfavorable disulfides). One possible explanation is that the rate of dilution was accelerated due to poor control of the flow rate. It is critical that the rate of dilution is slow. At high denaturant concentrations, the unfolded protein is well solvated and flexible. Rapidly altering solvent dynamics toward an aqueous environment forces the protein to collapse into a compact and rigid structure. Unfortunately, the resulting structure is often misfolded or aggregated and therefore insoluble. Gradual dilution allows for refolding at intermediate concentrations of urea, where the denaturant concentration is low enough to force protein molecules to collapse, yet allowing flexible motion enabling proteins to reorganize their structures and stay in solution. Therefore, it may be beneficial to alter the refolding strategy so to

PAGE 33

25 provide a slower and more controlled rate of denaturant dilution. Alternative refolding strategies include, but are not limited to: one-step dialysis, step-wise dialysis, and buffer-exchange by gel filtration.53 Although expressing recombinant Zps1p from E. coli is advantageous due to high protein yield, it is possible the protein will not properly refold after solubilization from inclusion bodies. If future efforts to refold and purify Zps1p from E. coli inclusion bodies are unsuccessful, it may be necessary to purify Zps1p directly from S. cerevisiae.

PAGE 34

CHAPTER 3 CONCLUSIONS The work presented represents initial steps toward characterization of Zps1p from S. cerevisiae. ZPS1 expression is regulated by extracellular pH and zinc-deficiency, environmental signals known to elicit Zps1p-like antigen production in Candida and Aspergillus spp., respectively. These results suggest that the regulation, and consequently function, of the cell wall proteins is conserved among these fungi. This hypothesis is supported by their high sequence homology. Because of their localization within the fungal cell wall and the observed binding of Pra1 and Aspnd1 to host molecules, the Zps1p-like proteins in C. albicans and Aspergillus spp. are believed play a role in establishing infection. The Zps1p-like proteins may function as virulence factors by mediating critical host-parasite interactions or through involvement in morphological processes. Therefore, future characterization of Zps1p may include investigating the proteins potential role in fungal cell-cell adhesion (flocculation) or adherence to host ligands (e.g.., ECM or serum proteins). The partial purification of recombinant Zps1p from bacterial inclusion bodies is an important first step toward characterization of Zps1p. Once purified, a wealth of information can be obtained by studying both Zps1p structure and function. Due to similarities between the Zps1p-like proteins and zinc-dependent metalloproteases, future work using purified Zps1p ought to include metal binding studies and testing for proteolytic activity towards a variety of substrates. 26

PAGE 35

CHAPTER 4 MATERIALS AND METHODS Growth Media For standard growth of E. coli, LB medium was used. The recipe per liter is 10 g NaCl, 10 g Bactotryptone, and 5 g Yeast Extract. When required, ampicillin was added to a final concentration of 200 g/mL. In preparation of plates, 15 g of agar was added per liter. YPD medium was used for routine, non-selective yeast growth. The recipe per liter is 10 g Yeast Extract, 20 g Bactopeptone, and 20 g Dextrose. In preparation of plates, 15 g of agar was added per liter. For maintenance of recombinant yeast strains, selective (SD) medium was used. The base recipe per liter is 5 g (NH4)2SO4, 20 g Dextrose, and 1.7 g Yeast Nitrogen Base without amino acids or (NH4)2SO4 (Difco; Sparks, MD). To satisfy the auxotrophic strains used in this study, the medium was supplemented with 0.1 g L-Histidine, 0.1 g L-Leucine, and 0.1 g L-Lysine per liter. Although the strains required Uracil, this was omitted from the medium for selective growth. This medium will be referred to as SD-Ura. For plates, 15 g of agar was added per liter. To limit zinc and iron availability, Chelex-treated synthetic defined medium (CSD) was used. The recipe per liter, using H2O at 18 M purity, is 20 g Dextrose, 5.1 g Yeast Nitrogen Base without amino acids or divalent cations or potassium phosphate (Bio101; Vista, CA), and 0.1 g each of L-Histidine, L-Leucine, and L-Lysine. Again, for selective purposes, Uracil was omitted from the medium. To remove metals from the 27

PAGE 36

28 media, 25 g of Chelex-100 ion exchange resin (Sigma) was added, and the mixture was stirred for a minimum of 2 h. After removal of the resin, 10 mL of potassium phosphate monobasic (100 g/L) was added and the pH was adjusted to 4.0 using HCl. Next, divalent metal ions were added to the medium to the following concentrations (as recommended by Bio101): 0.4 mg/L MnSO4, 0.04 mg/L CuSO4, 100 mg/L CaCl2, and 500 mg/L MgSO4. The resulting solution was filter sterilized into a polycarbonate flask washed with Acationox detergent (Baxter Scientific Products; McGraw Park, IL). The resulting solution contains residual zinc and iron at concentrations less than 100 nM (approximate value), and is referred to as CSD-Ura(-Zn/-Fe). For zinc or iron replete medium, the desired metal is added back to the medium to a final concentration of 10 M. Solutions and Buffers for Yeast Transformations and -Galactosidase Assays 10x TE (250 mL): 100 mM Tris and 10 mM EDTA pH 7.5. Sterilize. LiTE solution (50 mL): 5 mL sterile 10x TE, 5 mL sterile 1 M Lithium Acetate 40 mL sterile H2O. PEG-LiTE solution (50 mL): 5 mL sterile 10x TE, 5 mL sterile 1 M Lithium Acetate, 40 mL sterile 44% (w/v) PEG-3350

PAGE 37

29 Carrier DNA (10 mL): 100 mg salmon testes DNA and 10 mL ultrapure H2O. Shear DNA by drawing the mixture up into a 10 mL syringe with an 18g needle 15 times, boil, and restore volume to 10 mL. Store as 1 mL aliquots at -20oC. Z-buffer, pH 7.0 (1 L): 0.06 M Na2HPO4, 0.04 M NaH2PO4-H2O, 0.01 M KCl, 0.001 M MgSO4. Bacterial and Yeast Strains Listed below are the bacterial and yeast strains used in this work. Table 4-1. Yeast strains used for the work described. Yeast Strain Mutation Source Genotype BY4742 Wild type MAT ; his3; leu2; ura 3; lys2 Y11367 zap1 EUROSCARF MAT ; his3; leu2; ura 3; lys2 zap1::kanMX4 Y10936 rim101 EUROSCARF MAT ; his3; leu2; ura 3; lys2 rim101::kanMX4 Table 4-2: Bacterial strains used for the work described. E. coli Strain Genotype TOP 10 FmcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 deoR recA1 araD139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG BL21(DE3) FompT hsdSB(rB-mB-) gal dcm (DE3)

PAGE 38

30 Yeast Transformations Using the lithium acetate method, yeast strains of interest were transformed with a plasmid containing the ZPS1-lacZ fusion (with Ura+ selection), which was previously constructed34 in YEp35354 by gap repair.55 This was accomplished by growing the yeast in 5 mL YPD at 30oC at 250 rpm overnight. The following day, 300 L of the overnight culture was transferred to a new tube containing 5 mL YPD and incubated for 2 hr. at 30oC at 250 rpm. Next, the cells were harvested by centrifugation at 3500 rpm for 3 min and the supernatant was decanted. The remaining cell pellet was washed by adding 5 mL LiTE solution and vortexing. Again, the cells were harvested by centrifugation (as described above). The cells were resuspended in residual LiTE solution by vortexing and 50 L of the cell suspension was transferred into a sterile 1.5 mL centrifuge tube. Added to the centrifuge tube containing cells were 2 L (~ 400 g) of plasmid DNA containing the ZPS1-lacZ fusion and 10 L (~ 10 g) of salmon sperm carrier DNA (boiled for 5 min and flash cooled on ice prior to use). Next, 500 L of PEG-LiTE solution was added. The mixture was vortexed briefly and incubated at 30oC at 250 rpm for 30 45 min. After incubation, the sample was heat shocked for 10 15 min at 42oC. The cells were pelleted at 4000 rpm for 1 min in a microcentrifuge. The supernatant was aspirated and 500 L LiTE solution was added to the pellet and subsequently vortexed. Finally, 50 200 L of transformant was plated on SD-Ura plates to (select for the YEp353 plasmid) and incubated at 30oC for 3 5 days. Plates which grew colonies were stored at 4oC for future use.

PAGE 39

31 -Galactosidase Assays A single colony of yeast transformed with the ZPS1-lacZ fusion plasmid was transferred to 5 mL of SD-Ura and incubated at 30oC at 250 rpm overnight. This liquid culture was used to inoculate metal-free 14 mL polystyrene tubes containing 5 mL of CSD-Ura with the appropriate combinations of zinc and iron as follows: -Zn/-Fe, 45 L cell culture; -Zn/+Fe, 30 L cell culture; +Zn/-Fe, 30 L cell culture; +Zn/+Fe, 20 L cell culture. The cultures were grown for 12 h and then stored on ice for approximately 20 min. Next, the cells were harvested by centrifugation for 3 min at 3500 rpm at 4oC. The supernatant was discarded and the resulting pellet was washed by adding 5 mL cold Z-buffer and vortexing. The cells were harvested by centrifugation (as above) and the supernatant was discarded. The cells were resuspended in 2 mL cold Z-buffer and 1 mL of the cell suspension was transferred to a 5 mL glass assay tube containing 50 L CHCl3 and 50 L 0.1% SDS. The contents of the tube were vortexed to permeablize the cells and then incubated at 30oC for 10 min to equilibrate. After incubation, the tube was vortexed vigorously for 3 sec and its contents (principally CHCl3) were allowed to settle for approximately 10 se. before transferring 100 L of the suspension to a 96-well plate (in triplicate). The assay reaction was initiated by adding 20 L of 4 mg/mL o-nitrophenyl--D-galactopyranoside (ONPG). The sample was mixed and the reaction was allowed to proceed until the darkest samples were an intense yellow color. The reaction was stopped by adding 50 L of 1 M Na2CO3 and the reaction time (min) was noted. The absorbance at 420 nm was measured (reference wavelength, 600 nm) using a SAFIRE microplate reader (Tecan) and XFLUOR software. The absorbance at 600 nm (OD600) of

PAGE 40

32 the remaining cell suspension (from above) was also measured using a BIO-RAD SmartSpecTM 3000 bench-top spectrophotometer. -galactosidase activity was measured in Miller Units using the method of Guarente,56 and activity units were calculated as follows: (A420 x 1000)/(min x mL of culture used x OD600). Cloning of ZPS1 and Construction of an E. coli Expression Plasmid The gene that encodes the mature form of Zps1p (lacking the signal peptide) was PCR cloned from S. cerevisiae strain BY4724 genomic DNA using forward and reverse primers containing Nde I and EcoR I restriction sites, respectively. The primers were obtained from Integrated DNA Technologies, Inc. (Coralville, IA) and were designed as follows: ZPS1 for: 5AAC TTT AAG AAG GAG ATA TAC ATA TGC CTG TCA CTT ACG ACA CCA A -3 ZPS1 rev: 5CAA GCT TGT CGA CGG AGC TCG AAT TCT TAC AAG TTA CCT AGA CAG C -3 The PCR reaction was catalyzed using Taq DNA polymerase and the thermocycling conditions employed were as follows: one cycle at 95oC for 3 min; and 25 cycles at 95oC for 30 sec, 50oC for 30 sec, 72oC for 1.5 min; and a final extension at 72oC for 8 min. The PCR product was digested for 4 h at 37oC using the restriction enzymes Nde I and EcoR I (New England Biolabs; Beverly, MA). The pET-22b(+) vector was obtained from Novagen (La Jolla, CA). The pET-22b(+) plasmid was also digested using Nde I and EcoR I (as described above). Following restriction digestion, the cut PCR and pET-22b(+) samples were subjected to agarose gel electrophoresis (0.8% agarose) and

PAGE 41

33 purified using the QIAquick Gel Extraction Kit, following the manufactures protocol (QIAGEN Inc.; Valencia, CA). These purified samples were subsequently used to ligate the cloned ZPS1 gene into the pET-22b(+) vector between the Nde I and EcoR I restriction sites using T4 DNA ligase (New England Biolabs), incubated overnight at 16oC. The ligation product was used to transform electrocompetent E. coli TOP10 cells by electroporation following standard procedures.57 The E. coli transformant (10 150 L) was plated on LB agar plates containing ampicillin (for plasmid selection) and incubated at 37oC overnight. Plates that grew colonies were stored at 4oC for future use. To obtain large quantities of the pET-22b(+)-ZPS1 construct, a single colony from the transformation product was used to inoculate 5 mL of LB medium containing ampicillin. The cells were grown at 37oC at 250 rpm overnight. Using the Promega (Madison, WI) Wizard Plus Miniprep DNA purification system, the pET-22b(+)-ZPS1 plasmid was purified from the overnight culture according to the manufactures directions. Using the purified plasmid, the sequence of the cloned ZPS1 gene was confirmed by the ICBR DNA sequencing core laboratory at the University of Florida. Expression of Zps1p in E. coli To obtain Zps1p using the T7 expression system, the pET-22b(+)-ZPS1 plasmid was transformed into BL21(DE3) E. coli by electroporation using standard methods. The E. coli transformant (10 150 L) was plated on LB agar plates containing ampicillin and incubated at 37oC overnight. Plates that grew colonies were stored at 4oC for future use. A single colony from the transformation product was used to inoculate 15 mL of LB medium containing ampicillin. The cells were grown at 37oC at 250 rpm overnight and 10 mL of culture was used to inoculate 1 L of LB medium containing ampicillin. The 1

PAGE 42

34 L culture was incubated at 37oC at 250 rpm for approximately 2 h until reaching an OD600 of 0.4 1.0. At this time, Zps1p expression was induced by addition of IPTG (Isopropyl--D-thioglactoside) to a final concentration of 1 mM. The culture was then incubated at 30oC at 250 rpm for 8 h. Finally, the cells were harvested by centrifugation (3000 rpm for 15 min at 4oC) and washed two times using 50 mM Tris (pH 7.4). The resulting pellet was stored frozen at -20oC overnight. Estimation of Protein Purity by SDS-PAGE SDS-PAGE gels containing 14% (w/v) polyacrylamide were prepared and analyzed by standard methods. Samples were prepared by adding equal volumes of 2x Laemmli sample buffer, boiling for 10 min, followed by centrifugation at 14,000 rpm in a microfuge for 1 min to pellet any insoluble debris. The gels were run at 70 V, using a Tris-glycine electrode buffer. All gels were stained with Coomassie blue.

PAGE 43

LIST OF REFERENCES 1) W. L. Chaffin; J. L. Lopez-Ribot; M. Casanova; D. Gozalbo; J. P. Martinez Microbiol Mol Biol Rev 1998, 62, 130-180. 2) F. De Bernardis; F. A. Muhlschlegel; A. Cassone; W. A. Fonzi Infect Immun 1998, 66, 3317-3325. 3) D. Davis Curr Genet 2003, 44, 1-7. 4) D. R. Soll Curr Top Med Mycol 1985, 1, 258-284. 5) N. D. Rawlings; E. O'Brien; A. J. Barrett Nucleic Acids Res 2002, 30, 343-346. 6) B. Banerjee; P. A. Greenberger; J. N. Fink; V. P. Kurup Infect Immun 1998, 66, 5175-5182. 7) J. A. Calera; M. C. Ovejero; R. Lopez-Medrano; M. Segurado; P. Puente; F. Leal Infect Immun 1997, 65, 1335-1344. 8) M. Sentandreu; M. V. Elorza; R. Sentandreu; W. A. Fonzi J Bacteriolo 1998, 180, 282-289. 9) A. Bouali; R. Robert; G. Tronchin; J. Senet J Gen Microbiol 1987, 133, 545-551. 10) J. L. Lopez-Ribot; P. Sepulveda; A. M. Cervera; P. Roig; D. Gozalbo; J. P. Martinez FEMS Microbiol Lett 1997, 157, 273-278. 11) M. Casanova; J. L. Lopez-Ribot; C. Monteagudo; A. Llombart-Bosch; R. Sentandreu; J. P. Martinez Infect Immun 1992, 60, 4221-4229. 12) F. M. Klis; P. Mol; K. Hellingwerf; S. Brul FEMS Microbiol Rev 2002, 26, 239-256. 13) R. Teshima; H. Ikebuchi; J. Sawada; S. Miyaachi; S. Kitani; M. Iwama; M. Irie; M. Ichinoe; T. Terao J Allergy Clin Immunol 1993, 92, 698-706. 14) B. Banerjee; V. P. Kurup; P. A. Greenberger; D. R. Hoffman; D. S. Nair; J. N. Fink J Allergy Clin Immunol 1997, 6, 821-827. 15) J. A. Calera; M. C. Ovejero; R. Lopez-Medrano; R. Lopez-Aragon; P. Puente; F. Leal Microbiology 1998, 144, 561-567. 35

PAGE 44

36 16) B. Banerjee; V. P. Kurup; S. Phadnis; P. A. Greenberger; J. N. Fink J Lab Clin Med 1996, 127, 253-262. 17) P. Sepulveda; J. L. Lopez-Ribot; A. Murgui; E. Canton; D. Navarro; J. P. Martinez Int Microbiol 1998, 1, 209-216. 18) A. Viudes; S. Perea; J. L. Lopez-Ribot Infect Immun 2001, 69, 2909-2919. 19) G. Tronchin; K. Esnault; G. Renier; R. Filmon; D. Chabasse; J. P. Bouchara Infect Immun 1997, 65, 9-15. 20) M. C. Penalver; J. E. O'Connor; J. P. Martinez; M. L. Gil Infect Immun 1996, 64, 1146-1153. 21) M. L. Gil; M. C. Penalver; J. L. Lopez-Ribot; J. E. O'Connor; J. P. Martinez Infect Immun 1996, 64, 5239-5247. 22) D. Davis; R. B. Wilson; A. P. Mitchell Mol Cell Biol 2000 20, 971-978. 23) D. Davis; J. E. Edwards, JR.; A. P. Mitchell; A. S. Ibrahim Infect Immun 2000, 68, 5953-5959. 24) M. Segurado; R. Lopez-Aragon; J. A. Calera; J. M. Fernandez-Abalos; F. Leal Infect Immun 1999, 67, 2377-2382. 25) S. H. Denison Fungal Genet Biol 2000, 29, 61-71. 26) J. A. Tayek; G. L. Blackburn Am J Med 1984, 81-88. 27) V. Braun; T. Focareta Crit Rev Microbiol 1991, 18, 115-158. 28) A. Alsina; N. Rodriguez-del Valle Sabouraudia: Journal of Medical and Veterinary Mycology 1984, 22, 1-5. 29) R. H. K. Eng; R. Drehmel; S. M. Smith; E. J. C. Goldstein Sabouraudia: J Med Vet Mycol 1984, 22, 403-407. 30) J. K. Byron; K. V. Clemons; J. H. McCusker; R. W. Davis; D. A. Stevens Infect Immun 1995, 63, 478-485. 31) H. Tershima; S. Fukuchi; K. Nakai; M. Arisawa; K. Hamada; N. Yabuki; K. Kitada Curr Genet 2002, 40, 311-316. 32) M. J. Lafuente; C. Gancedo Yeast 1999, 15, 935-943. 33) H. Zhao; D. J. Eide Mol Cell Biol 1997, 17, 5044-5052. 34) T. J. Lyons; A. P. Gasch; L. A. Gaither; D. Botstein; P. O. Brown PNAS 2000, 97, 7957-7962.

PAGE 45

37 35) T. M. Lamb; W. Xu; A. Diamond; A. P. Mitchell J Biol Chem 2001, 276, 1850-1856. 36) P. Uetz; L. Giot; G. Cagney; T. A. Mansfield; R. S. Judson; J. R. Knight; D. Lockshon; V. Narayan; M. Srinivasan; P. Pochart; A. Qureshi-Emili; Y. Li; B. Goodwin; D. Conover; T. Kalbfleisch; G. Vijayadamodar; M. Yang; M. Johnson; S. Fields; J. M. Rothberg Nature 2000, 403, 623-627. 37) J. C. Rutherford; S. Jaron; E. Ray; P. O. Brown; D. R. Winge PNAS 2001, 98, 14322-14327. 38) Y. Yamaguchi-Iwai; A. Dancis; R. D. Klausner Embo J 1995, 14, 1231-1239. 39) N. Fushimi; C. Ewe Ee; T. Nakajima; E. Ichishima J Biol Chem 1999, 274, 24195-24201. 40) K. Matsumoto; M. Yamaguchi; E. Ichishima Biochim Biophys Acta 1994, 1218, 469-472. 41) M. V. Ramesh; T. D. Sirakova; P. E. Kolattukudy Gene 1995, 165, 121-125. 42) M. J. Orbach; L. Farrall; J. A. Sweigard; F. G. Chumley; B. Valent Plant Cell 2000, 12, 2019-2032. 43) K. E. McAuley; Y. Jia-Xing; E. J. Dodson; J. Lehmbeck; P. R. Ostergaard; K. S. Wilson Acta Cryst 2001, D57, 1571-1578. 44) T. Nonaka; N. Dohmae; Y. Hashimoto; K. Takio J Biol Chem 1997, 272, 30032-30039. 45) J. Kim; Y. S. Kim Biosci Biotechnol Biochem 1999, 63, 2130-2136. 46) T. M. Chang; C. C. Liu; M. S. Chang Gene 1997, 199, 225-229. 47) U. Wagner; B. K. Gudmunsdottir; K. Drossier J Appl Microbiol 1999, 87, 620-629.

PAGE 46

38 48) A. C. R. da Silva; J. A. Ferro; F. C. Reinach; C. S. Farah; L. R. Furian; R. B. Quaggio; C. B. Monteiro-Vitorello; M. A. Van Sluys; N. F. Almelda; L. M. C. Alves; A. M. do Amaral; M. C. Bertolini; L. E. A. Camargo; G. Camarotte; F. Cannavan; J. Cardozo; F. Chambergo; L. P. Clapina; R. M. B. Ciarelli; L. L. Coutinho; J. R. Cursino-Santos; H. El-Dorry; J. B. Faria; A. J. S. Ferreira; R. C. C. Ferreira; M. I. T.Ferro; E. F. Formighieri; M. C. Franco; C. C. Greggio; A. Gruber; A. M. Katsuyama; L. T. Kishi; R. P. Leite; E. G. M. Lemos; M. V. F. Lemos; E. C. Locali; M. A. Machado; A. M. B. N. Madeira; N. M. Martinez-Rossi; E. C. Martins; J. Meidanis; C. F. M. Menck; C. Y. Miyaki; D. H. Moon; L. M. Moreira; M. T. M. Novo; V. K. Okura; M. C. Oliveira; V. R. Oliveira; H. A. Pereira; A. Rossi; J. A. D. Sena; C. Silva; R. F. de Souza; L. A. F. Spinola; M. A. Takita; R. E. Tamura; E. C. Teixeira; R. I.D. Tezza; M. Trindade dos Santos; D. Truffi; S. M. Tsai; F. F. White; J. C. Setubal; J. P. Kitajima Nature 2002, 417, 459-463. 49) T. Hori; T. Kumasaka; M. Yamamoto; T. Nonaka; N. Tanaka; Y. Hashimoto; T. Ueki; K. Takio Acta Cryst 2001, D57, 361-368. 50) Y. Doi; B. R. Lee; M. Ikeguchi; Y. Ohoba; T. Ikoma; S. Tero-Kubota; S. Yamauchi; K. Takahashi; E. Ichishima Biosci Biotechnol Biochem 2003, 67, 264-270. 51) M. Bradford Anal Biochem 1976, 72, 248-254. 52) D. R. Marshak; J. T. Kadonaga; R. R. Burgess; M. W. Knuth; W. A. Brennan, Jr.; S. Lin Strategies for Protein Purification and Characterization: A Laboratory Course Manual; Cold Spring Harbor Laboratory Press: Plainview, NY, 1996; pp 209-218. 53) K. Tsumoto; D. Ejima; I. Kumagai; T. Arakawa Protein Expression & Purification 2003, 28, 1-8. 54) A. M. Myers; A. Tzagaloff; D. M. Kinney; C. J. Lusty Gene 1986, 45, 299-310. 55) S. Kunes; H. Ma; K. Overbye; M. S. Fox; D. Botstein Genetics 1987, 115, 73-81. 56) L. Guarente Methods Enzymol 1983, 101, 181-191. 57) J. Sambrook; D. W. Russell Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001; pp A8.40-A8.47

PAGE 47

BIOGRAPHICAL SKETCH Stephanie L. Drobiak is from Brooklyn, Connecticut. She graduated from Wheaton College in Norton, Massachusetts, in 2001 with a Bachelor of Arts in biochemistry. In the fall of 2001, she entered the graduate program in the Department of Chemistry at the University of Florida. Upon completion of her masters, she will continue working towards her PhD at the University of Florida. 39


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20110115_AAAAEV INGEST_TIME 2011-01-16T01:49:08Z PACKAGE UFE0008961_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 68194 DFID F20110115_AADGMC ORIGIN DEPOSITOR PATH drobiak_s_Page_27.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
8121dc23395bfa6af5d930bbe9423e4b
SHA-1
b62f542c893ef5cbffe7ed7185369ddaef19add6
1053954 F20110115_AADGHF drobiak_s_Page_07.tif
804334b93a7b24522ddd014eb990c111
5b2721e650555e0876a6364f1f1429ffc391e9d5
6318 F20110115_AADGRA drobiak_s_Page_21thm.jpg
2090266c68219816da5a0734ddbf2974
c989cf735b207536ac604a50ce5f267aaa3251f0
5069 F20110115_AADGMD drobiak_s_Page_36thm.jpg
1671a461ccbb40b9c3839588b5338d84
4d51a06868d8b8bc1c5620310fcead7f1ce1b1c3
59641 F20110115_AADGHG drobiak_s_Page_35.jpg
9bf341cdb7bd34c6c831f9e6be92d0ca
04e94ab8f67ae63e2481da68c2da5324f1c1d198
21002 F20110115_AADGRB drobiak_s_Page_26.QC.jpg
8917807c827ac85f7ff733bf97fc18f1
d7630e7c8fc75a3a08ffeaf1c82052b878846195
F20110115_AADGME drobiak_s_Page_39.tif
b113580ac0d7b1c7ac26ecb04b5b31f3
c3113157d896edcb504e8c2096297c75340edc58
68308 F20110115_AADGHH drobiak_s_Page_31.jpg
686dc89b09b46933d5b8cd04c9a43d65
c233b226dfe2f3d42a50e5adf3ec8f03c036eb98
5896 F20110115_AADGRC drobiak_s_Page_26thm.jpg
f6e486df48beeccd8601820c41aedfc1
22c70c178abe79b6bfc91719e87f079ebbd87ece
63045 F20110115_AADGMF drobiak_s_Page_37.jp2
dad88cb17599cbd40028d0b8c7cd42cd
cc25c7294e18bda2e3ede112b5ec9a533f3efefa
21032 F20110115_AADGHI drobiak_s_Page_27.QC.jpg
fc8d40fe32612c9ee6c554b33c48b23d
cc608a2b0bf5e45fc29afb7c15b8a447ebeec14a
6089 F20110115_AADGRD drobiak_s_Page_27thm.jpg
32501858b934cbc1004b0069ec2ae48f
7efa71dccc7e7149933401b58de9c199935eefca
72601 F20110115_AADGMG drobiak_s_Page_16.jpg
57bc8a094e6eca51f61b5fda66ec4a24
99c11a5fa095750395314626836e18d29c95b799
110460 F20110115_AADGHJ drobiak_s_Page_12.jp2
001e336fad41548097bdeffa87ef3a1f
bea808200b7c2201f30c4b5cec9a45736b409a0d
19762 F20110115_AADGRE drobiak_s_Page_29.QC.jpg
d575f6c4a1fdfc849eec9296077e1ab1
d5835af73fe168bf32c3b7cc697f1ca3a2fd91a8
1051899 F20110115_AADGMH drobiak_s_Page_27.jp2
28b9f91f99a4d967ab9040e15929c3db
4885cd8ee5b449b4eed1070b4f20addc01fb67b3
6527 F20110115_AADGHK drobiak_s_Page_17thm.jpg
84ffb270ea8b82bbcb39c1616bc8db62
0c89ad928c8562bf7cd4428c8ae0a0e65244b006
6310 F20110115_AADGRF drobiak_s_Page_30thm.jpg
781afec6c3acf6e13ce292f208139eb2
82485e655d673b889436eac6850a762b78620758
38840 F20110115_AADGMI drobiak_s_Page_07.pro
22108fcdc08896e924a5a2c4e310b556
dba041a06e4ed514722716550a7d15b694b02f7a
16813 F20110115_AADGHL drobiak_s_Page_36.QC.jpg
e09ba80b17fde68baddef139939941ab
0da26dc1b9dd0a4c7b0df2ffef1edb66343e1a21
23213 F20110115_AADGRG drobiak_s_Page_32.QC.jpg
82be7e492bc47a2bc49b777ae0494dc8
0713a00b6881928b12e486c18e078a1c51833f94
52296 F20110115_AADGMJ drobiak_s_Page_28.jpg
8f7a6fb10318385d4b56331a3a241b31
a5fce0b5294dc5eebbaae37007d0ac7e95bf7c5f
17829 F20110115_AADGHM drobiak_s_Page_05.QC.jpg
b9f67bfef811004cfcbc263c55e6353d
a4c79af1fb1d6677853ef856222ce7dd0384c49e
19532 F20110115_AADGRH drobiak_s_Page_35.QC.jpg
ef6d0ec426e97f7298130f1a1b51a837
64b19faf2902222b50a518c5be7da5b6ff6945b5
5874 F20110115_AADGMK drobiak_s_Page_06thm.jpg
e663a860c41c5351732aabbb7fb944a4
b216a11f11057b602f4a3d97295ed8eb7804161a
70006 F20110115_AADGHN drobiak_s_Page_30.jpg
df08880e2ddf62124b9c85642854cd39
fb7ad7e0d907cb9e90b8ebb8fcd7109bd31a6eab
13454 F20110115_AADGRI drobiak_s_Page_42.QC.jpg
2d1479a0d7c11ae3bbeeb94b044ec4b6
01893823002154aab037fe0349851a2800331a61
79467 F20110115_AADGML drobiak_s_Page_06.jpg
b526d68547ebd60c6d8f8306c5f67a4b
56d8b96d0e9d65c04270e3d5100a677360f58d3a
3854 F20110115_AADGRJ drobiak_s_Page_42thm.jpg
54b469b3873b1c0853079a149825ea54
0a38035d6f65e51f63fce0c317d881a97d70f003
17993 F20110115_AADGMM drobiak_s_Page_24.QC.jpg
fc52203bf1ecc7c0909ba1bd07e24dcb
28f4dc7c1ce6c25a8a7de24e30f1e485a6a26b99
20968 F20110115_AADGHO drobiak_s_Page_38.QC.jpg
52b7ebf1287b3e7bd2c11112cefafbb2
63010dbf69bec16ca17b163e8b998e7ed700f871
38813 F20110115_AADGMN drobiak_s_Page_27.pro
79ea818b0443ecde9af3113db10343bd
8272a2386b65045cf85b97f8c639719858a91e0a
21286 F20110115_AADGHP drobiak_s_Page_09.QC.jpg
870b0213383a41df645fd1c76f5ced18
fd23ce58518a5795108d63d9a40d443b104d2cbf
22276 F20110115_AADGRK drobiak_s_Page_44.QC.jpg
8b7adf016e8c395087d35bee78feee02
9c9d73d4a16dbadf858a289db0b88e8adb55d702
F20110115_AADGMO drobiak_s_Page_13.tif
f2471a89a403af3e8d3ac0645095bb40
63631803d019e6cb9b85ecccedc2e6a542e7997f
100396 F20110115_AADGHQ drobiak_s_Page_44.jp2
fe0efa9fd0a0545fc80ebf6284a009b8
c81ea0490f2795e02fb46a8b0a8e3baaf365e57a
57189 F20110115_AADGRL UFE0008961_00001.mets FULL
deeb3f2a8df788ae500c40fbe812742c
a7db84be4b51c93b262c6611b2878f780f3a5bb1
6379 F20110115_AADGMP drobiak_s_Page_32thm.jpg
5e1a4a3ac39f3fc5f6dfc175e6a3b804
08ed3744a3e26fe114c204d022ab7f2b3804a117
40130 F20110115_AADGHR drobiak_s_Page_35.pro
06db2a2556842f1ff4abb0d5f726695f
4a7f14e0b08dd46651ccec7515d21550e836d9b8
5407 F20110115_AADGMQ drobiak_s_Page_04.QC.jpg
36be36b22e3392cc0c24047cc8f6915f
210fe564043f092957baf53180f08485faa0b9bd
1715 F20110115_AADGHS drobiak_s_Page_43.txt
edbdcc7e2a7e0725c5daa1c0ab90e87d
7972c32d867964af8fbc2e40797a9815a20a39bc
1051932 F20110115_AADGMR drobiak_s_Page_31.jp2
9a1b0045fd621d375bcc2c43327872ba
723059591e688cad0edde8876df9d5c887f04b6c
22856 F20110115_AADGHT drobiak_s_Page_30.QC.jpg
19542c21594eedeadd0c9521b1e1c7cd
4548beedadd8a5ed05be735eecdc866d5e0fed07
65340 F20110115_AADGMS drobiak_s_Page_05.jpg
3dab323e53da56c36874c17c9ddeb7a4
817e84750daf96d4be590f3f31d2cfd241244470
F20110115_AADGHU drobiak_s_Page_37.tif
570f28a21cdc2c39f2ac4e28f627acec
192502c1ba7b5b3d4c3a4a90feaf5c0535da4286
1590 F20110115_AADGHV drobiak_s_Page_03thm.jpg
b12964b6f940fecb8eafca49a28234ce
44760fcc6be03518d64e8425c579266518635990
F20110115_AADGMT drobiak_s_Page_18.tif
b47f58afeb4597225668585b0b666f16
c2f1003450c2ed2d0739df764915fa31cf37892f
205 F20110115_AADGHW drobiak_s_Page_03.txt
b427f676f789bc18fd25482fabc35585
5565b236fe541a24ad24630ef0abd04a3231da44
1627 F20110115_AADGMU drobiak_s_Page_24.txt
9e053da7d0e6c493c143d99a77be87e3
140675d9cedf6a711466b66ef8a6ecf33ab2e461
25271604 F20110115_AADGHX drobiak_s_Page_22.tif
e69ec804851669ed71e210f2a5fbb4df
ff5916220702f18be8f69596cd5466a36725e50e
909734 F20110115_AADGMV drobiak_s_Page_26.jp2
676aa4b9c7eb3e0e51f232632a8fb1d1
7f1198b6fbf890bc19499c38beb0e52db7e42387
22574 F20110115_AADGFA drobiak_s_Page_20.QC.jpg
af3d25b5482d8d1a71070ccd23266dd0
120da96cf14d3e6c3597841654f124828605e7d0
23760 F20110115_AADGHY drobiak_s_Page_16.QC.jpg
0f2fa5cca0acc221208b9b8c327e0a06
86d124511141db7ef6abe1e59c10a58e404dff13
1219 F20110115_AADGMW drobiak_s_Page_23.txt
5a86a1d6eeea9ec20532ac4ec902aab5
76f2bf2a83894a90576d8862c386711f4398c60e
26752 F20110115_AADGFB drobiak_s_Page_11.QC.jpg
302d9cdab17c1ddc5ae32d015387adc5
24f35885ec06586c49efb3d12afb4509d3587b23
8103 F20110115_AADGHZ drobiak_s_Page_01.pro
a35fd26d34f6a5fef8a6e1df25007f12
6a8b3903b7bf50ebd0a111ac2252be8e6559309f
2062 F20110115_AADGMX drobiak_s_Page_08thm.jpg
14ab026dfca7357a68525c44fc43e79c
f1e0b15264cfab85fc783bb7e2a37218cc785c87
6077 F20110115_AADGFC drobiak_s_Page_04.pro
d16d76e1088eb7e2f2e19c5b70ecb786
93b2f5c247deeb60f3157d6fea65b02df396f840
21505 F20110115_AADGMY drobiak_s_Page_40.QC.jpg
f8c5aa42960c99d391f5a291b318efd2
95c0bd50b5bca890d6c89929f2a037d0ab5c19c4
6521 F20110115_AADGFD drobiak_s_Page_39thm.jpg
3453941aa103482efc2dac539b8630a5
7a315e78f55299421de76aa5dd96c63dd6d004d0
16415 F20110115_AADGKA drobiak_s_Page_28.QC.jpg
651a949b6e170367b6c264e69ece9d0b
25316d7f1b09f0abdee010ce55508e54d2d20896
40900 F20110115_AADGMZ drobiak_s_Page_42.jpg
2f0bb770b50dca123d2993b3ddb3949a
2bb90152ca1e160c9064118e21ec7050e042b90e
73416 F20110115_AADGFE drobiak_s_Page_41.jpg
0d445d2889982fb89e7bd9c80e96d1b4
e01a2582d3dfb3321c4091ec41b327673b3541df
6962 F20110115_AADGKB drobiak_s_Page_11thm.jpg
7ed6f4d0e4bad2254f6e377c7dffde81
cb51e28063a78f276a7a7ce6b4f0a66b4752c63a
41397 F20110115_AADGFF drobiak_s_Page_43.pro
2454d16f96c775aca1b1438bec6c620a
b0d051a4df9dadc0814ee4872371510a5ba01c5f
104524 F20110115_AADGKC drobiak_s_Page_25.jp2
f48468f3c7efd86c1a8c48a0d9af1d92
4d2d2ba0e72d811bbfaddfc7680d131e5d244df6
72435 F20110115_AADGFG drobiak_s_Page_12.jpg
b0126505be957772300584a9134ab80f
ab400ccf564da0081c3b7db0ecf7968fe734566c
F20110115_AADGPA drobiak_s_Page_33.tif
e8010f0a039865cd48c9427412da36d9
a4c3b807f4cccddee5818931c1b5cd4d88fa2c9d
6671 F20110115_AADGKD drobiak_s_Page_44thm.jpg
fcae9bebc76d5017e03ba17f7b89b731
e22124d7dde644e3b97cd6c6a00c1e252229c8f1
111694 F20110115_AADGFH drobiak_s_Page_10.jp2
36ffbfb5f507d00459658c874e8ad8b0
166c9ce0668fed47f1cc2c0daa33348a429977f7
F20110115_AADGPB drobiak_s_Page_38.tif
8e0ab61db5efd43d1a1f506fd4b8a16a
eaaf710b01e99587d4e715e005442f6a4bb82fb6
1701 F20110115_AADGKE drobiak_s_Page_22.txt
0ae627f12d9650c119fb86fe68684c95
812ffe3ac20746f9caff9481b75b73ef371f5d80
18707 F20110115_AADGFI drobiak_s_Page_07.QC.jpg
f705f2564dd667e693d80a12f135cc8f
70e02cf1e89c427ca42c76bb9ae81ab3ef4cdf69
F20110115_AADGPC drobiak_s_Page_43.tif
db1156755951b9bd568ad04001866900
fc6fb697d818a3c6b910697ecc8dddc04ed6c1db
22401 F20110115_AADGKF drobiak_s_Page_46.QC.jpg
266649ec5600229415eee064a77a8a2d
5d3682da396df522336dcfaa5f044381eb5a7544
1051868 F20110115_AADGFJ drobiak_s_Page_32.jp2
9e42a21c5ed427083bff7d19d01f5898
bbd1d120efd495daf697710fc1a61a6d188ffb3f
F20110115_AADGPD drobiak_s_Page_45.tif
8dfbe84f274fe2b132bb48a800a9049d
ff278c08bd576810deab6fece0d2c85a1f37efd8
5953 F20110115_AADGKG drobiak_s_Page_31thm.jpg
50cadfaa59839e8f8151eb5f138911e7
30c7a559509e063a31c1577efd0e2ee2f5fbdd07
F20110115_AADGFK drobiak_s_Page_25.tif
8065c3a60a5bc8e967e784513ef1a3f5
c8c0b66d8624bc569e675167d2c1df9750d79c1e
F20110115_AADGPE drobiak_s_Page_46.tif
969f91b545f357dc82a4fd7245f7fd26
fda3e4846b42a8170606065ca27d318b2733dbb4
1908 F20110115_AADGKH drobiak_s_Page_13.txt
19d95eb9d97be6deb645e3a78d0e9067
6db3345e0da2103e23375ca43e1d4d255f3b881c
53909 F20110115_AADGFL drobiak_s_Page_22.jpg
e5a73317a0bf74819e4f6da13fc29355
792fa0c185aabb41b13625f2105ac841df7da50d
1304 F20110115_AADGPF drobiak_s_Page_02.pro
939c7f9d716a3a6944bf0a931a32e32d
e3b5f350a2a3b0929d516ed28fd2b1bc2b5288e7
71950 F20110115_AADGKI drobiak_s_Page_36.jp2
c71eb26de63fa69f591f1eef40d77fdc
4816e242e1368a741ac60cf42dd8335617c444c8
3187 F20110115_AADGPG drobiak_s_Page_03.pro
104bb97eeea5f87f7c3a112813556150
43f3889caffdcb10302e3c5a2eedff2e67053515
10467 F20110115_AADGKJ drobiak_s_Page_02.jpg
6a8a12d9dfb5ab13ab6f2c89b143ed38
07818cee983fcc0057f6d5e0d0942fb61c97d813
1731 F20110115_AADGFM drobiak_s_Page_07.txt
6149e23d4a3fb4c5c25be10da5ede872
1bb16599e49c0538195bfbc089e25ce725cf29d1
55968 F20110115_AADGPH drobiak_s_Page_06.pro
4c3b87a43f89103fa9f6dce8bb2ebe96
4dd0b16538063393c011403c65e5076662b4b135
80865 F20110115_AADGKK drobiak_s_Page_19.jpg
cb46389a7e6691c45ddf998f136dad9e
b0842213b7a63d402db6b1cf387abbeea99a22bb
2274 F20110115_AADGFN drobiak_s_Page_05.txt
13ab63da5db0d057ff2c3ce3936ee538
f7b5a9f02b6dc875726ddaa0d247276208a20668
45351 F20110115_AADGPI drobiak_s_Page_09.pro
dd50a10357024af50bbb7418763ce557
99d3e407c83319d63bbeb316ca140301152377e3
F20110115_AADGKL drobiak_s_Page_36.tif
30a54f77629111391708d3c8541ec6a7
9510f5fb76b1a59b75319aeba60b265a6efa7e7b
10787 F20110115_AADGFO drobiak_s_Page_47.pro
97763c6091d1effe1229b1ea7ddf917b
e10042e88c15e7f4fee0921b65c32d9e24e1cdb8
70370 F20110115_AADGPJ drobiak_s_Page_11.pro
5f25921dad10ab23a3e5a67de294056a
105d0de802906cc063f0d70bd22699e885024cea
67487 F20110115_AADGKM drobiak_s_Page_44.jpg
d92af96fb7665fb4c92db9dee7dbe16f
f4c64554363238e6a7de42fde093f163d93688af
F20110115_AADGFP drobiak_s_Page_32.tif
e43ad5682475f9305c38f8f9e1192a9a
b33072db29575a78363b48d5f30d153d60403c47
50196 F20110115_AADGPK drobiak_s_Page_12.pro
25e4a3378d56cfbe952c6b040c093e76
52f33a6c28d454aef2dc90bf1c3232901220d0fd
2748 F20110115_AADGKN drobiak_s_Page_33thm.jpg
7670a4589da01a492dbe61ae1654fda5
0c6340c7d3fdb1c3a277fc4ad94bd6152392aa6a
108627 F20110115_AADGFQ drobiak_s_Page_41.jp2
e9e0dbdf880a0baef61e5d40222e2a21
a4e613b30be632b3626be852700b5996a8c51e2a
48475 F20110115_AADGPL drobiak_s_Page_13.pro
acc29572999bd428a58fb79f151118cf
315c6b7fb397652c35f287b9322ef7edc695373c
F20110115_AADGKO drobiak_s_Page_34.tif
68d1d3c86f308bb39654add3766d5ed2
b2b497db2b98e2fbfb8217317b438763b92b3108
70401 F20110115_AADGFR drobiak_s_Page_13.jpg
a98a9844193451022b82dcef3b363c60
b76d22ad6ca00c0aa3c9e83276c188ace2005386
50151 F20110115_AADGPM drobiak_s_Page_19.pro
2e3a99c8d4dcec5be23c053bb0bfab7c
cef2e82580d514d4053584f33f7d1a5179ad8736
F20110115_AADGKP drobiak_s_Page_10.tif
98433cbffab3e6ac29d500ed34f296b3
4bd50672f527f4bafca257c155960053b7d147e6
97117 F20110115_AADGFS drobiak_s_Page_11.jpg
c67c8e646bd669e48e3299f1d4ed7ce8
6b8698933d2f5aa9b1dbe40828098dd8377605af
35986 F20110115_AADGPN drobiak_s_Page_22.pro
e003150f3ff59553656cfc33368905a4
33474a900727312b2066afcb528bf1210b7ef45c
F20110115_AADGKQ drobiak_s_Page_28.tif
e8cda50be49d0abe44a8cf1f69bf345d
2063fdafc399d6cdeaabec99749646911c5d64f7
50470 F20110115_AADGFT drobiak_s_Page_36.jpg
f544687511619b1ab4cfe3559a618525
7c5ff7931c8e2757821d4b568530dadb3d7c2800
47526 F20110115_AADGPO drobiak_s_Page_25.pro
e55dd8f1dc8e8cb35d3dfbfb1257001f
571154f32d78e7a1e4079becc03d4e5e6e662c32
1992 F20110115_AADGFU drobiak_s_Page_16.txt
5a976ce096ff1c063a815fd473e4518f
58dfddc3806131a6290a79ea09a20d2b6e3a4afc
40331 F20110115_AADGPP drobiak_s_Page_26.pro
07aad8bb5ccfe4f80e95af660c0c2c9d
5e88e5fe2f252124fe043d7361c47d3c0f2f5eda
F20110115_AADGKR drobiak_s_Page_23.tif
8be5b78e0984215c762c5b8a639471b4
727eccb20edecccfd97ddfd022e133fb65b87de7
F20110115_AADGFV drobiak_s_Page_40.tif
459f381aef72bdb03ab9ba6ffeacb349
8e8bf2aabf381a31f60c8563812eaec9c7dadd52
22379 F20110115_AADGPQ drobiak_s_Page_28.pro
a03a6b1846e770d9382b90a35925ec3b
6e16ac032811a68c601f2d62bfa87fdc13016106
715107 F20110115_AADGKS drobiak_s_Page_22.jp2
172ae51e39f8a351ff4ce444e55a668f
80fee342d2ac828e1a64684dcbbf2b0281d5e41b
5259 F20110115_AADGFW drobiak_s_Page_45thm.jpg
f48e3ccb85b94bd76461cb75fd94dce9
686db4752d502137eb1b466e1fc2684d627c1f9e
34514 F20110115_AADGPR drobiak_s_Page_29.pro
de8fd06204ad275ffee72afeb7289ff5
d2fb25e48f1b6cdd5e22e4c1b528b24329c9e3b7
25175 F20110115_AADGKT drobiak_s_Page_23.pro
be524d64a0683823ce7e4840e82fa452
a7b12e59b685b14663b3f882f67f413e47ef94e3
5595 F20110115_AADGFX drobiak_s_Page_34thm.jpg
17a291df263a850947944da00447691d
07725a63488c7ff6f087d9635defec16d7a9b7b2
48151 F20110115_AADGPS drobiak_s_Page_30.pro
aeb27e5071d65b94854c7a248b55ac0b
bb2a2a11feee574d9d8ce55a4ab58926917ce63d
59497 F20110115_AADGKU drobiak_s_Page_07.jpg
c7a436fe8749f14c6cde0bf3658f11af
2fcdef5d928b5ba9ebfad5b70b4520f69a329a98
12975 F20110115_AADGFY drobiak_s_Page_03.jpg
1dace24c5153ad49d733162b0ae18071
79e89b20e9b5cc9722f2c2ce1cfa7f7ea050e466
39515 F20110115_AADGPT drobiak_s_Page_31.pro
c25b0e94c55f5858960c32fc4b9282c1
362d19c0951c27c7a1a4a0631ac047674331434d
1355 F20110115_AADGKV drobiak_s_Page_36.txt
6cd94502c02b3ba1c65f30b1d8ea8e4a
17be77324bf99d1e6b09155b0fb01f7c61c59a5d
15022 F20110115_AADGPU drobiak_s_Page_33.pro
50365a320b9fdd6769a5795bd334ebe2
13b0153153837163e98a2ab91fcc6852a0fee94f
17126 F20110115_AADGKW drobiak_s_Page_22.QC.jpg
08d2340595ccc8ff1a2179ff9df5c62b
d88ce3c602684a0c0c83a60399cff6a3224f9e39
2545 F20110115_AADGFZ drobiak_s_Page_47thm.jpg
3cf7a4ae88f6cbc06eaf734775d4c51f
23e87f839fd923c5636d43d800284a33fa1c4ca9
41319 F20110115_AADGPV drobiak_s_Page_40.pro
7aba6f7eabc1591958583724db8271f7
6fa015fa12e1f01c48d4b58d098da143a320ea29
1667 F20110115_AADGKX drobiak_s_Page_40.txt
ff0601c49510b50d0b3257da32a6e4c1
9c470752990d31623c5a97f7481d86869b1e3f77
F20110115_AADGIA drobiak_s_Page_09.tif
e49cefb3ad3a003c307174454193a7d6
be5d0afe748190a3ffcf8feeacbea80a7bed5321
F20110115_AADGKY drobiak_s_Page_14.tif
f73e09699e8523892566e62a91d07664
a3b56ece310089ff13df3c4b2e33e28211be57a5
35023 F20110115_AADGPW drobiak_s_Page_45.pro
94999ad6801ba6f9473ab88dcd8db99a
8a84fa600d02cd7af458964eb513bfad0dbc68f2
49925 F20110115_AADGIB drobiak_s_Page_17.pro
77c956f8e84f7f3f78610e21d508cd53
67554eb6007aa8a2d990eabf102e3559af052830
1389 F20110115_AADGKZ drobiak_s_Page_02thm.jpg
081c479c49be8711948f57c65f01b6c0
b4ad5804128a2ab06f0619b7182751999b58a6ed
458 F20110115_AADGPX drobiak_s_Page_01.txt
cb7c7f1b18f9540fb06f6ca449293530
437aaa00c7dc54acedc004761628548f75dff525
50527 F20110115_AADGIC drobiak_s_Page_16.pro
30891709965e58950d9cd3c7f365e747
c4a9011040d04eda6d140907fd0b138040555284
2006 F20110115_AADGPY drobiak_s_Page_12.txt
2c54fc89f5b397d7a81188384be92349
6edaff99796208b549b161ebcb7fa5c3f79547b8
281 F20110115_AADGID drobiak_s_Page_08.txt
d254367d4b2d629ad3ef1d3b24c81954
3f6809a04480b8058897b93728d51ed7645344c6
4540 F20110115_AADGNA drobiak_s_Page_05thm.jpg
6ac643e6a00fdff566675ff861710d92
cceeb35b5074ae2682eed36a2e41808a99b8f457
1794 F20110115_AADGPZ drobiak_s_Page_14.txt
380f7462f51cf70e8792389a959034dc
c234cd7dc75a91ac010aad171b4470f8b7c4e4f8
16971 F20110115_AADGIE drobiak_s_Page_04.jpg
4458d9f14b824db110653b58f09c8125
ea9686aef5b72e169413e8b16017aaf055a88fc3
70703 F20110115_AADGNB drobiak_s_Page_15.jpg
399ad4627429fc56568df91c49172ec3
2949b88aeda46999d018a4cec779ba48dd878bfb
46244 F20110115_AADGIF drobiak_s_Page_44.pro
c319d454418a27c0b2de951c2616dcf0
437355bec27a67ff2adda8b8331326eefeee8cea
79858 F20110115_AADGNC drobiak_s_Page_46.jpg
ac7d61ae71ce181a6fa7474fbe174e3a
420a5120c10d02d453afa35bd1db142d8e250cc7
F20110115_AADGIG drobiak_s_Page_44.tif
e9df29eb412f7e40a1dc7be1193bc3c7
2f6e4cc623b29566fce0bec485765d6fd87b1168
24158 F20110115_AADGND drobiak_s_Page_41.QC.jpg
662492db7f1ca92ddd227461ecb0ce20
11cc8b5d98c60c6ee8b15caa0bbca0f92de11a3f
23689 F20110115_AADGIH drobiak_s_Page_12.QC.jpg
68d59118000f08d049a213c88213c1f7
c5238d2a9f094cac8547a2cd7ebe964e2a64002a
7024 F20110115_AADGNE drobiak_s_Page_08.pro
bf23d7b00d6a67b271285155ba47dcdb
5e3feb4696e198c63f4c56f97a8475e605f3960e
1051728 F20110115_AADGII drobiak_s_Page_28.jp2
0c30c455ab6c760f48096ccbb9b01b05
7abc46fe65925c895e63ee5198c5ef75d7e4c46c
5739 F20110115_AADGNF drobiak_s_Page_46thm.jpg
2b51f1cc41574fffe5df3d02d82ce007
8755d03583321442528cf4ffc2afb042854cc862
106100 F20110115_AADGIJ drobiak_s_Page_30.jp2
1090cad2a9e5be0e89c2f1b12fa9bb47
14d01033b8fa5440774c11e610d1496b8cb1c999
5987 F20110115_AADGNG drobiak_s_Page_08.QC.jpg
edb6f5ac14fcdb8aad2f475a15d9c6d4
fdb06af149bca2005b0259837513115c56c99709
4018 F20110115_AADGIK drobiak_s_Page_23thm.jpg
0be81d42b1535241b02e88db66fabd91
819ad65e78399fc56faf2c991034b0c0753b28f9
25946 F20110115_AADGNH drobiak_s_Page_37.pro
e3ac1983737d6a0936aa783a422ad356
8415bd5ccff020783b3b8d4fe77682fdc7741872
2239 F20110115_AADGIL drobiak_s_Page_46.txt
aa45751874bbf5ba7cf01248d08272fc
f8c6dec0c3e4dde109213a1d4e9a818aff826ce4
1861 F20110115_AADGNI drobiak_s_Page_09.txt
3e8cf7fd70632649fd8b5205210bc052
e3c60ed27777f27e77053a233479af9f8c3328d7
49110 F20110115_AADGIM drobiak_s_Page_15.pro
f32ab5b6bdfc72e2ec645c84dd545752
94a8ec1b8f2fd76817ae880d901fc7d3a2f6924c
22745 F20110115_AADGNJ drobiak_s_Page_01.jpg
75cfe41666e3ada9fe798e18406513c2
767d936d697615225eb42712e4b89e8dd52d3178
2021 F20110115_AADGIN drobiak_s_Page_10.txt
8aacc2996dc5ef2ea4e52167f2864031
c951eb709cbdfc60f5d222640151926080433705
F20110115_AADGNK drobiak_s_Page_06.tif
dd2f89a63a8ba986dac44c3624f2a478
6ae36c865891baee77ec3e1671bdcd1fb0876207
34961 F20110115_AADGIO drobiak_s_Page_33.jp2
04ea156a09096ac6d61fca3bdf1e8e7d
38701c050d87b86180c00f31cac36bac0db8dd78
F20110115_AADGNL drobiak_s_Page_24.tif
26fb8783f23fc8bf4cd0312c78644110
60f34a12d6ae286de2863e2ffd19fc5ede7eb35e
70609 F20110115_AADGNM drobiak_s_Page_17.jpg
291a21194c167ad670d566bb0fac4070
345bcbb929d2d5d8aa455172f87d9460e9fa118b
6673 F20110115_AADGIP drobiak_s_Page_41thm.jpg
e9462ccba2312b6000448853b4e2c9f0
c413b27fb5aa88e8198d949c187cbcf898716eb1
9093 F20110115_AADGNN drobiak_s_Page_33.QC.jpg
78b63d1976f036bb99b5dd1434d8c97b
cf7a29d63a915128018ef6c8591c151c080b8dea
65797 F20110115_AADGIQ drobiak_s_Page_14.jpg
28c9f2c064dc3df75aa6682c869f9f17
b7c583a6890b5ff9d96ad066a627d19e9ee6e5d8
66434 F20110115_AADGNO drobiak_s_Page_38.jpg
ddc9aff4c4b0a0d9dac7590aa9806a84
64435248b615df2766b380e55c15db4643d47baf
5690 F20110115_AADGIR drobiak_s_Page_35thm.jpg
5c284f033f09b27db2d3ffc0de419ee3
6f83a94e8671a56f4cc709056cdf61098ff098b8
F20110115_AADGNP drobiak_s_Page_16.tif
b2861d274402d7158a2c70bfa14335ba
d213323803b654e95e090b26efcf5271d7cf7930
16555 F20110115_AADGIS drobiak_s_Page_04.jp2
60a7dd79847691ce675c468bc73ffa3c
c23c6b3882d0c158a0105614d51fc573bfa36460
F20110115_AADGNQ drobiak_s_Page_30.tif
5b7f0dccd7a09527b9c2426d94734b19
c7059604d8a0c4e1b869a8cd36847cd65c510810
43330 F20110115_AADGIT drobiak_s_Page_14.pro
e36ebffc8681fe96b49bc3d10dd78f4e
8255fa259b05edb56a9f06dfe6d469c30fb1fe1d
54672 F20110115_AADGNR drobiak_s_Page_46.pro
da4310e5c868fcd023e74eaf6dfd0c98
616ec2cf75b514821c40c4d1a71271e5ef9320ac
943 F20110115_AADGIU drobiak_s_Page_28.txt
26e14688c3e8c95f081177f0dc5daf76
dc13c331b40e602f39145b9fe156938cbd0914ab
35195 F20110115_AADGNS drobiak_s_Page_24.pro
f3aba7e43b4c3fa51e3cf8405d7f46a8
5a470b55c16bb0516cd095bbc49b8ab6f74c8506
14958 F20110115_AADGNT drobiak_s_Page_37.QC.jpg
5c6f624cb2c1653940c32c5088639912
b3f0dec1c036298f64cbbf7c3e7978091db03513
119 F20110115_AADGIV drobiak_s_Page_02.txt
2ef8ef84ded994be22b6b183ee64b90d
3ac3b158094ae376992d338d1894eafadb7b5ef5
F20110115_AADGIW drobiak_s_Page_18thm.jpg
47de3ee1a25b0c9ca10db62dbbf7ff56
b325de476e0133c93774d0acd7cb8d67bc3e67f8
1976 F20110115_AADGNU drobiak_s_Page_19.txt
f7d1d94f2d454d499a61e7ec197401c4
d207ae9440f1dd18fbd74195e7f46faa2e3ebb2c
F20110115_AADGIX drobiak_s_Page_15.tif
aac94959ab3613584bf82a760b094b08
89f854bf965d0a29bf458bee154cfbd2ab3e8a25
73717 F20110115_AADGNV UFE0008961_00001.xml
9aaa8b5999f1887cbf8d20acf659d891
40ed77cd036df706861c94d5ecf394309bea3a53
F20110115_AADGGA drobiak_s_Page_41.tif
e9ae19a0993dcf5bc6dd70693116069b
bc8216e1909d1e4f576818e94d0b9aa749cd3791
56193 F20110115_AADGIY drobiak_s_Page_42.jp2
05d2475bd769a6b2f10e1056a8f0ded7
deb8cc1a35cecdc02053e90fd6636270b30d26fa
22909 F20110115_AADGGB drobiak_s_Page_25.QC.jpg
3bc0fe9ec012df0891dc25ae33093ba6
97639fb89ab7c2fd523ea5e4d817ede816399316
22620 F20110115_AADGIZ drobiak_s_Page_39.QC.jpg
3826376907b3e6ce7a303e769c43db3a
a3eaeed4a1e2502cf2d85f67089eb226659df549
48534 F20110115_AADGGC drobiak_s_Page_39.pro
1280035679c3002f6e8e605b345d3245
f8979a8798105d40b6a278c4e5172834a48ded3e
1471 F20110115_AADGLA drobiak_s_Page_45.txt
0c94810f552eb79417ee1c7d6e09e274
8eaeb45ff943410b3eb4362c696d566298e02d3e
17432 F20110115_AADGNY drobiak_s_Page_08.jpg
bd09ffd8e0dd08addf3d58cf379418a0
7132fc091ccc939740cf344f657ba21643bc0323
6564 F20110115_AADGGD drobiak_s_Page_10thm.jpg
03682a4c8cac856ea69206a29eaee21c
feb34e2fe0ec0b19da7ff4288ab77a2cdbfbb957
22195 F20110115_AADGLB drobiak_s_Page_06.QC.jpg
408a003faf07deb2f2ddfce3f37f90bd
701631ac56ca102b8f63b04a0812acd615376d38
70369 F20110115_AADGNZ drobiak_s_Page_18.jpg
881114d963b797a2c6036c5a3d87885f
24358ef2cb0e4401ca963476faee4e23d4081077
F20110115_AADGGE drobiak_s_Page_08.tif
07b503c58919236901367eb850f4ae67
cf89e8a94e3159f5030dd83bd60bc6243c9bbf4d
22413 F20110115_AADGLC drobiak_s_Page_21.QC.jpg
d204eb51808bd0c9daaaa02a7d897657
6c0eeda63b182c1800fcc03e390445b055ad5d58
34475 F20110115_AADGGF drobiak_s_Page_21.pro
0a31229527314a2f0a02ed51bc90f8ae
8ae2dc8423ed4bcbd02803d2b16e6d0700f2c37a
1699 F20110115_AADGLD drobiak_s_Page_35.txt
660dac9efbaa71664de3afe9b5feac0d
ea2843276af06770102a4bca8452d415e00d942e
20040 F20110115_AADGGG drobiak_s_Page_34.QC.jpg
d191fdb72ece3af4e2914956e4aa2bf1
355f29d9ea8a610c6189808de06338f192adbada
1871 F20110115_AADGQA drobiak_s_Page_20.txt
7597b0ae85a7d32e3e9730294afaf247
994ca39d59ff7c2f092469337b90692095007f0f
110428 F20110115_AADGLE drobiak_s_Page_46.jp2
412efdb6bfb05a9711f9295fecce62eb
d587fb5ebb3b89a92fa4f1cd2bf12439fc3dd26c
51340 F20110115_AADGGH drobiak_s_Page_05.pro
b5ea749cb2055ee5a4b3783fc60c4ebf
7bde8031edd29cdf49095eb18d43ff3e27732ce6
1393 F20110115_AADGQB drobiak_s_Page_21.txt
bd822e12cdef814e2f8c4bd7823dc54f
70279e85258811203bcf0a9837c1bcc088ee0bad
97072 F20110115_AADGLF drobiak_s_Page_38.jp2
65fd295f523feff69a538fa8c4cd2cf6
70a08f652b11f1acafbd0b99fa050e9e2bf940fa
292 F20110115_AADGGI drobiak_s_Page_04.txt
c9284ce39346556e03a3b698c4fa1777
77a23d647dc6bfe4dcde7daabedc43d9697ff46b
1898 F20110115_AADGQC drobiak_s_Page_25.txt
2fa4ba85dbd45c6b58197ccefc99f549
e3d2e798b626bfe931868e039aed1db99483172c
66733 F20110115_AADGLG drobiak_s_Page_26.jpg
6833c62680d65a1eb151bc11e180d573
3cc6c4cbbd88842ff4b010d17ee7fb4c06b82c81
46903 F20110115_AADGGJ drobiak_s_Page_20.pro
a73a5fc0dfbdbed6d1b58c98333ba5a5
e3e77000591517356c2a1ffec65e1bd7da9867af
1732 F20110115_AADGQD drobiak_s_Page_26.txt
9e0e82f7d13921a2e93f9ab22ad35cd4
8784858456214d7e9f456f284c18210f28dd731c
F20110115_AADGLH drobiak_s_Page_02.tif
c535b3885aaf30f2e352be3edc493acd
0d7b0c986d60c685fd02ee5445c7e39e989b4363
5957 F20110115_AADGGK drobiak_s_Page_09thm.jpg
fcec2936f7d43a6e6cdc41f7e0068ace
c62d6ff527a4464c9771f5c505d5c3ad94f4c6d2
1615 F20110115_AADGQE drobiak_s_Page_27.txt
e92aa6ddc68fa2d845031c4681d27655
d35f607fd47946c86e451ccaa7f52113f56fc3e0
56832 F20110115_AADGLI drobiak_s_Page_45.jpg
96db6d25e503741d3bd9a68bf326a3e8
8c8a472cc47b437f617c0e1727755e0ec7597214
F20110115_AADGGL drobiak_s_Page_29.tif
1bdd198cd75e211a8bb3ee22788707c9
bbd9cfe431fa2cdd9b5b6b38852116d81b510c09
1900 F20110115_AADGQF drobiak_s_Page_30.txt
db9e117e73abe27dc59ee5b37ed0150c
a6dcea5c982349ddd39b335a8e63ff208069d3ba
F20110115_AADGLJ drobiak_s_Page_31.tif
2bdb26a65171b3394ac7fe557b74efb0
1753414e9b9e027470d191c69436af8e9c3577e8
1051944 F20110115_AADGGM drobiak_s_Page_06.jp2
1659c12814fdbc59d43636063b544087
9a1e68121a49da39ee4f40bebd0a87b9da174caa
1624 F20110115_AADGQG drobiak_s_Page_31.txt
2953de0dce1055e2d75ba30fd9ead98c
6e4f0a6ed277a09488c20640fb183fe66642fd47
18271 F20110115_AADGLK drobiak_s_Page_45.QC.jpg
b9f12ba8261e08b7ce296e095b6ddfcb
646792429b11ce6cf2d80fc8dd8bf1d6a3d05667
F20110115_AADGQH drobiak_s_Page_32.txt
4e896db37e46208543a2f4c2ba643386
40bda94113c6fcdd8eb75315a13e4659994acb59
5871 F20110115_AADGLL drobiak_s_Page_29thm.jpg
8d30ed845d60eff371b022d3c117b5f2
5b3ba497b7eabed45acb811399d53cf8351816b2
31546 F20110115_AADGGN drobiak_s_Page_36.pro
2b33e3f7027a336f2d11f84c0ff79e60
ad926ad72ec150a7e7695d982f34c975a30d0ded
1728 F20110115_AADGQI drobiak_s_Page_34.txt
17f2e05dc9020122fb50ebd132cf3510
299fb6e8982e473d7f83b072f4cac2b79fcdc1bf
5377 F20110115_AADGLM drobiak_s_Page_24thm.jpg
85ccfbddb41b42d48290d47109ce09ec
9e4cd022d52fe417a2a08a4b48e99066c2a725bf
F20110115_AADGGO drobiak_s_Page_21.tif
c140efff1ac0b4f975d349c2274afb9d
e6e5d1a1cbe3a9dac4fdee08687d62694ddb9c34
1420 F20110115_AADGQJ drobiak_s_Page_37.txt
428ca70fc635315f3b9742a082e4abf6
e47373cfc6cd1701adca2697722e543248f39989
784461 F20110115_AADGLN drobiak_s_Page_24.jp2
e3dec08469190a05cf7a06bbb68fc061
ffbf6708d552ee401243cae6ca0d5af8630af2a5
4870 F20110115_AADGGP drobiak_s_Page_28thm.jpg
5db1b2dc15efb90f0d6d8434b985eb2b
eb2fcf900ac4209dfdbc2676a21d6dc1183eddc9
1789 F20110115_AADGQK drobiak_s_Page_38.txt
f19e7bf085433de2d5c532e95ba75d6e
11d930a736c2e515652d66ff325a50d0f6875315
44270 F20110115_AADGLO drobiak_s_Page_23.jpg
aa22aebcda33d283b36bcc17b0e81e59
1a0114dba45c0eb20fa55edb7e5daa30ba2bfe43
50824 F20110115_AADGGQ drobiak_s_Page_10.pro
b08be7e3add91fcc34d129c6cdf22433
d1bb85b56b84fb9eec8376bdbc32af75f1825c70
1983 F20110115_AADGQL drobiak_s_Page_41.txt
a829f7599b0e069652bef46d36d6a60b
5d251bd234eff50c2eefc85bfa8b45e80873c79a
1543622 F20110115_AADGLP drobiak_s.pdf
2d220bf3f67090ebcd72ce63ffd1b345
225c996cf00332f7c62466b6d6bc39d137e0f717
44338 F20110115_AADGGR drobiak_s_Page_38.pro
580ef36473bd55a28f0162e21e587de2
9edb66058049f01f41b5c8cae380ef93e2f2c7dd
996 F20110115_AADGQM drobiak_s_Page_42.txt
163f6a1226d9406782d65f473be3b4e5
b7fedf333ef79a6d582e872e8da40b431946047e
89594 F20110115_AADGLQ drobiak_s_Page_43.jp2
f2b194d52852b65ce0be360a1c9da5f7
5ef635bdadbf9929ff6f4103a4718cff10ae169f
F20110115_AADGGS drobiak_s_Page_11.tif
1a3cb185fcf6d96e4a50f84c77ea3c33
4c098b7ace40fe8055a8fd8356aa2eb5821ead37
2281 F20110115_AADGQN drobiak_s_Page_01thm.jpg
10689126fba7b03a704956d6e74afbff
8f815c8fb9d51a9937ccdbf3f85e19e074858f6f
6234 F20110115_AADGLR drobiak_s_Page_40thm.jpg
d8141b682ac9bbda6ef5917ddb87e646
44bc913187f28017bf8d010671e71cedd8bedd5c
1945 F20110115_AADGGT drobiak_s_Page_39.txt
ea81b5142eec74b5394852c9c21f575e
bfa923cf1004109f146dfcdbd0f3354915268480
3338 F20110115_AADGQO drobiak_s_Page_02.QC.jpg
0eb24d308d22f37e25e078262c73a580
d4e4a69fb9be8e966ebb0ef361eb7dae7075e7b5
F20110115_AADGGU drobiak_s_Page_01.tif
eeb8308b57d5843bcc5244028e0066fc
10b4ec5c4789110fa1b303eb7f3802d521ad2425
3784 F20110115_AADGQP drobiak_s_Page_03.QC.jpg
a1de350a2d45d478296db73f056a4f45
f58098bc29257da468df0280e187acbf68e421c7
24344 F20110115_AADGGV drobiak_s_Page_42.pro
a6dda69f3ec45b9e39ad462319a2224a
7402d67210109baf10c13c156b4f38861cdf5455
1961 F20110115_AADGQQ drobiak_s_Page_04thm.jpg
bafe95dcb4d879dcec9d3399b23c06c0
bb126015c0ca163c71887c5d53baa2f38ef3c13d
19920 F20110115_AADGLS drobiak_s_Page_43.QC.jpg
52c6846dca59b4ca409f3524f3ccba6f
d68578f915d660c4941083b93472404c082f8cbb
F20110115_AADGGW drobiak_s_Page_11.txt
945a3e43799c036c4b2dd669aa03942b
c0367bef6790eb44add176b501f7c04a488f3608
5359 F20110115_AADGQR drobiak_s_Page_07thm.jpg
fe37e981b5696c79af308295423e4fa9
7a329b8596e4a8e12482249e490dea8f6678282a
21483 F20110115_AADGLT drobiak_s_Page_31.QC.jpg
db677cc75a3898108be7e9a8f250cf3a
c03ba5cee2ec946af1ad60c716b4e40326356c17
106222 F20110115_AADGGX drobiak_s_Page_15.jp2
c67186aed9054f3d0d6985e1dfe56d84
ff9578ded08b239be3fca17d590e1075068e2fb2
24358 F20110115_AADGQS drobiak_s_Page_10.QC.jpg
e1bdf60eab4659258241311cfbd5680b
db2808a7afb6f2aeb8f8cfd5c00088cca7853f79
6967 F20110115_AADGLU drobiak_s_Page_19thm.jpg
4196728e472c3c3b1c4a7ae099812de3
95ad19395bac25021fa592177e3507b4c8fe1179
95664 F20110115_AADGGY drobiak_s_Page_40.jp2
af469fb901e45671aaae50074c011120
fdbf5c6505dd4f6f549b25dc4efb0e05a6a12f5c
5848 F20110115_AADGQT drobiak_s_Page_14thm.jpg
0fbd8cfa551b816c01cb8a9ef1539a54
8b75d35bd7b39bce924f8235062c8b9f5ad1d65d
73533 F20110115_AADGLV drobiak_s_Page_10.jpg
d41dd83b65eeea81a8abbf8f3749a705
966a621913c86eb21b01f966974b918112c9f5be
23171 F20110115_AADGGZ drobiak_s_Page_13.QC.jpg
169a0abb830876fe61541fafc353837b
148b2f7b082ab29a1e1b361cfc239485cc4c7509
22700 F20110115_AADGQU drobiak_s_Page_15.QC.jpg
eb19dcd8b4036e13651ceb43bc4a37ed
679d176c0f0360551cf2a7a4571459e02a520bf8
6561 F20110115_AADGLW drobiak_s_Page_16thm.jpg
226ed5930d2f2cdbd5773117ae9de459
8e20ea4bff752d4e7cb7c6dd228212fbc96d0edd
6242 F20110115_AADGQV drobiak_s_Page_15thm.jpg
a346ce64cbffc001c63b5b7300b383d7
d5509ee7a6ef1d490e18ef7dfb4e57fdd7f6c046
108958 F20110115_AADGLX drobiak_s_Page_17.jp2
e95cbf57c616d6827a5cebab16311e84
eecf3082b4cfff4b9cbcf249c9d823b0463a3c54
23011 F20110115_AADGQW drobiak_s_Page_17.QC.jpg
e9b594919667d95a9490d46619eeeff9
3c64fce86b312403c06e9232872174555830def9
13991 F20110115_AADGJA drobiak_s_Page_23.QC.jpg
0ef5c2dc7bed152395aeee04a810c47a
b1b6c97acac73316f5b19612215864c00756d79e
69693 F20110115_AADGLY drobiak_s_Page_25.jpg
1c1fe104bb24da438af455a3bcbbc7aa
1d5172210916aa9f67adae53586550b317220383
F20110115_AADGJB drobiak_s_Page_27.tif
0b93ebddc46c2455c4d6ee3bf334f981
5e2400ac2ba5bc46a9fc1ad089ea3789ff6e474e
1445 F20110115_AADGLZ drobiak_s_Page_29.txt
8f48f2fecd5ac84aa480291b69d5e1ec
33dee5053cdb48687443c38fe72d602edc21fbbb
23198 F20110115_AADGQX drobiak_s_Page_18.QC.jpg
db3f763d262d442e017fec6e27008e48
0f9590d7cd9507442193077bb7cfa24c9badce70
91616 F20110115_AADGJC drobiak_s_Page_34.jp2
aaecc3dec443d8c3b9888ccadcc5a643
5b4bcd5292b56e60d192f5dc6e3d832596d4de76
75167 F20110115_AADGOA drobiak_s_Page_32.jpg
6640f7bafd0be6ea0a74ddec49156556
acf4abe3361a357c1edbd79e6e24d1f13c96c74d
25332 F20110115_AADGQY drobiak_s_Page_19.QC.jpg
d9db96660127271101a9e8b07c641541
54efb2b16ecd0f2efa9d0d75685a07bf75520752
69062 F20110115_AADGJD drobiak_s_Page_20.jpg
27459d0d8de5d186e836713d88c0fbd5
c460718fda84e3949ba9fee662d721cc6b1822b7
61360 F20110115_AADGOB drobiak_s_Page_34.jpg
b0654a6db0e0a9f491ef105c157820c8
3e5b71e4c5e8b0ebd3ee97e30de0b2ffdc87c2ea
6339 F20110115_AADGQZ drobiak_s_Page_20thm.jpg
b41622d5ae676a7b02d5e4a3aebd9b7a
8cf9d5d59619d3e808c75b487a138e225c1e19ca
41654 F20110115_AADGJE drobiak_s_Page_34.pro
df39bf7865b50bb36449a770f55cd192
f39a8d6f35f29419bdb1ae39af99905fe2c1419a
46560 F20110115_AADGOC drobiak_s_Page_37.jpg
6feb605e5fd3c7153e6ba3f5fb3037fd
edb7d23a2c40f1f08a6508d83710a98c524d5656
9884 F20110115_AADGJF drobiak_s_Page_03.jp2
0e2ecb04472aeb9285917a677a297101
a705e0cd5df8f44546d6802a04cfe6e9c1b7c8e0
70974 F20110115_AADGOD drobiak_s_Page_39.jpg
dc70b9144a5b12339308535da9b790fa
15c9562b5f78ff88a3ee153437f238c9878d7f95
6478 F20110115_AADGJG drobiak_s_Page_12thm.jpg
3afee6d0f0224a06dfa4ab552a3bbe17
ab4395833d595739cea836fb2acba1c82d5bca93
64334 F20110115_AADGOE drobiak_s_Page_40.jpg
efe3cb4470f91fcb44ea7b8567348d19
bc5e8dc388f4b32a37a7fee9b3c61d6852713ec1
2296 F20110115_AADGJH drobiak_s_Page_06.txt
446a24215afce36f515c3e1b93080ab8
2cf9744392bf59074a2e40c9c62458ccb25e362c
23393 F20110115_AADGOF drobiak_s_Page_47.jpg
b0b1112dda23ac0cd5340b3ae7e83146
45b5f91a8cd7e08234748e86fe99199b41e76e85
6356 F20110115_AADGJI drobiak_s_Page_25thm.jpg
6e37ad898b07946b14a53cca2b3ca7da
60ddd823ab2cd91e905d13fd84981e265397e102
F20110115_AADGOG drobiak_s_Page_02.jp2
a8d828db1629bfebed2305caafa238fa
409353385668337a2c1e513fb49be10dd8e4dac6
65407 F20110115_AADGJJ drobiak_s_Page_29.jpg
b2e87939868b26202e228561f8d283b8
b0f0a7f54ac20d493ca92b342a492a656e25db65
1051981 F20110115_AADGOH drobiak_s_Page_05.jp2
ab8d8f781c0fb9f2fd868718d182cc7a
0cf31aebcaa66b836d0535b28c960a8a30af8d03
1869 F20110115_AADGJK drobiak_s_Page_44.txt
8b38d1e073ca4b0b8450565e79cad4ca
cb23fc628967c1d0fa0fd0abea416ba8a4ea7022
87347 F20110115_AADGOI drobiak_s_Page_07.jp2
47027a81a8298f997af21b9a4394a2af
0cc7964cb3fda2e0e4cb262492cf36454eba662b
F20110115_AADGJL drobiak_s_Page_26.tif
fffbe55b8a2e9aafd401af7150029f4e
f37f8e327109c5400f25067b91f8369483a41743
97932 F20110115_AADGOJ drobiak_s_Page_09.jp2
e5a90166c827766a765175753ebbc0fa
d338293020282e30e280baa441b8e0c84a92537f
F20110115_AADGJM drobiak_s_Page_35.tif
4503bf6e3b9cfd8dced3d2bcf06479a4
3d824a7d88e8568996fc9e5fceb7dbdf114933ca
1051956 F20110115_AADGOK drobiak_s_Page_11.jp2
607e1064d4fcb4af4aedbc2f11f54e5e
456b81bdf749196ad27d63bc33947b441a547967
F20110115_AADGJN drobiak_s_Page_47.tif
52053d2443cc174e3aad6fcb5fbbee90
04d76b1f902b97b65b22bee914726f4d897726bc
93739 F20110115_AADGOL drobiak_s_Page_14.jp2
fc30759c9942ad9f05e6aae91ad22758
545ea4972e538c9421732b4baba0e24a1a20e698
26848 F20110115_AADGJO drobiak_s_Page_33.jpg
36052dc18875948bfe72258c3132bdbc
be7feed8a902e2b01b938fdf222e1f51fb9a4b09
1966 F20110115_AADGEQ drobiak_s_Page_17.txt
c76b989d070b96573e084f7bfc13368e
f7f427c0f144b4868eb1355c3edcb6aa457cd2be
109041 F20110115_AADGOM drobiak_s_Page_16.jp2
e2ac7609c61b89b0216e3feb8b00cc95
2d8e7098efcfe46449e47c4cb92222a71db9a98e
4593 F20110115_AADGJP drobiak_s_Page_37thm.jpg
8f488e8dc2979493c9460d466526bdfc
6c0ab299f5a9cb9e607d8ea8b3f2f2bb3f7a6645
7688 F20110115_AADGER drobiak_s_Page_47.QC.jpg
3f29fcd007c3a9f4841d0e5e00f079b5
95b8849874f269cb252d49a979cee3a43c8f3f07
107699 F20110115_AADGON drobiak_s_Page_18.jp2
ebb00f8c3ef227797ae209e03144ef22
a371e274f50f74e44cdab8e4d2d56d812a3040ab
640 F20110115_AADGES drobiak_s_Page_33.txt
93cf0535685a9bd908c1102619916cb1
b8e250a71b508c4bfd1910ec8874ecc7f4ac81a7
105725 F20110115_AADGJQ drobiak_s_Page_13.jp2
2a4dccec1311668f33c92733cfe14805
3151d14984842b0fe12bdbed19021e79d1530aed
48976 F20110115_AADGET drobiak_s_Page_18.pro
049a075b873dc8d37f59d60658d7a50c
73a679e40201bb1ecaf740fa992d423514c53628
1051953 F20110115_AADGOO drobiak_s_Page_19.jp2
02927efd956127ae80a5096f756858fe
b18afae4ca03f33ca9fee13f4bc361b651af6fde
F20110115_AADGJR drobiak_s_Page_42.tif
1afe4e110578ef2776faf469c1ffa9ae
7aca43bc567ddc99d058f964edf52f16d83a6c7a
F20110115_AADGEU drobiak_s_Page_17.tif
7d06b91ed18a1a4cbcacb00ce6d401a3
ef261ea4f13ffa9bfc958e329baa9fe3918d03de
104432 F20110115_AADGOP drobiak_s_Page_20.jp2
02f0e8c171debc5c8fd41cccdca8e94b
43322646dcdce16a880578379c02887c845e8301
64670 F20110115_AADGJS drobiak_s_Page_09.jpg
f40853d62ec0b47ac236e993d38182c9
f646e9794780e6efd15df70c862c147de9d51288
1956 F20110115_AADGEV drobiak_s_Page_18.txt
b5d63c82dadf853e5022486b8e28bcac
ec65cb3a577d66811ac37f3126382fc5bd85107c
636945 F20110115_AADGOQ drobiak_s_Page_23.jp2
e78d10d6bf6f1aa28ef5af5bb22d8dd2
e337f8d387d064b6d16423c480a9db80f7c89aef
5282 F20110115_AADGJT drobiak_s_Page_22thm.jpg
dbcb33313d7c9c5e475990cea83424c0
fec04e5f9908fe05b28c6d497d046f0618e6b244
6336 F20110115_AADGEW drobiak_s_Page_13thm.jpg
b92a27db651348270a0c1cb95540ce5d
0a5671d04b2e9949ec9306cab81c0e2bfc51ebe7
1051979 F20110115_AADGOR drobiak_s_Page_29.jp2
2e6a129e4021cd9615ec837cc05275b7
e402a101f721edb9ef6a6b0e6284ed3b2c00bf2d
F20110115_AADGJU drobiak_s_Page_12.tif
9483eeef1378e5b0c317fda3c3d29c26
8118a22a3b0ee552d614ef8213c5e7399ba2b4a8
58588 F20110115_AADGEX drobiak_s_Page_24.jpg
791d4d5716b1afece1c7e2617d77e74f
c89d6ce56a4f7b2431be4a43f301e3beb23c7d4c
87159 F20110115_AADGOS drobiak_s_Page_35.jp2
7d643c7c889b4ce25a2e5c633e448e05
05856adb4e26f86b38a3c07ab033553748a24b5c
5862 F20110115_AADGJV drobiak_s_Page_43thm.jpg
c1f6bc0d85343da9980381f14632d2e0
afd1138feb758da0d78b4dc1b63adaf6ae3c01ae
1937 F20110115_AADGEY drobiak_s_Page_15.txt
1688ba3510710dc0bd6b69507aa5f1a0
6a5f0685daa1099f221139bd061b26c557756506
105365 F20110115_AADGOT drobiak_s_Page_39.jp2
79dc6f14a09968c7fb4cb7425ca1fcc4
f5e9aa21560278d97873d2280106e58d4ddcf862
479 F20110115_AADGJW drobiak_s_Page_47.txt
436091d4a6b92f03e30c1dfdce582798
eccf36bc90b84ce204bb483832a7bb85c5e1cf7b
1051973 F20110115_AADGEZ drobiak_s_Page_21.jp2
44d2b379049243ac12f85380d2592ca8
02256f20724f09c0de117bcb43d65ba70766b996
78762 F20110115_AADGOU drobiak_s_Page_45.jp2
512e83125dc03b20a03f980e8990d1da
f60a828e7fbeaff3e7b7814fb9c3550be2b50a25
49795 F20110115_AADGJX drobiak_s_Page_41.pro
94acbb40436af6fb76930a61b0ee0e4a
65d43558c4fb3b9d8a3f87fb42e6372b62d61519
6031 F20110115_AADGHA drobiak_s_Page_38thm.jpg
056441fe979c1a3746c8b4dd0d4d6e00
6ac140550e15c9a73523240a636af14e0cf03b51
61658 F20110115_AADGJY drobiak_s_Page_43.jpg
f3087cea499acb703f86acfdaaca5f9e
0da95daf3d714cfd4de4f21a6a86b929575fd9c4
27179 F20110115_AADGOV drobiak_s_Page_47.jp2
52a14517c8f19da5409ac33c4059134f
0b3928b0b3245152fc5961b9455e03ff5b6ce81e
21082 F20110115_AADGJZ drobiak_s_Page_14.QC.jpg
ca5e5b5012ac1ccf4f9fd1a5c5e06e43
3974f24c8a4d896176f8543e1dc8e2654dc235d5
F20110115_AADGOW drobiak_s_Page_04.tif
940e943e333d85019719b11462474390
b664cd0ddc7dee4ba549b793811f6f56ae5ace85
23455 F20110115_AADGHB drobiak_s_Page_01.jp2
d9212c6c923f2586159d8c4a8641f66d
643ed02b488ba34a7ba7d9f343ef29c3e0b93c7d
F20110115_AADGOX drobiak_s_Page_05.tif
36987f3a2c6e286ccd7ebb0143d39148
51ff3ddf86aa5eab48f34abf34166c9900a56f9c
7169 F20110115_AADGHC drobiak_s_Page_01.QC.jpg
a35e4941e80864a81bacd4899e2d57d2
13818236b51fed36f7d84aac77ef0d4fc6f231bb
F20110115_AADGMA drobiak_s_Page_03.tif
06bd158044a36377c937d900e89cd594
338ec55ffb9afc64bc97c9b8ed0d097565bfe3bb
F20110115_AADGOY drobiak_s_Page_19.tif
9ef8a623f8121f190653416b7b334404
ba2847f7aa1d58b63df79d5c781379ca33532a85
19069 F20110115_AADGHD drobiak_s_Page_08.jp2
30d9b2772bcd3a59c3bed1b4f29e77ff
0d0f2a9ed99b4f62ec53d0fe59c137d288177ad3
73986 F20110115_AADGMB drobiak_s_Page_21.jpg
8f476b7967bc3e8da0e92fcacd15eb40
300e3233966ce4a3d6cda76938bf722127120cb1
F20110115_AADGOZ drobiak_s_Page_20.tif
b078a16bb8f488166e6334a3e3d56d40
4d6cd1896253c7dc6e232b87540a6aad06ae2307
45585 F20110115_AADGHE drobiak_s_Page_32.pro
482d0384a47b8058425824708ab88929
cb18087b0e675dac5716e152a94c5bcde54edd23


Permanent Link: http://ufdc.ufl.edu/UFE0008961/00001

Material Information

Title: Characterization of the Zps1p Cell Wall Protein from Saccharomyces cerevisiae
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008961:00001

Permanent Link: http://ufdc.ufl.edu/UFE0008961/00001

Material Information

Title: Characterization of the Zps1p Cell Wall Protein from Saccharomyces cerevisiae
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0008961:00001


This item has the following downloads:


Full Text












CHARACTERIZATION OF THE ZPS1P CELL WALL PROTEIN FROM
Saccharomyces cerevisiae
















By

STEPHANIE L. DROBIAK


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

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Stephanie L. Drobiak

































This document is dedicated to my fiance and my family for all their help and support
during the last few years.















ACKNOWLEDGMENTS

I would like to thank my future husband and my family for their moral support,

my lab mates for their continuous help and friendship, and my mentor, Dr. Thomas

Lyons, for his endless guidance and patience.
















TABLE OF CONTENTS
Page

ACKNOW LEDGM ENTS ........................................ iv

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

ABSTRACT.................. .................. vii

1 INTRODUCTION ................................................. ..............

Zpslp-like Proteins from Candida albicans and Aspergillus spp. ...................... 2
Zpsip from Saccharomyces cerevisiae............... ................... 10
Zinc-dependent Metalloproteases of the M35 Clan....................... ........ 12
Comparison of the Zpslp-like Proteins and the M35 Metalloproteases............ 14

2 RE SU LTS AN D D ISCU SSION ................................................................. ......16

Regulation of ZPSI Gene Expression........................... .............. 16
Partial Purification of Zpslp from Inclusion Bodies .......................................... 17

3 CONCLUSIONS..........................................................26

4 M ATERIALS AND M ETHODS................................ .................. 27

Growth Media................. ... .. ..... ................27
Solutions and Buffers for Yeast Transformations and P-Galactosidase Assays... 28
Bacterial and Yeast Strains ................................. ......................... .. .... 29
Y east T ransform nations ....................................................................... 30
P-G alactosidase A ssays................... ... .................................... 3 1
Cloning of ZPS1 and Construction of an E. coli Expression Plasmid.................. 32
Expression of Zpslp in E. coli..................... ............ .............. 33
Estimation of Protein Purity by SDS-PAGE ............... ..... ................. 34

LIST OF REFERENCES ....................... ......... ........35

BIOGRAPHICAL SKETCH .................................................. ............... 39








v
















LIST OF FIGURES


Figure page

1-1. Multiple sequence alignment of fungal cell wall proteins with related
metalloproteases. ................................................3

1-2. Active-site residues of deuterolysin.43........... ............................ ............ 13

1-3. Basic structural features of the Zpslp-like proteins and the metalloproteases in the
M 35 clan....................................... ................................ ......... 14

1-4. Active site structures. On the right is the known active site of the aspzincins,
deduced from the crystal structures of deuterolysin43 and G/MEP.49 On the left is a
possible structure of an active site within the Zpslp-like proteins. .......................15

2-1. Zinc and iron responsiveness of the ZPS]-lacZ reporter. P-Galactosidase activity in
wild-type cells and zap] mutant cells grown in CSD. ..........................................16

2-2. Zinc and iron responsiveness of the ZPS]-lacZ reporter. 3-Galactosidase activity in
wild-type cells and riml01 mutant cells grown in CSD. .......................................18

2-3. SDS-PAGE analysis of E. coli transformants containing the pET-22b(+)-ZPS]
expression vector ............. ................ ........ .........19

2-4. SDS-PAGE analysis of soluble and insoluble components of the cell lysate obtained
from breakage of E. coli expressing Zpslp.................. .......................20

2-5. SDS-PAGE analysis of the soluble and insoluble products obtained after
solubilization and refolding of the inclusion body pellet...................................20

2-6. SDS-PAGE analysis of the major protein peak collected after SEC (combined
fractions 9 12)..................................... ......... 21

2-7. SDS-PAGE analysis of the washed inclusion body pellet, solubilized in Buffer A
containing 8 M Urea. .......... ... .. ........................23

2-8. SDS-PAGE analysis of the soluble and insoluble products obtained after
solubilization and refolding of the inclusion body pellet................ .................24
















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

CHARACTERIZATION OF THE ZPS1P CELL WALL PROTEIN FROM
Saccharomyces cerevisiae

By

Stephanie L. Drobiak

December 2004

Chair: Thomas Lyons
Major Department: Chemistry

Fungal cell wall proteins are involved in establishing infection through interaction

with host ligands and by mediating morphological changes that enhance pathogenicity.

In recent years, research has focused on a family of fungal cell wall proteins that are

structurally related to zinc-dependent metalloproteases of the M35 clan. Members of this

protein family include Zpslp from Saccharomyces cerevisiae, Pral from Candida

albicans, CpAspf2 from Coccidioides posadassii, Aspndl from Aspergillus nidulans, and

Aspf2 from Aspergillusfumigatus. The proteins from C. albicans and Aspergillus spp.

are known cell-surface antigens during fungal infections, and both Pral and Aspf2 bind

specific ligands within mammalian hosts. Although expression of these proteins during

fungal infection is well documented, their biological function remains unknown. In this

thesis, we report preliminary work toward characterization of Zpslp from S. cerevisiae.

Results indicate expression of ZPSI to be regulated in response to zinc- and iron-

limitation, as well as extracellular pH. In addition, we present the partial purification of









recombinant Zpslp from bacterial inclusion bodies. Analysis of Zpslp is intended to

provide the framework for future expression, purification, and characterization of the

Zpslp-like proteins from the medically important fungi C. albicans and Aspergillus spp.














CHAPTER 1
INTRODUCTION

Many fungi are responsible for both superficial and systemic infections in man.

Immunocompromised individuals are susceptible to fungal infections caused by a variety

of pathogens, including Candida albicans and several Aspergillus species. Relevant

diseases caused by these species include candidiasis, aspergilloma, invasive aspergillosis,

and allergic bronchopulmonary aspergillosis (ABPA). Although these mycoses are well

documented, many factors contributing to fungal pathogenesis are still not well

understood. Efforts to better understand virulence factors often focus on components of

the fungal cell wall.

The fungal cell wall is a complex mixture of carbohydrates (80 to 90%), proteins

(6 to 25%), and minor amounts of lipid (1 to 7%).1 As the outermost part of the cell, the

wall initiates physical interaction between the microorganism and the environment,

including the host. The host-parasite interaction, resulting in adhesion, is the first critical

step in establishing infection and modulation of the host immune response. In addition,

the cell wall mediates fungal cell-cell adhesion flocculationn), a first step in the

morphological change from a unicellular yeast to growth as multicellular filaments

(mycelia or hypha). Formation of mycelia enhances pathogenicity, allowing the invasion

of host tissues, and is influenced by environmental variables including extracellular pH2'3

and nutritional status.4 For these reasons, fungal cell wall proteins (CWPs) have been of

heightened interest. Not only are CWPs involved in intercellular binding, many possess

enzymatic activity involved in cell wall biosynthesis and maintenance, and acquisition of









extracellular nutrients.1 When the actions of CWPs negatively impact the viability of the

host, the proteins are considered virulence factors that advance the establishment of

infection. Due to their accessibility at the cell surface, and their critical role in

intercellular interactions, CWPs are ideal targets for the development of antifungal drugs.

A family of cell wall proteins from various fungi has been the focus of much

research in recent years. Members of this family include Zpslp from Saccharomyces

cerevisiae, Pral from Candida albicans, CpAspf2 from Coccidioides posadasii, and

Aspndl and Aspf2 from Aspergillus nidulans and Aspergillusfumigatus, respectively.

These CWPs share a number of key structural features, have high sequence homology,

and exhibit significant similarity to a family of zinc-dependent metalloproteases of the

M35 clan, known as the aspzincins5 (Figure 1-1). At present, the biochemical function of

these proteins remains unknown.

The focus of this research is to characterize the Zpslp cell wall protein from the

yeast Saccharomyces cerevisiae. This document entails the preliminary work toward

characterization of Zpslp through study of its structure, function, and gene regulation in

S. cerevisiae. Analysis of Zpslp is intended to provide the framework for future

expression, purification, and characterization of the Zpslp-like proteins from the

medically important fungi C. albicans (Pral) and Aspergillus spp. (Aspndl and Aspf2).

The relevance of the Zpslp-like proteins is discussed below.

Zpslp-like Proteins from Candida albicans and Aspergillus spp.

The homologues from A. fumigatus and A. nidulans are known as Aspf26 and

Aspndl,7 respectively. In C. albicans, the homologue is known by many names: Pral

(pH regulated antigen),8 FBF (fibrinogen binding factor),9 FBP1 (fibrinogen binding

protein),10 and mp58 (58-kDa fibrinogen-binding mannoprotein).11













NPII Secondary Structure NX(
ellp MKFSSGKS l FATIASLALSAPVTY-. T Y .- t. FI T -1
SPutetu eC teavame SIcu t- F5
T T -
RED T
SNxT = putting Nzlyryslation rite PVY Y rD Y N P I HG DL -
Zn=Zinligand i n -
XMiCUM important active siresidue VVNG E
XC TX Boxing/Shading = 45% identity MVNG i
Boldboxing=invarriantritkilreidue -M S ;AJ L A
p disulfidebond AT FVG SA ROTLNA SO NIA
afinep Secondary Structure -

-a- -p- --- -a-
1p r NYGVDDVYYIKR F 3ANGS IFTVMG VFE QLMEASKGA LMR D I01 A A N GHHROSAA P TV D Y 5E TTS K
1 'EET I







-a- "B l -[ -
RIC 33 So Y F I T T t. .AL
ITa a UK IR KVY V R E













r= r1e TTT IA GDG TA I
3 r. A I



--c --a- -P- -P- -- t -a









XAnt arrd etght c e ei ir
-L 1LA3 NCLO1
te i O- g t o i a io .lo o i A LK [ N Q











Apnd1 33lacks [J F a Thr- Nich -- egiIon.. H v t C-t regio o .Ap:
T.a S E N T i I













bchemal ca r funcT iF or all thets excep Zprthe m rep hant d
Asergillus ai o n ag ng-------aie- OT
AA rre N
function.TTG- T 1 - - - r . . .





A 1s D S- a- -How r th C-tri ri o Asp
ioc h mica l Sf o orrQ Rih Ceil Wall Bindingt Domain Metap Bindinf Site?
As. rI ll -- I.m. .d m .. i fTn i
N Multiple sequence alignment of funga I antigens with related metalloproteases.
WXAEB i7e N For all proteins except Zps Ip the mature peptide is shown.
gr1p i7 s Secondary structure based on crystal structures for NPII (top) and gfmep (bottom).


Figure 1-1. Multiple sequence alignment of fungal cell wall proteins with related
metalloproteases.


Aspf2, Aspndl, and Pral are all secreted proteins with four N-glycosylation sites (Asn-


X-Ser/Thr) and eight cysteine residues perfectly conserved, suggesting a similar


function.6711 Both Pral and Aspf2 contain a serine- and threonine-rich region of


potential 0-glycosylation close to the C-terminus, a purported cell wall binding domain


(CWBD).12 Studies have shown that Pral does indeed contain O-linked sugar moieties."


Aspndl lacks this Ser- and Thr- rich region. However, the C-terminal region of Aspndl


is glutamate- and glutamine-rich, the purpose of which is unclear. Although their


biochemical function remains unknown, these three proteins from Candida and


Aspergillus are immunodominant antigens in fungal infections.









Purification and characterization of various Aspergillus spp. cell wall proteins for

use in immunodiagnosis of ABPA and other diseases led to the identification of Aspf2

and Aspndl as proteins that elicit a strong immune response.6'7'13'14 These discoveries

were made by immunoblotting water-soluble extracts of Aspergillus spp. with sera from

patients with ABPA. Sera from those infected with different forms of aspergillosis

contain elevated levels of immunoglobulin G (IgG) and immunoglobulin E (IgE)

antibodies specific for Aspergillus antigens, including Aspndl and Aspf2. These

antigens are consistently recognized by serum samples from aspergilloma patients, but

not with sera from control or healthy individuals.7'14 Deglycosylated forms of the

purified proteins remain reactive to the antibodies, suggesting the N-glycosidic groups

are not required for recognition by the aspergillosis serum samples tested. These data

indicate that the epitopes recognized are located mainly in the polypeptide region.7;13

This hypothesis is further supported by antibody reactivity with the recombinant forms of

Aspndl and Aspf2 which, when over-expressed in the prokaryote E. coli, lack the

glycosidic moieties. 1516 Furthermore, all reactivity was abolished following protease

7;13
treatment.7

During characterization of the Aspndl antigen from A. nidulans, Calera et al.

observed sera that reacted with Aspndl also consistently reacted with antigens from A.

fumigatus. Therefore, they tested the reactivity of purified anti-Aspndl specific IgG with

several different A. fimigatus antigens including Aspf2 (also known as gp55). The

various antigens reacted with the anti-Aspndl, suggesting a close relationship between

the immunodominat antigens from the two Aspergillus species, possibly through the

existence of common peptide epitopes.7 Likewise, Banerjee et al. detected binding of









anti-Aspf2 antibodies by Aspndl. The relatedness of Aspndl and Aspf2 is supported

through analysis of their primary structure. The similarity between the two proteins

suggests they share several epitopes and should therefore elicit the formation of IgG and

IgE antibodies able to recognize both antigens. To answer this question, Banerjee et al.

compared the reactivity of purified Aspndl and Aspf2 toward IgE antibodies from ABPA

serum. The mean IgE binding of purified Aspf2 was almost three fold higher than

binding of Aspndl. Differences in Aspnd and Aspf2 binding to IgE may result from

differences in posttranslational modifications or the tertiary structures of these proteins.6

Comparison of IgE binding by both native and recombinant forms of these antigens

indicates that the recombinant forms most likely have structures functionally comparable

to the native proteins, since IgE binding is dependent on proper three-dimensional

structure.15;16

Similarly, Pral from C. albicans elicits a strong immune response with sera from

patients infected with candidasis. Antibodies against Pral are present in sera from

patients with systemic candidasis,17;18 and Pral itself has been detected in the cell wall of

clinical isolates of C. albicans.17 Deglycoslyation of Pral did not affect reactivity with

anti-Pral antibodies, again suggesting the epitopes recognized are located mainly in the

polypeptide region. An extensive epitope-scanning study, employing a complete set of

overlapping dodecapeptides deduced from the Pral sequence, identified several

immunoreactive continuous B-cell epitopes within the protein sequence. Six regions of

elevated reactivity were identified, including four internal regions and both the amino and

carboxy termini of the mature polypeptide. Of these regions, the C-terminal domain was









highly reactive towards the anti-Pral antibodies and therefore subjected to further epitope

mapping.18

Analysis of the epitopic region at the C-terminal domain of the Pral polypeptide

identified the nonapeptide 290HTHADGEVH298 as the minimal region required to retain

antibody-binding activity. Researchers further probed the significance of this epitopic

region by synthesizing a synthetic peptide corresponding to the last ten amino acid

residues at the C-terminus. The synthetic peptide, coupled to keyhole limpet hemocyanin

(KLH), was used to immunize two mice. The serum samples obtained from the two

immunized mice were able to recognize Pral from cell wall extracts of C. albicans with

high specificity. Interestingly, this C-terminal sequence (CHTHxxGxxHC) is conserved

in both Aspndl and Aspf2, as are the four internal epitope regions (Table 1-1). The

conservation of linear epitopes within this family of cell wall proteins from various

fungal pathogens provides additional support suggesting their role in the host-parasite

interaction.18

Table 1-1. Sequence homology of five of the identified IgG epitopes. 18
Pral Antigen Sequence
epitope sequence Aspnd1 Aspf2

LRFGSK LRWGNE LRWGNE
RKYF RKYF RKYF
NDGWAGYW LEGWGGHW LEGWGGHW
DVYA EVYA EAYA
HTHADGEVH HTHEGGELH HTHEGGQLH

Not only do these fungal antigens interact with antibodies within the host, but

Aspf2 and Pral also bind specific host ligands. Fungal adhesion to host cells and tissues

initiates establishment of infection and is considered a potential virulence factor. Aspf2









binds the extracellular matrix protein laminin,6;19 and Pral binds the serum protein

fibrinogen.9; 11

Proteins in the extracellular matrix (ECM) are known to bind to A. fumigatus

conidia (an infectious airborne form of the fungus). Binding of conidia to ECM ligands

is believed to be a crucial first step initiating aspergillosis, and specific recognition of

these ligands may greatly influence pathogenicity.2021 One component of the ECM is

laminin, a multidomain glycoprotein and major component of the basement membrane.

Interaction of laminin with cell surface ligands facilitates cell-cell adhesion, cell

migration and cell differentiation.1 As reported by Banerjee et al., laminin shows a dose-

dependent interaction with Aspf2. Both native and recombinant Aspf2 demonstrate high

binding affinity to laminin, with greater affinity observed by the native protein. With

specific binding to laminin, and significant homology to Pral (which binds fibrinogen),

the involvement of Aspf2 in fungal adherence to the ECM may play an important role in

establishing pathogenicity.6

Blood serum proteins (e.g., serum albumin, transferring, fibrinogen, complement

fragments C3d and iC3b) are additional targets for fungal binding. Interactions of C.

albicans with fibrinogen have been well characterized.1 In 1987, Bouali et al. identified

a fibrinogen binding factor (FBF) on the surface of C. albicans germ-tubes and

mycelium, the fungal forms most often found in infected tissues.9 Five years later, in

1992, Casanova et al. identified this FBF as a 58-kDa fibrinogen-binding mannoprotein

(mp58), which is now known to be Pral. Binding of Pral to fibrinogen is apparently

specific, since binding to other mammalian proteins tested (laminin, fibronectin, C3d,

type IV collagen) was not observed. O-deglycosylated Pral was unable to interact with









fibrinogen, implying this carbohydrate domain may play a role in binding. The in vivo

production of Pral during candidasis and its ability to bind fibrinogen suggest a role in

infection.1'1

Another factor supporting an active role of Pral in candidasis is its differential

expression in response to pH.8 The ability of C. albicans to grow and differentiate over a

broad pH range is critical for its survival in a variety of environments and host tissues

(e.g., blood, pH ~ 7.2; vaginal tract, pH ~ 4.5).3 Extracellular pH is an environmental

signal that regulates the yeast-to-mycelia transition in vivo, a morphological change that

greatly enhances invasion of host tissues.2 In C. albicans, gene expression is regulated

by the RimlOlp transcription factor in response to alkaline pH.22 Studies have shown the

C. albicans RimlOlp pH response pathway to be required for several host-pathogen

interactions, and therefore essential for pathogenesis.23 Pral is a RimlOlp target gene

maximally expressed at neutral pH, with no detectable expression below pH 6.0.

However, ambient pH is not the sole factor influencing expression. When cultured in

rich medium (YPD) buffered at pH 7.0, no Pral production was detected. This result

implies partial regulation by nutritional status, a hypothesis that remains to be tested.8

Although the effect of nutritional status on Pral expression has not been assessed,

the nutrient regulation of the Apsergillus spp. antigens has been investigated.

Researchers recognized that production of Aspndl and Aspf2 only occurred when the

fungi are grown in certain conditions, especially in Czapek-Dox (CD) medium (3g

NaNO3, 0.5g MgS04'7H20, 0.5g KC1, 55mg FeS04, Ig KH2PO4, and 30g sucrose per

liter). Therefore, the various components of CD medium were tested to determine which

is influencing antigen production. Elucidation of the regulatory elements responsible for









Aspndl and Aspf2 expression may provide clues to their function and potential roles in

virulence.24

Variations of CD medium were quantitatively and qualitatively tested against a

control medium, AMM (1% glucose, 0.6% NaNO3, 0.052% MgS04, 0.052% KC1, 0.15%

KH2PO4, and traces ofFeS04 and ZnS04), known not to stimulate Aspndl or Aspf2

production under normal conditions. The type and amount of carbon or nitrogen source

did not affect antigen production, nor did addition of iron to the CD medium. However,

addition of [[-molar concentrations of zinc eliminated antigen synthesis in CD, while

removal of zinc from AMM medium induced antigen production. Addition of other

divalent metals (Co2+, Ni2+, CU2+, Ca2+) had no inhibitory effects, with the exception of

Cd2+ and Mn2+ (only slight inhibition). Currently, researchers are attempting to identify

and characterize potential zinc response elements (ZREs) in the promoter regions of

ASPND1 and ASPF2.24 Detected in the promoter region ofASPND] are at least five

potential PacC binding sites.7 PacC is a pH responsive transcription factor in Aspergillus

spp. and is homologous to C. albicans RimlOlp.25 The presence of putative PacC sites

suggests possible regulation of Aspndl expression in response to ambient pH.

Regulation of Aspndl and Aspf2 expression by zinc deficiency may play an

important role in pathogenesis. During infection, the host environment is one of

nutritional limitation. In efforts to starve invading pathogens, part of the acute-phase

response of the human immune system is to redistribute micronutrients like iron and zinc

to the liver.26 Therefore, this regulation may illustrate the role of zinc's nutritional status

as a signal for fungal pathogens of a host environment, initiating transcription of genes

involved in zinc acquisition and transport or commencement of pathogenesis. Bacterial









hemolysins offer precedent for the metalloregulation of virulence factors.27 The partial

regulation of Pral expression by nutritional status may also have important implications

in pathogenesis. The effect of zinc limitation on Pral expression may be worth

investigation, since zinc deficiency has been shown to induce mycelium formation in

several dimorphic yeasts.4:28

Zpsip from Saccharomyces cerevisiae

Within the family of cell wall proteins, the homologue from Saccharomyces

cerevisiae is Zpslp (Yoll54w). Although S. cerevisiae is typically nonpathogenic, it is

able to infect immunocompromised individuals29 and colonize complement factor five-

deficient mice.30 Zpslp is a secreted cell wall protein,31 with two putative N-

glycosylation sites and six cysteine residues conserved with respect to Pral, Aspndl, and

Aspf2. Unlike the homologues from Candida and Aspergillus spp., Zpslp has a

truncated C-terminal region lacking the potential cell wall binding domain and the

conserved antigenicity determinant. Like its related fungal antigens, the function of

Zpslp is also unknown. Disruption of the ZPS1 gene failed to reveal any strong

phenotype and resulted in a viable strain, indicating that Zpslp is non-essential.32

Although the function of Zpslp is unknown, many research groups have provided

information about its regulation. Interestingly, Zpslp expression is regulated by some of

the same factors as the other fungal antigens, including zinc limitation and extracellular

pH.

In S. cerevisiae, the transcription factor Zaplp is activated by zinc deficiency.33

DNA microarray data has shown Zaplp regulates expression of 46 genes in S. cerevisiae

under zinc deficient conditions. Of these genes, one of the most heavily induced is ZPS1.

This result was confirmed by measuring zinc-regulation of a ZPS]-lacZ reporter









construct, resulting from fusion of the ZPS1 promoter region (-1000 bp to ATG) to the

lacZ reporter gene. Zaplp activates gene transcription during zinc deficiency by binding

to a zinc response element (ZRE) upstream of the target gene's start codon. The

consensus ZRE recognized by Zaplp is ACCTTNAAGGT. Within the ZPS1 promoter

region are two putative ZREs between -300 and -340 bp upstream of the start codon:

ACCTTCAGGGT (-328 to -318) and ACCCTGAAGGT (-313 to -303). DNA

microarray data indicated that ZPS1 was induced 14 fold by zinc deficiency, while ZPS1-

lacZ fusion constructs were 10 times more inducible.34

Expression of Zpslp is also affected by alkaline pH. This regulation is dependant

on the S. cerevisiae RimlOlp transcription factor, which is homologous to the RimlOlp

and PacC transcription factors of Candida and Aspergillus, respectively. Using the

ZPS]-lacZ construct, researchers observed a 100-fold increase in expression at pH 8

compared to pH 4, while alkaline induction did not occur in a rim]01A strain. In

addition, ZPS1 is more highly expressed in yeast strains harboring a hyperactive allele of

RimlOlp at pH 4.35 This direct regulation of ZPS1 expression by RimlOlp is intriguing,

for not only is RimlOlp structurally similar to Zaplp, but these two proteins also interact

in vivo,36 suggesting they may co-regulate ZPS1 expression.

Potential regulation of ZPS1 by environmental iron status has also been implied in

work studying iron-regulatory systems in yeast.37 In S. cerevisiae, iron homeostasis is

regulated by the Aftlp transcription factor in response to low-iron conditions.38 In

addition, S. cerevisiae contains a homologue of Aftlp, known as Aft2p, which regulates

transcription of many of the same genes as Aftlp during iron deficiency.37 In strains

harboring a hyperactive allele of Aft2p, activation of ZPS1 was increased over 8 fold









when compared to wild type strains. This effect is dependent on Zaplp, suggesting Aft2p

activity affects zinc metabolism.37 The data obtained through study of ZPS1 regulation

further support its similarity to the fungal antigens from C. albicans and Aspergillus spp.

Zinc-dependent Metalloproteases of the M35 Clan

Based on sequence comparison and structural predictions, the Zpslp-like proteins

show similarity to zinc-dependent metalloproteases of the M35 clan5 (known as the

aspzincins). Included in this subfamily of secreted metalloendopeptidases (MEPs) are

deuterolysin (neutral proteinase, NPII, aspzincin) from Aspergillus oryzae,39

penicilloysin (PlnC) from Penicillium citrinum,40 mep20 from both Aspergillus fumigatus

and Aspergillus flavus,41 and the AVR Pi-ta avirulence determinant from Magnaporthe

grisea.42 Many of these species are known pathogens. This protein family is

characterized by a leader sequence directing the protein into the secretary pathway, a

long pro-peptide that is cleaved during secretion, a mature polypeptide that contains three

disulfide bonds, and two highly conserved motifs: HExxH and GTxDDxxYG.43 A

crystal structure of deuterolysin,43 supported by site directed mutagenesis studies,39

indicated the two histidine residues of the HExxH motif, the second aspartate residue of

the GTxDDxxYG motif, and two water molecules were the zinc binding ligands. The

conserved glutamate is a catalytic residue, promoting the nucleophilic attack of a water

molecule on the carbonyl moiety of the substrate. The conserved tyrosine residue

interacts with the second zinc bound water molecule, possibly stabilizing the transition

state by hydrogen bonding interactions.43 Figure 1-2 shows the crystal structure of the

active site residues.





















More distantly related members of the M35 clan include GfMEP from Grifola

frondosa, PoMEP from Pleurotus ostreatus,44 AmMEP from Armillariella mella,45 eprAl

from Aeromonas hydrophila,46 asaPi from Aeromonas salmonicida,47 XAC2763 from

Xanthomonas axonopodis, and XCC2062 from Xanthomonas campestris.48 Many of

these species are also pathogenic. Furthermore, AmMEP from the edible mushroom A.

mella is known to hydrolyze fibrinogen.45 The crystal structure of GJMEP has been

solved. Despite one less disulfide bond, GJMEP possesses a near identical fold and

active site as deuterolysin, suggesting a conserved mechanism.49

The substrate specificities of deuterolysin, PlnC, and mep20 are toward basic

polypeptides. Both deuterolysin and PlnC show high activities on the basic nuclear

proteins histone, protamine, and salmine, but very low activities on milk casein,

hemoglobin, albumin, and gelatin.39 Further analysis of deuterolysin's substrate

specificity indicates high proteolytic activity toward the peptide bonds next to pairs of

basic residues.50 GJMEP and PoMEP have strict specificity toward acyl-lysine bonds,

also basic in nature.44 Analysis of the GJMEP structure reveals and electrostatically

negative region that attracts a positively charged lysine side chain of a substrate.49










Comparison of the Zpslp-like Proteins and the M35 Metalloproteases

Although Zpslp and the related fungal antigens possess similarities to the M35

clan (i.e., secretary signal, conserved cysteine residues), they differ in their most highly

conserved motifs. Figure 1-3 illustrates the basic structural features of the Zpslp-like

proteins and the M35 proteases.


Zpsip x Ci M HH ExxE C


Pral,Aspf2 I IZC HRMxH DKOED C CS.T-rigCHi
CpAspf2








GfMEP dZ.i.
in the M35 clan.











The Zpslp-like proteins lack the HExxH and GTxDDxxYG motifs found in the
metallo-proteases. However, they contain highly conserved CTRxxH and D/ExxD/E
















the metalloproteases in the M35 clan with a possible structure in the Zpslp-like proteins.
AVA Pi-1.
GfMIP zn2+
PMEP
e.,AlI Cgta? pr~WiMUp< CLC C HBExH GTxDDlfVGI

XCC2I02 I = leader peptide (scretory signal) ZL2+

Figure 1-3. Basic structural features of the Zpslp-like proteins and the metalloproteases
in the M3 5 clan.

The Zpslp-like proteins lack the HExxH and GTxDDxxYG motifs found in the

metallo-proteases. However, they contain highly conserved HRxxH and D/ExxD/E

motifs, which may serve as functional replacements enabling metal binding and

potentially proteolytic activity. Figure 1-4 compares the known active site structure of

the metalloproteases in the M35 clan with a possible structure in the Zpslp-like proteins.












Ei29 R187

IH H H 1
V- I Y146 Ito I
4128 --D HI0 EZa

D 0143 < E205

H132 H 190

Aspzincins Zpslp-like proteins

Figure 1-4. Active site structures. On the right is the known active site of the aspzincins,
deduced from the crystal structures of deuterolysin43 and GJMEP.49 On the
left is a possible structure of an active site within the Zpslp-like proteins.

Despite their similarity to the M35 metalloproteases, it is quite possible that the

Zpslp-like proteins do not act as metal-binding proteins or possess proteolytic activity.

However, the HRxxH and D/ExxD/E motifs highly conserved within the Zpslp family of

cell wall proteins may act as peptide binding ligands, enhancing potential virulence

within a host. The significance of these motifs can be thoroughly probed through study

of purified Zpslp structure and function.

















CHAPTER 2
RESULTS AND DISCUSSION

Regulation of ZPS1 Gene Expression

ZPS1 regulation in S. cerevisiae was studied using the ZPS]-lacZ reporter


construct. P-Galactosidase activity, reported in Miller units, was measured as a function

of growth condition. To confirm the dependence of Zaplp on ZPS1 regulation, we

monitored the responsiveness of the ZPS]-lacZ reporter to zinc deficiency.

Simultaneously, we further probed the apparent regulation by iron status by measuring

activity of the ZPS]-lacZ reporter in response to growth under iron deficient conditions

and combined zinc- and iron-limitation (Figure 2-1).


4500
4000
S3500
3000-
E 2500 H WT
2 2000 *zapl
U 1500 -
9 1000 -
500 -
0
Fe-/Zn- Fe+/Zn- Fe-/Zn+ Fe+/Zn+
Growth Condition

Figure 2-1. Zinc and iron responsiveness of the ZPS]-lacZ reporter. P-Galactosidase
activity in wild-type cells and zap] mutant cells grown in CSD with or
without 10[M iron and/or zinc added.

The ZPS]-lacZ reporter construct was indeed regulated by zinc in a Zaplp

dependent manner, with no detectable expression in a zap] knockout strain. Induction

was only observed when the yeast were grown under zinc-deficient conditions, with no

measurable increase in ZPS]-lacZ activity when the yeast were grown under solely iron









deficient conditions. When the yeast were grown under both zinc- and iron-limitation, a

significant increase in ZPS]-lacZ activity was observed. As the literature previously

suggests, the increased ZPS1 expression by iron-deficiency may be due to Aft2p.37 To

further investigate this hypothesis, future work may involve monitoring ZPS1 expression

in strains lacking Aft2p, Aftlp, or both.

Previously, reports have described ZPS1 regulation by RimlOlp in response to

alkaline pH.35 Therefore, we attempted to study ZPS]-lacZ activity in response to iron

and/or zinc deficiency at both acidic and alkaline pH. Under standard growth conditions,

the Chelex-treated synthetic defined medium (CSD) used to limit zinc and iron

availability is at pH 4 (optimal for yeast growth). When the CSD medium was buffered

to pH 8.0, the metals in the medium became insoluble and precipitated out of solution.

Therefore we were limited to monitoring the effects of RimlOlp on ZPS]-lacZ

expression at acidic pH (Figure 2-2). When compared to wild type yeast, strains lacking

RimlOlp exhibit a significant decrease in ZPS]-lacZ activity, which remained a function

of zinc-deficiency. This result suggests that RimlOlp affects ZPS1 expression even at

acidic pH, possibly by enhancing Zaplp regulation of this gene. These observations

further support the hypothesis that Zaplp and RimlOlp co-regulate ZPS1.

Partial Purification of Zps1p from Inclusion Bodies

We are currently attempting to purify recombinant Zps1p from Escherichia coli

for use in characterizing Zpslp structure and function. Although Zpslp is not native in

E. coli, expression of yeast proteins in bacterial systems has several advantages,

including high yield and lack of glycosylation moieties, which may complicate protein

purification.











4500
4000-
.5 3500-
S3000-
C 2500- m WT
'V 2000- riml01
1500-
( 1000
500
0
Fe-/Zn- Fe+/Zn- Fe-/Zn+ Fe+/Zn+
Growth Condition

Figure 2-2. Zinc and iron responsiveness of the ZPS]-lacZ reporter. 3-Galactosidase
activity in wild-type cells and riml01 mutant cells grown in CSD (pH 4.0)
with or without 10[M iron and/or zinc added.

As described in the Materials and Methods section, ZPS1 (lacking the leader

peptide sequence) has been cloned by the polymerase chain reaction (PCR) and inserted

into the pET-22b(+) expression vector for isopropyl-P-D-thioglactopyranoside (IPTG)

inducible expression by bacteriophage T7 RNA polymerase in BL21(DE3) E. coli. When

expressed, the mature form of Zpslp should have an apparent molecular weight of ~ 25.5

kDa. Approximately eight hours of induction by IPTG is required for optimum Zpslp

yield (Figure 2-3).

Following large scale induction (as described in Methods section), the resulting

pellet was thawed and resuspended in 20 mL of cold 50 mM Tris(hydroxymethyl)

aminomethane (Tris), buffered at pH 7.4, containing 1 mM phenylmethanesulfonly

fluoride (PMSF), a protease inhibitor used to prevent degradation of Zpslp. The cells

were lysed using several cycles of French press at 4oC. Multiple rounds of French press

were required to adequately break the large cell pellet resulting from 8 h of growth. The

resulting lysate was centrifuged at 19,000 rpm for 20 min at 4oC and the supernatant was

decanted and saved.









M C1 2h 4h 6h 8h C2





29 kDa-
22 kDa-





Figure 2-3. SDS-PAGE analysis of E. coli transformants containing the pET-22b(+)-
ZPS1 expression vector not induced (lane C) or induced with IPTG for the
number of hours indicated (lanes 2 8 h). For comparison, products from
non-induced cells after 8 h growth are also shown (lane C2). In lane M is a
molecular weight marker.

At this time, the soluble supernatantt) and insoluble (pellet) components of the lysate

were analyzed by SDS-PAGE to determine the location of Zpslp (Figure 2-4). Zpslp

was present in the insoluble fraction, indicating the protein accumulates as inclusion

bodies (dense aggregates of misfolded polypeptide). Formation of recombinant Zpslp

inclusion bodies is not unexpected, given that expression of recombinant Aspndlp in E.

coli also results in inclusion body formation.15

To solubilize the inclusion bodies, 5 mL of 50 mM Tris (pH 7.4) containing 8 M

Urea was used to denature the Zpslp aggregates by gentle mixing overnight. Next, we

attempted to refold the denatured protein by single-step dilution. This entailed slowly (>

24 h) dripping 50 mL of buffer into the sample so to gradually decrease the concentration

of Urea to ~ 0.7 M. Dilution was followed by dialysis to remove all traces of the

denaturant and the sample was centrifuged at 8,000 rpm for 20 min at 40C to collect any

insoluble material. The soluble and insoluble components were analyzed for Zpslp










content by SDS-PAGE (Figure 2-5). The results indicated Zpslp was successfully

solubilized, with only trace amounts in the insoluble fraction.


C I


S P


SDS-PAGE analysis of soluble and insoluble components of the cell lysate
obtained from breakage of E. coli expressing Zpslp. Lane M, molecular
weight marker; Lane C, non-induced E. coli (8 h growth); Lane I, IPTG
induced E. coli (8 h growth); Lane S, soluble fraction; Lane P, insoluble
fraction. In attempts to load the maximum sample volumes to each lane,
runoff into neighboring lanes occurred (Lanes X).


Soluble





1. A&.


Insoluble


Figure 2-5. SDS-PAGE analysis of the soluble and insoluble products obtained after
solubilization and refolding of the inclusion body pellet. Lane M, molecular
weight marker.


29 kDa
22 kDa


Figure 2-4.
















29 kDa-
22 kDa-









Next, we attempted to purify the soluble Zpslp by size-exclusion chromatography

(SEC). The protein sample was concentrated, applied to a column containing Sephadex

G-75 (Sigma) size-exclusion resin (molecular weight cutoff ca. 80 kDa), with a bed

volume of approximately 310 mL. The protein was eluted using 50 mM Tris (pH 7.4).

After elution of the void volume, 2 mL fractions were collected and analyzed for protein

using the method of Bradford.51 The Bradford protein assay indicated that the protein

eluted as one major peak (fractions 9 12) shortly after collection of the void volume.

Fractions 9 12 were combined and the content was analyzed by SDS-PAGE (Figure

2-6).

M C I E





29 kDa

22 kDa







Figure 2-6. SDS-PAGE analysis of the major protein peak collected after SEC
(combined fractions 9 12). Lane C, non-induced E. coli (8 h growth); Lane
I, IPTG induced E. coli (8 h growth); Lane E, protein eluate from SEC
column.

The results indicated poor separation of Zpslp from contaminating proteins. The

lack of separation may result from aggregation of Zpslp with other peptides, possibly due

to unfavorable disulfide bridging involving one ofZpslp's six cysteine residues.

Therefore, use of a reducing agent such as dithiothreitol (DTT) during the refolding,









concentrating, and chromatographic steps may prove effective in decreasing unfavorable

disulfide bond formation.

To reduce the concentration of contaminating proteins that could unfavorably

interact with Zpslp forming aggregates, a purification strategy was adapted from a

published method.52 This method involves washing the inclusion body pellet with the

detergent sodium deoxycholate (DOC) to remove impurities. First, the frozen cell pellet

obtained after IPTG induction was thawed and resuspended in 20 mL of cold Buffer A

(5% Glycerol, 50 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl) containing 1 mM PMSF

and 0.1 mM DTT. The cells were lysed using several cycles of French press at 40C.

Next, DOC was added to the lysate to give a concentration of 0.2% (approximately 240

[IL of a 20% DOC stock), which is used to help liberate slightly insoluble proteins. The

solution was mixed well, allowed to stand for 10 min at room temperature, and

centrifuged at 13,000 rpm for 10 min at 40C. The supernatant was decanted and saved for

future analysis (Supernatant 1).

Following collection by centrifugation, the inclusion body pellet appears as a

white bull's-eye, which is the inclusion body protein, surrounded by a brownish layer.

The brownish layer consists of contaminating cellular debris that can be effectively

solubilized by washing the pellet with 2% DOC. Therefore, the inclusion body protein

was washed by resuspending the pellet in 18 mL of Buffer A (containing 1 mM PMSF

and 0.1 mM DTT) and 2 mL of 20% DOC. The solution was allowed to stand for at least

10 min at room temperature before being centrifuged at 13,000 rpm for 10 min at 40C.

The supernatant was decanted and saved for future analysis (Supernatant 2). The

remaining pellet was washed one additional time and, after centrifugation, the









supernatant was decanted and saved for future analysis (Supernatant 3). At this time, the

washed inclusion body pellet was solubilized by resuspending in 5 mL Buffer A

containing 8 M Urea and gently agitated overnight at 40C. Prior to refolding by single-

step dilution, the protein purity was assessed by SDS-PAGE to determine the

effectiveness of the DOC wash (Figure 2-7). The gel showed few major bands, one being

Zpslp, thus demonstrating the value of the DOC wash in purifying the inclusion body

protein.

M C I P





29kDa --
22 kDa -





Figure 2-7. SDS-PAGE analysis of the washed inclusion body pellet, solubilized in
Buffer A containing 8 M Urea (Lanes P). Lane M, molecular weight
marker; Lane C, non-induced E. coli (8 h growth); Lane I, IPTG induced E.
coli (8 h growth).

Next, we attempted to refold the solubilized inclusion body protein by single-step

dilution using Buffer A. As before, this procedure involved slowly decreasing the

concentration of Urea by dilution followed by dialysis. In an attempt to discourage

unfavorable disulfide bridging, 0.1 mM DTT was added to the buffer during the refolding

process. After dialysis, the sample was centrifuged at 8,000 rpm for 20 min at 40C to

pellet insoluble materials, and the supernatant was decanted and concentrated. The

supernatant and pellet collected after refolding were analyzed by SDS-PAGE for protein

content, as were the soluble fractions (Supernatant 1 3) collected after each treatment









with DOC (Figure 2-8). Unfortunately, refolding was unsuccessful and Zpslp was

present in the insoluble fraction.

M C 1 2 3 S P





29 kDa -
22 kDa -






Figure 2-8. SDS-PAGE analysis of the soluble and insoluble products obtained after
solubilization and refolding of the inclusion body pellet. Also shown are the
supernatant fractions collected after each purification step. Lane M,
molecular weight marker; Lane C, non-induced E. coli (8 h growth); Lane I,
IPTG induced E. coli (8 h growth); Lane 1, Supernatant 1 (post-lysis); Lane
2, Supernatant 2 (after first DOC wash); Lane 3, Supernatant 3 (after second
DOC wash); Lane S, soluble fraction (after refolding); Lane P, insoluble
fraction (after refolding).

It is unclear why the solubilized inclusion body protein failed to refold despite its

improved purity and the addition of DTT (to prevent unfavorable disulfides). One

possible explanation is that the rate of dilution was accelerated due to poor control of the

flow rate. It is critical that the rate of dilution is slow. At high denaturant concentrations,

the unfolded protein is well solvated and flexible. Rapidly altering solvent dynamics

toward an aqueous environment forces the protein to collapse into a compact and rigid

structure. Unfortunately, the resulting structure is often misfolded or aggregated and

therefore insoluble. Gradual dilution allows for refolding at intermediate concentrations

of urea, where the denaturant concentration is low enough to force protein molecules to

collapse, yet allowing flexible motion enabling proteins to reorganize their structures and

stay in solution. Therefore, it may be beneficial to alter the refolding strategy so to






25


provide a slower and more controlled rate of denaturant dilution. Alternative refolding

strategies include, but are not limited to: one-step dialysis, step-wise dialysis, and buffer-

exchange by gel filtration.53 Although expressing recombinant Zpslp from E. coli is

advantageous due to high protein yield, it is possible the protein will not properly refold

after solubilization from inclusion bodies. If future efforts to refold and purify Zpslp

from E. coli inclusion bodies are unsuccessful, it may be necessary to purify Zpslp

directly from S. cerevisiae.














CHAPTER 3
CONCLUSIONS

The work presented represents initial steps toward characterization of Zpslp from

S. cerevisiae. ZPS1 expression is regulated by extracellular pH and zinc-deficiency,

environmental signals known to elicit Zpslp-like antigen production in Candida and

Aspergillus spp., respectively. These results suggest that the regulation, and

consequently function, of the cell wall proteins is conserved among these fungi. This

hypothesis is supported by their high sequence homology. Because of their localization

within the fungal cell wall and the observed binding of Pral and Aspndl to host

molecules, the Zpslp-like proteins in C. albicans and Aspergillus spp. are believed play a

role in establishing infection. The Zpslp-like proteins may function as virulence factors

by mediating critical host-parasite interactions or through involvement in morphological

processes. Therefore, future characterization of Zpslp may include investigating the

protein's potential role in fungal cell-cell adhesion flocculationn) or adherence to host

ligands (e.g.., ECM or serum proteins).

The partial purification of recombinant Zpslp from bacterial inclusion bodies is

an important first step toward characterization ofZpslp. Once purified, a wealth of

information can be obtained by studying both Zpslp structure and function. Due to

similarities between the Zpslp-like proteins and zinc-dependent metalloproteases, future

work using purified Zpslp ought to include metal binding studies and testing for

proteolytic activity towards a variety of substrates.














CHAPTER 4
MATERIALS AND METHODS

Growth Media

For standard growth of E. coli, LB medium was used. The recipe per liter is 10 g

NaC1, 10 g Bactotryptone, and 5 g Yeast Extract. When required, ampicillin was added

to a final concentration of 200 [tg/mL. In preparation of plates, 15 g of agar was added

per liter.

YPD medium was used for routine, non-selective yeast growth. The recipe per

liter is 10 g Yeast Extract, 20 g Bactopeptone, and 20 g Dextrose. In preparation of

plates, 15 g of agar was added per liter.

For maintenance of recombinant yeast strains, selective (SD) medium was used.

The base recipe per liter is 5 g (NH4)2SO4, 20 g Dextrose, and 1.7 g Yeast Nitrogen Base

without amino acids or (NH4)2SO4 (Difco; Sparks, MD). To satisfy the auxotrophic

strains used in this study, the medium was supplemented with 0.1 g L-Histidine, 0.1 g L-

Leucine, and 0.1 g L-Lysine per liter. Although the strains required Uracil, this was

omitted from the medium for selective growth. This medium will be referred to as SD-

Ura. For plates, 15 g of agar was added per liter.

To limit zinc and iron availability, Chelex-treated synthetic defined medium

(CSD) was used. The recipe per liter, using H20 at 18 MQ purity, is 20 g Dextrose, 5.1 g

Yeast Nitrogen Base without amino acids or divalent cations or potassium phosphate

(BiolOl; Vista, CA), and 0.1 g each of L-Histidine, L-Leucine, and L-Lysine. Again, for

selective purposes, Uracil was omitted from the medium. To remove metals from the









media, 25 g of Chelex-100 ion exchange resin (Sigma) was added, and the mixture was

stirred for a minimum of 2 h. After removal of the resin, 10 mL of potassium phosphate

monobasic (100 g/L) was added and the pH was adjusted to 4.0 using HC1. Next,

divalent metal ions were added to the medium to the following concentrations (as

recommended by BiolOl): 0.4 mg/L MnS04, 0.04 mg/L CuS04, 100 mg/L CaCl2, and

500 mg/L MgS04. The resulting solution was filter sterilized into a polycarbonate flask

washed with Acationox detergent (Baxter Scientific Products; McGraw Park, IL). The

resulting solution contains residual zinc and iron at concentrations less than 100 nM

(approximate value), and is referred to as CSD-Ura(-Zn/-Fe). For zinc or iron replete

medium, the desired metal is added back to the medium to a final concentration of 10

[IM.

Solutions and Buffers for Yeast Transformations and P-Galactosidase Assays

10x TE (250 mL):

100 mM Tris and 10 mM EDTA pH 7.5. Sterilize.

LiTE solution (50 mL):

5 mL sterile 10x TE, 5 mL sterile 1 M Lithium Acetate 40 mL sterile

H20.

PEG-LiTE solution (50 mL):

5 mL sterile 10x TE, 5 mL sterile 1 M Lithium Acetate, 40 mL sterile

44% (w/v) PEG-3350









Carrier DNA (10 mL):

100 mg salmon testes DNA and 10 mL ultrapure H20. Shear DNA by

drawing the mixture up into a 10 mL syringe with an 18g needle 15 times,

boil, and restore volume to 10 mL. Store as 1 mL aliquots at -200C.

Z-buffer, pH 7.0 (1 L):

0.06 M Na2HPO4, 0.04 M NaH2PO4-H20, 0.01 M KC1, 0.001 M

MgSO4.

Bacterial and Yeast Strains

Listed below are the bacterial and yeast strains used in this work.

Table 4-1. Yeast strains used for the work described.
Yeast
Strain Mutation Source Genotype
MAT u; his3; leu2; ura 3;
BY4742 Wild type lys2
MAT a; his3; leu2; ura 3;
Y11367 zapl EUROSCARF lys2
zapl::kanMX4
MAT u; his3; leu2; ura 3;
Y10936 riml01 EUROSCARF lys2
rim]01::kanMX4

Table 4-2: Bacterial strains used for the work described.
E. coli
Strain Genotype

TOP 10 F mcrA A(mrr-hsdRMS-mcrBC) 4801acZAM15 AlacX74
deoR recAl araD139 A(ara-leu)7697 galU galK rpsL (StrR)
endAl nupG

BL21(DE3) F- ompT hsdSB(rBm,-) gal dcm (DE3)









Yeast Transformations

Using the lithium acetate method, yeast strains of interest were transformed with a

plasmid containing the ZPS]-lacZ fusion (with Ura+ selection), which was previously

constructed34 in YEp35354 by gap repair.55 This was accomplished by growing the yeast

in 5 mL YPD at 30'C at 250 rpm overnight. The following day, 300 |pL of the overnight

culture was transferred to a new tube containing 5 mL YPD and incubated for 2 hr. at

30'C at 250 rpm. Next, the cells were harvested by centrifugation at 3500 rpm for 3 min

and the supernatant was decanted. The remaining cell pellet was washed by adding 5 mL

LiTE solution and vortexing. Again, the cells were harvested by centrifugation (as

described above). The cells were resuspended in residual LiTE solution by vortexing and

50 |pL of the cell suspension was transferred into a sterile 1.5 mL centrifuge tube. Added

to the centrifuge tube containing cells were 2 |pL (~ 400 [tg) of plasmid DNA containing

the ZPS]-lacZ fusion and 10 |pL (~ 10 [tg) of salmon sperm carrier DNA (boiled for 5

min and flash cooled on ice prior to use). Next, 500 |pL of PEG-LiTE solution was

added. The mixture was vortexed briefly and incubated at 300C at 250 rpm for 30 45

min. After incubation, the sample was heat shocked for 10 15 min at 420C. The cells

were pelleted at 4000 rpm for 1 min in a microcentrifuge. The supernatant was aspirated

and 500 |pL LiTE solution was added to the pellet and subsequently vortexed. Finally, 50

- 200 |pL of transformant was plated on SD-Ura plates to (select for the YEp353 plasmid)

and incubated at 300C for 3 5 days. Plates which grew colonies were stored at 40C for

future use.









p-Galactosidase Assays

A single colony of yeast transformed with the ZPS]-lacZ fusion plasmid was

transferred to 5 mL of SD-Ura and incubated at 300C at 250 rpm overnight. This liquid

culture was used to inoculate metal-free 14 mL polystyrene tubes containing 5 mL of

CSD-Ura with the appropriate combinations of zinc and iron as follows: -Zn/-Fe, 45 |pL

cell culture; -Zn/+Fe, 30 |pL cell culture; +Zn/-Fe, 30 |pL cell culture; +Zn/+Fe, 20 |pL

cell culture. The cultures were grown for 12 h and then stored on ice for approximately

20 min. Next, the cells were harvested by centrifugation for 3 min at 3500 rpm at 40C.

The supernatant was discarded and the resulting pellet was washed by adding 5 mL cold

Z-buffer and vortexing. The cells were harvested by centrifugation (as above) and the

supernatant was discarded.

The cells were resuspended in 2 mL cold Z-buffer and 1 mL of the cell

suspension was transferred to a 5 mL glass assay tube containing 50 |pL CHCl3 and 50 |pL

0.1% SDS. The contents of the tube were vortexed to permeablize the cells and then

incubated at 300C for 10 min to equilibrate. After incubation, the tube was vortexed

vigorously for 3 sec and its contents (principally CHCl3) were allowed to settle for

approximately 10 se. before transferring 100 |pL of the suspension to a 96-well plate (in

triplicate). The assay reaction was initiated by adding 20 |pL of 4 mg/mL o-nitrophenyl-

P-D-galactopyranoside (ONPG). The sample was mixed and the reaction was allowed to

proceed until the darkest samples were an intense yellow color. The reaction was

stopped by adding 50 |pL of 1 M Na2CO3 and the reaction time (min) was noted. The

absorbance at 420 nm was measured (reference wavelength, 600 nm) using a SAFIRE

microplate reader (Tecan) and XFLUOR software. The absorbance at 600 nm (OD600) Of









the remaining cell suspension (from above) was also measured using a BIO-RAD

SmartSpecTM 3000 bench-top spectrophotometer. 3-galactosidase activity was measured

in Miller Units using the method of Guarente,56 and activity units were calculated as

follows: (AA420 x 1000)/(min x mL of culture used x OD600).

Cloning of ZPS1 and Construction of an E. coli Expression Plasmid

The gene that encodes the mature form of Zpslp (lacking the signal peptide) was

PCR cloned from S. cerevisiae strain BY4724 genomic DNA using forward and reverse

primers containing Nde I and EcoR I restriction sites, respectively. The primers were

obtained from Integrated DNA Technologies, Inc. (Coralville, IA) and were designed as

follows:

ZPS1 for: 5'- AAC TTT AAG AAG GAG ATA TAC ATA

TGC CTG TCA CTT ACG ACA CCA A -3'

ZPS1 rev: 5'- CAA GCT TGT CGA CGG AGC TCG AAT

TCT TAC AAG TTA CCT AGA CAG C -3'

The PCR reaction was catalyzed using Taq DNA polymerase and the

thermocycling conditions employed were as follows: one cycle at 95oC for 3 min; and

25 cycles at 95oC for 30 sec, 50oC for 30 sec, 72oC for 1.5 min; and a final extension at

72oC for 8 min.

The PCR product was digested for 4 h at 37oC using the restriction enzymes Nde

I and EcoR I (New England Biolabs; Beverly, MA). The pET-22b(+) vector was

obtained from Novagen (La Jolla, CA). The pET-22b(+) plasmid was also digested using

Nde I and EcoR I (as described above). Following restriction digestion, the cut PCR and

pET-22b(+) samples were subjected to agarose gel electrophoresis (0.8% agarose) and









purified using the QIAquick Gel Extraction Kit, following the manufacture's protocol

(QIAGEN Inc.; Valencia, CA). These purified samples were subsequently used to ligate

the cloned ZPS1 gene into the pET-22b(+) vector between the Nde I and EcoR I

restriction sites using T4 DNA ligase (New England Biolabs), incubated overnight at

16oC. The ligation product was used to transform electrocompetent E. coli TOP10 cells

by electroporation following standard procedures.57 The E. coli transformant (10 150

1L) was plated on LB agar plates containing ampicillin (for plasmid selection) and

incubated at 37oC overnight. Plates that grew colonies were stored at 4oC for future use.

To obtain large quantities of the pET-22b(+)-ZPS1 construct, a single colony from the

transformation product was used to inoculate 5 mL of LB medium containing ampicillin.

The cells were grown at 37oC at 250 rpm overnight. Using the Promega (Madison, WI)

Wizard Plus Miniprep DNA purification system, the pET-22b(+)-ZPS1 plasmid was

purified from the overnight culture according to the manufacture's directions. Using the

purified plasmid, the sequence of the cloned ZPS 1 gene was confirmed by the ICBR

DNA sequencing core laboratory at the University of Florida.

Expression of Zpslp in E. coli

To obtain Zpslp using the T7 expression system, the pET-22b(+)-ZPS] plasmid

was transformed into BL21(DE3) E. coli by electroporation using standard methods. The

E. coli transformant (10 150 [LL) was plated on LB agar plates containing ampicillin

and incubated at 370C overnight. Plates that grew colonies were stored at 40C for future

use. A single colony from the transformation product was used to inoculate 15 mL of LB

medium containing ampicillin. The cells were grown at 370C at 250 rpm overnight and

10 mL of culture was used to inoculate 1 L of LB medium containing ampicillin. The 1









L culture was incubated at 370C at 250 rpm for approximately 2 h until reaching an OD600

of 0.4 1.0. At this time, Zpslp expression was induced by addition of IPTG (Isopropyl-

P-D-thioglactoside) to a final concentration of 1 mM. The culture was then incubated at

30'C at 250 rpm for 8 h. Finally, the cells were harvested by centrifugation (3000 rpm

for 15 min at 40C) and washed two times using 50 mM Tris (pH 7.4). The resulting

pellet was stored frozen at -200C overnight.

Estimation of Protein Purity by SDS-PAGE

SDS-PAGE gels containing 14% (w/v) polyacrylamide were prepared and

analyzed by standard methods. Samples were prepared by adding equal volumes of 2x

Laemmli sample buffer, boiling for 10 min, followed by centrifugation at 14,000 rpm in a

microfuge for 1 min to pellet any insoluble debris. The gels were run at 70 V, using a

Tris-glycine electrode buffer. All gels were stained with Coomassie blue.
















LIST OF REFERENCES

1) W. L. Chaffin; J. L. Lopez-Ribot; M. Casanova; D. Gozalbo; J. P. Martinez
Microbiol Mol Biol Rev 1998, 62, 130-180.

2) F. De Bernardis; F. A. Muhlschlegel; A. Cassone; W. A. Fonzi Infect Immun 1998,
66, 3317-3325.

3) D. Davis Curr Genet 2003, 44, 1-7.

4) D. R. Soil Curr Top MedMycol 1985, 1, 258-284.

5) N. D. Rawlings; E. O'Brien; A. J. Barrett Nucleic Acids Res 2002, 30, 343-346.

6) B. Banerjee; P. A. Greenberger; J. N. Fink; V. P. Kurup Infect Immun 1998, 66,
5175-5182.

7) J. A. Calera; M. C. Ovejero; R. Lopez-Medrano; M. Segurado; P. Puente; F. Leal
Infect Immun 1997, 65, 1335-1344.

8) M. Sentandreu; M. V. Elorza; R. Sentandreu; W. A. Fonzi JBacteriolo 1998, 180,
282-289.

9) A. Bouali; R. Robert; G. Tronchin; J. Senet J Gen Microbiol 1987, 133, 545-551.

10) J. L. Lopez-Ribot; P. Sepulveda; A. M. Cervera; P. Roig; D. Gozalbo; J. P.
Martinez FEMS Microbiol Lett 1997, 157, 273-278.

11) M. Casanova; J. L. Lopez-Ribot; C. Monteagudo; A. Llombart-Bosch; R.
Sentandreu; J. P. Martinez Infect Immun 1992, 60, 4221-4229.

12) F. M. Klis; P. Mol; K. Hellingwerf, S. Brul FEMSMicrobiol Rev 2002, 26, 239-
256.

13) R. Teshima; H. Ikebuchi; J. Sawada; S. Miyaachi; S. Kitani; M. Iwama; M. Irie; M.
Ichinoe; T. Terao JAllergy Clin Immunol 1993, 92, 698-706.

14) B. Banerjee; V. P. Kurup; P. A. Greenberger; D. R. Hoffman; D. S. Nair; J. N. Fink
JAllergy Clin Immunol 1997, 6, 821-827.

15) J. A. Calera; M. C. Ovejero; R. Lopez-Medrano; R. Lopez-Aragon; P. Puente; F.
Leal Microbiology 1998, 144, 561-567.









16) B. Banerjee; V. P. Kurup; S. Phadnis; P. A. Greenberger; J. N. Fink JLab Clin
Med 1996, 127, 253-262.

17) P. Sepulveda; J. L. Lopez-Ribot; A. Murgui; E. Canton; D. Navarro; J. P. Martinez
IntMicrobiol 1998, 1, 209-216.

18) A. Viudes; S. Perea; J. L. Lopez-Ribot Infect Immun 2001, 69, 2909-2919.

19) G. Tronchin; K. Esnault; G. Renier; R. Filmon; D. Chabasse; J. P. Bouchara Infect
Immun 1997, 65, 9-15.

20) M. C. Penalver; J. E. O'Connor; J. P. Martinez; M. L. Gil Infect Immun 1996, 64,
1146-1153.

21) M. L. Gil; M. C. Penalver; J. L. Lopez-Ribot; J. E. O'Connor; J. P. Martinez Infect
Immun 1996, 64, 5239-5247.

22) D. Davis; R. B. Wilson; A. P. Mitchell Mol Cell Biol 2000 20, 971-978.

23) D. Davis; J. E. Edwards, JR.; A. P. Mitchell; A. S. Ibrahim Infect Immun 2000, 68,
5953-5959.

24) M. Segurado; R. Lopez-Aragon; J. A. Calera; J. M. Fernandez-Abalos; F. Leal
InfectImmun 1999, 67, 2377-2382.

25) S. H. Denison Fungal Genet Biol 2000, 29, 61-71.

26) J. A. Tayek; G. L. Blackburn Am JMed 1984, 81-88.

27) V. Braun; T. Focareta Crit Rev Microbiol 1991, 18, 115-158.

28) A. Alsina; N. Rodriguez-del Valle Sabouraudia: Journal of Medical and
Veterinary Mycology 1984, 22, 1-5.

29) R. H. K. Eng; R. Drehmel; S. M. Smith; E. J. C. Goldstein Sabouraudia: JMed Vet
Mycol 1984, 22, 403-407.

30) J. K. Byron; K. V. Clemons; J. H. McCusker; R. W. Davis; D. A. Stevens Infect
Immun 1995, 63, 478-485.

31) H. Tershima; S. Fukuchi; K. Nakai; M. Arisawa; K. Hamada; N. Yabuki; K. Kitada
Curr Genet 2002, 40, 311-316.

32) M. J. Lafuente; C. Gancedo Yeast 1999, 15, 935-943.

33) H. Zhao; D. J. Eide Mol Cell Biol 1997, 17, 5044-5052.

34) T. J. Lyons; A. P. Gasch; L. A. Gaither; D. Botstein; P. 0. Brown PNAS 2000, 97,
7957-7962.






37


35) T. M. Lamb; W. Xu; A. Diamond; A. P. Mitchell JBiol Chem 2001, 276, 1850-
1856.

36) P. Uetz; L. Giot; G. Cagney; T. A. Mansfield; R. S. Judson; J. R. Knight; D.
Lockshon; V. Narayan; M. Srinivasan; P. Pochart; A. Qureshi-Emili; Y. Li; B.
Goodwin; D. Conover; T. Kalbfleisch; G. Vijayadamodar; M. Yang; M. Johnson;
S. Fields; J. M. Rothberg Nature 2000, 403, 623-627.

37) J. C. Rutherford; S. Jaron; E. Ray; P. 0. Brown; D. R. Winge PNAS 2001, 98,
14322-14327.

38) Y. Yamaguchi-Iwai; A. Dancis; R. D. Klausner Embo J 1995, 14, 1231-1239.

39) N. Fushimi; C. Ewe Ee; T. Nakajima; E. Ichishima JBiol Chem 1999, 274, 24195-
24201.

40) K. Matsumoto; M. Yamaguchi; E. Ichishima Biochim Biophys Acta 1994, 1218,
469-472.

41) M. V. Ramesh; T. D. Sirakova; P. E. Kolattukudy Gene 1995, 165, 121-125.

42) M. J. Orbach; L. Farrall; J. A. Sweigard; F. G. Chumley; B. Valent Plant Cell
2000, 12, 2019-2032.

43) K. E. McAuley; Y. Jia-Xing; E. J. Dodson; J. Lehmbeck; P. R. Ostergaard; K. S.
Wilson Acta Cryst 2001, D57, 1571-1578.

44) T. Nonaka; N. Dohmae; Y. Hashimoto; K. Takio JBiol Chem 1997, 272, 30032-
30039.

45) J. Kim; Y. S. Kim Biosci Biotechnol Biochem 1999, 63, 2130-2136.

46) T. M. Chang; C. C. Liu; M. S. Chang Gene 1997, 199, 225-229.

47) U. Wagner; B. K. Gudmunsdottir; K. Drossier JApplMicrobiol 1999, 87, 620-629.









48) A. C. R. da Silva; J. A. Ferro; F. C. Reinach; C. S. Farah; L. R. Furian; R. B.
Quaggio; C. B. Monteiro-Vitorello; M. A. Van Sluys; N. F. Almelda; L. M. C.
Alves; A. M. do Amaral; M. C. Bertolini; L. E. A. Camargo; G. Camarotte; F.
Cannavan; J. Cardozo; F. Chambergo; L. P. Clapina; R. M. B. Ciarelli; L. L.
Coutinho; J. R. Cursino-Santos; H. El-Dorry; J. B. Faria; A. J. S. Ferreira; R. C. C.
Ferreira; M. I. T.Ferro; E. F. Formighieri; M. C. Franco; C. C. Greggio; A. Gruber;
A. M. Katsuyama; L. T. Kishi; R. P. Leite; E. G. M. Lemos; M. V. F. Lemos; E. C.
Locali; M. A. Machado; A. M. B. N. Madeira; N. M. Martinez-Rossi; E. C.
Martins; J. Meidanis; C. F. M. Menck; C. Y. Miyaki; D. H. Moon; L. M. Moreira;
M. T. M. Novo; V. K. Okura; M. C. Oliveira; V. R. Oliveira; H. A. Pereira; A.
Rossi; J. A. D. Sena; C. Silva; R. F. de Souza; L. A. F. Spinola; M. A. Takita; R. E.
Tamura; E. C. Teixeira; R. I.D. Tezza; M. Trindade dos Santos; D. Truffi; S. M.
Tsai; F. F. White; J. C. Setubal; J. P. Kitajima Nature 2002, 417, 459-463.

49) T. Hori; T. Kumasaka; M. Yamamoto; T. Nonaka; N. Tanaka; Y. Hashimoto; T.
Ueki; K. Takio Acta Cryst 2001, D57, 361-368.

50) Y. Doi; B. R. Lee; M. Ikeguchi; Y. Ohoba; T. Ikoma; S. Tero-Kubota; S.
Yamauchi; K. Takahashi; E. Ichishima Biosci Biotechnol Biochem 2003, 67, 264-
270.

51) M. Bradford Anal Biochem 1976, 72, 248-254.

52) D. R. Marshak; J. T. Kadonaga; R. R. Burgess; M. W. Knuth; W. A. Brennan, Jr.;
S. Lin Strategiesfor Protein Purification and Characterization: A Laboratory
Course Manual; Cold Spring Harbor Laboratory Press: Plainview, NY, 1996; pp
209-218.

53) K. Tsumoto; D. Ejima; I. Kumagai; T. Arakawa Protein Expression & Purification
2003, 28, 1-8.

54) A. M. Myers; A. Tzagaloff; D. M. Kinney; C. J. Lusty Gene 1986, 45, 299-310.

55) S. Kunes; H. Ma; K. Overbye; M. S. Fox; D. Botstein Genetics 1987, 115, 73-81.

56) L. Guarente Methods Enzymol 1983, 101, 181-191.

57) J. Sambrook; D. W. Russell Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2001; pp A8.40-A8.47















BIOGRAPHICAL SKETCH

Stephanie L. Drobiak is from Brooklyn, Connecticut. She graduated from

Wheaton College in Norton, Massachusetts, in 2001 with a Bachelor of Arts in

biochemistry. In the fall of 2001, she entered the graduate program in the Department of

Chemistry at the University of Florida. Upon completion of her master's, she will

continue working towards her PhD at the University of Florida.