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Eukaryotic Cell, April 2005, p. 685-693, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.685-693.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Surface Localization of the Yps3p Protein of Histoplasma capsulatum

Megan L. Bohse and Jon P. Woods*

Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin

Received 4 October 2004/ Accepted 25 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The YPS3 gene of Histoplasma capsulatum encodes a protein that is both resident in the cell wall and also released into the culture medium. This protein is produced only during the pathogenic yeast phase of infection and is also expressed differently in H. capsulatum strains that differ in virulence. We investigated the cellular localization of Yps3p. We demonstrated that the cell wall fraction of Yps3p was surface localized in restriction fragment length polymorphism class 2 strains. We also established that Yps3p released into the G217B culture supernatant binds to the surface of strains that do not naturally express the protein. This binding was saturable and occurred within 5 min of exposure and occurred similarly with live and heat-killed H. capsulatum. Flow cytometric analysis of H. capsulatum after enzymatic treatments was consistent with Yps3p binding to chitin, a carbohydrate polymer that is a component of fungal cell walls. Polysaccharide binding assays demonstrated that chitin but not cellulose binds to and extracts Yps3p from culture supernatants.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Histoplasma capsulatum is a pathogenic fungus with worldwide distribution. It is the causative agent of histoplasmosis, one of the most common fungal respiratory infections in the world, with an estimated 500,000 cases in the United States alone per year. The regions where histoplasmosis is endemic include the midwestern and southwestern United States as well as areas of South America. In the United States it reaches its highest levels along the Ohio and Mississippi river valleys, where skin test reactivity to H. capsulatum antigens indicates that more than 90% of the population has had primary histoplasmosis (36).

A thermally dimorphic fungus, Histoplasma capsulatum exists in the soil as a mold, but after inhalational infection of mammalian tissues it transforms into its pathogenic yeast phase. This dimorphism is essential for virulence; chemically treated mycelial cultures that are unable to make the transition to yeasts are avirulent (26). Dimorphism is also the best-studied system of H. capsulatum gene regulation, and both mold-phase-specific and yeast-phase-specific genes have been identified (7, 13, 15-18, 28, 32, 33).

YPS3 is a yeast-phase-specific gene originally identified in a differential hybridization screen (21). The encoded Yps3p protein is both found in the cell wall and secreted from cells (35). Beyond its phase specificity, Yps3p expression varies among H. capsulatum strains that differ in thermotolerance and virulence. Restriction fragment length polymorphism (RFLP) class 2 strains are the most virulent and thermotolerant and are predominantly North American isolates. In class 2 strains, YPS3 transcription initiates between 2 h and 1 day after a temperature shift from ambient to 37°C and remains continuous during the yeast phase of growth (21, 25, 34). In RFLP class 3 strains, strains of intermediate virulence and thermotolerance found predominantly in Central and South America, YPS3 transcription is initiated 3 days after a temperature shift, but expression drops off to become undetectable after approximately 12 days (21, 25, 34). YPS3 is not expressed in RFLP class 1 strains, which are the least virulent and thermotolerant and are geographically widely distributed but have only been found as clinical isolates in severely immunocompromised patients (21, 31, 34).

In the present study, we sought to characterize the cellular localization of Yps3p based on predicted sequence homology that we noted with Bad1, an established virulence factor of another dimorphic fungus, Blastomyces dermatitidis (6). Our results indicate that Yps3p is surface localized on class 2 strains of Histoplasma capsulatum and that a mechanism of localization is the loading of secreted Yps3p on the surface via an interaction with the cell wall polysaccharide chitin.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fungal strains and culture conditions. H. capsulatum strains G184AS, G184AR, G217B, and Downs have been described previously (1, 2, 14). Downs (ATCC 38904) and UCLA 531S are clinical isolates of RFLP class 1. G217B (ATCC 26032) and G222B (ATCC 26034) are clinical isolates of RFLP class 2. The clinical isolate G184AR (ATCC 26027) and its derivative G184AS are members of RFLP class 3. G186AS is a derivative of the class 3 clinical isolate G186AR (ATCC 26029). G184AS and G186AS are spontaneous smooth-colony morphology variants isolated from G184AR and G186AR, respectively. H. capsulatum was grown in Histoplasma-macrophage medium (HMM) broth, a rich defined medium (38), in a 5% CO2-95% air atmosphere. Most experiments were done with H. capsulatum grown as yeast cells at 37°C. Conversion to mycelial growth was achieved by incubating a culture at 28.5°C for 3 weeks. Under these conditions, yeast cells as well as mycelia are present.

Cloning, expression, and purification of recombinant Yps3p. We prepared H. capsulatum strain G217B genomic DNA as previously described (37). We PCR amplified the YPS3 open reading frame from G217B genomic DNA. This open reading frame is different from that reported previously (20); see Results and Discussion for details. To allow nickel affinity purification, we included codons for six histidines prior to the stop codon at the C terminus of the YPS3 open reading frame. The PCR-amplified YPS3 was cloned into the pTYB2 expression vector (New England Biolabs, Beverly, Mass.) and sequenced. The recombinant Yps3p plasmid was transformed into Escherichia coli strain ER2566, and expression was induced with isopropylthiogalactopyranoside (IPTG) according to the supplier's recommendations. Recombinant 6xHis-tagged Yps3p was purified with a Ni-nitrilotriacetic acid Superflow resin (Qiagen, Valencia, Calif.) under denaturing conditions and assessed for purity with silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

Antibodies. Rabbit antiserum was raised against nickel affinity-purified recombinant 6xHis-tagged Yps3p by repeated subcutaneous immunizations at multiple sites, as described previously (10). Immune serum was tested by Western immunoblotting against strain G217B as a positive control and strain Downs as a negative control. In addition to recognizing Yps3p exclusively on the G217B blots, there was some cross-reactivity with a high-molecular-weight antigen in both G217B and Downs. We removed cross-reactive antibodies by adsorbing the final antiserum against Downs culture supernatant. Nitrocellulose strips were incubated in concentrated Downs supernatant overnight at 4°C to allow antigen binding. Nonspecific protein binding sites were then blocked with phosphate-buffered saline (PBS; 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mM NaCl, pH 7.2) containing 5% bovine serum albumin for 1 h at 4°C. Rabbit antiserum diluted 1:10 in PBS containing 5% bovine serum albumin was incubated with the antigen-coated strips overnight at 4°C a total of three times. After adsorption, the high-molecular-weight cross-reactivity was eliminated from immunoblots of both Downs and G217B culture supernatants.

Flow cytometric detection of surface Yps3p. We washed 107 H. capsulatum yeast cells from log-phase HMM broth cultures and resuspended them at a concentration of 107 cells/ml in PBS containing 1% bovine serum albumin. After a 5-min blocking incubation, cells were stained indirectly with a 1:1,000 dilution of preimmune serum or a 1:10,000 dilution of Yps3p-specific rabbit antiserum followed by a 1:100 dilution of goat anti-rabbit immunoglobulin G-fluorescein isothiocyanate (FITC) (BD biosciences, San Jose, Calif.) for 30 min at 4°C. Stained cells were washed with PBS and fixed with 2% paraformaldehyde, pH 7.2, for 30 min at 4°C. Cells were analyzed with BD FACSCan or FACSCalibur flow cytometers (BD biosciences, San Jose, Calif.), and color analysis was performed with FlowJo analysis software (Treestar, Ashland, Oreg.). H. capsulatum cells were exposed to FITC-labeled wheat germ agglutinin (WGA) (Sigma, St. Louis, Mo.) at a concentration of 1 µg/ml in PBS for 30 min at 4°C, washed with PBS, and fixed with 2% paraformaldehyde, pH 7.2. In cases of preincubation with wheat germ agglutinin, cells were first incubated with 1 µg of unlabeled wheat germ agglutinin per ml in PBS for 1 h at 4°C. After incubation, cells were washed three times with PBS, followed by staining for flow cytometric analysis as described above.

Immunofluorescence microscopy. Cell staining for immunofluorescence microscopy was done as for flow cytometry, but cells were indirectly stained with a 1:100 dilution goat anti-rabbit immunoglobulin G-Alexa 488 (Molecular Probes, Eugene, Oreg.). Following fixation, a 50-µl aliquot was spread on a washed glass slide, air dried, and mounted under a glass coverslip with Prolong antifade reagent (Molecular Probes, Eugene, Oreg.). Images were collected on a Zeiss AxioPlan Iii microscope at 100x magnification and captured with a Zeiss AxioCam B&W charge-coupled device camera. These images were pseudocolored with OpenLabs 3.0 software (Improvision, Lexington, Mass.).

Enzymatic digestions. We heat killed 107 H. capsulatum yeast cells from log-phase HMM broth cultures by incubating them at 65°C for 1 h. Heat-killed cells were washed with PBS and treated with degradative enzymes (Sigma Aldrich, St. Louis, Mo.) specific for different structural components of the H. capsulatum cell wall. Chitinase treatment utilized 1 unit of chitinase in 200 mM potassium phosphate buffer, pH 6.0, with 2 mM CaCl2 at 25°C for 1 h. Lipase treatment utilized 1 unit of lipase in 400 mM NaCl, pH 7.4, with 5 mM CaCl2 at 37°C for 1 h. Phosphatidylinositol-specific phospholipase C (PIPLC) treatment utilized 1 unit of enzyme in 10 mM Tris-HCl, 144 mM NaCl, pH 7.4, with 0.02% bovine serum albumin at 30°C for 10 min. Proteinase K treatment utilized 2 mg of proteinase K per ml in 100 mM Tris-HCl, pH 7.5, with 2 mM CaCl2 at 37°C for 1 h. After treatment, cells were washed with PBS.

Binding assay. Supernatant from strain G217B log-phase cultures was collected, filter sterilized, and concentrated with Amicon stirred ultrafiltration cell model 8400 and NMWL 10,000 ultrafiltration membranes, Centricon Plus-80 NMWL 10,000, and Ultrafree-15 NMWL 10,000 filter devices (Millipore). We washed 107 Histoplasma capsulatum cells with PBS. Filtered concentrated G217B supernatant was added, and the cells were incubated at 37°C with rocking for various time intervals. In the time course assay, supernatant was added to the cells, and the cells were incubated for the amount of time indicated prior to washing with PBS. If the incubation time is not specifically noted in the assay, it was the default time of 1 h. In some cases, cells were enzymatically digested or heat killed as described prior to the addition of supernatant.

Polysaccharide binding assay. We washed 20 mg of practical-grade chitin or cellulose powder (Sigma) with PBS. Filtered concentrated supernatant was added to the polysaccharide and brought to a volume of 1 ml with PBS and incubated for 1 h at 4°C. After incubation, the tubes were centrifuged for 1 min at 13,000 x g, and the supernatant-PBS was removed from the polysaccharide pellet. The polysaccharide pellet was washed four times with PBS with centrifugation at 13,000 x g after each wash. These fractions were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, followed by transfer to nitrocellulose and Western immunoblotting.

For Western blot analysis, samples were denatured by incubation in 50 mM Tris-Cl containing 2% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol, 10% glycerol, and 0.1% bromophenol blue for 5 min at 95°C, electrophoresed in an SDS-12% polyacrylamide gel, and electroblotted onto a nitrocellulose membrane. This membrane was blocked for 1 h with Tris-buffered saline (TBS; 25 mM Tris, 123 mM NaCl, 2.7 mM KCl) containing 0.01% SDS, 0.05% Tween 20, and 5% dried milk. The blot was then incubated with a 1:10,000 dilution of Yps3p-specific antiserum. Nonspecific antibody was removed by washing three times for 20 min in 0.01% SDS-0.05% Tween 20 in TBS. The washed nitrocellulose membrane was incubated in a 1:6,000 dilution of horseradish peroxidase-labeled goat anti-rabbit immunoglobulin antibody (Bio-Rad, Hercules, Calif.). Bound antibody was removed from this membrane by submersion in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at 50°C for 30 min, washed in TBS for 30 min, and reblotted with anti-G217B supernatant antibody (11) as a specificity control.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Yps3p bears homology to Bad1p from Blastomyces dermatitidis. The nucleotide sequence from our G217B YPS3 clones differed from the published sequence (20) but matched sequence that subsequently appeared in the Histoplasma capsulatum genome sequence database (http://www.genome.wustl.edu/projects/hcapsulatum/). When we queried GenBank with our own Yps3p predicted amino acid sequence, we detected homology (2e–23) with the Blastomyces dermatitidis adhesin and virulence factor Bad1p (6). Alignments of the predicted protein products of these genes revealed homology in both an N-terminal signal sequence region and a cysteine-rich epidermal growth factor (EGF) domain similar to the EGF-like domain that characterizes the C terminus of Bad1 (Fig. 1). Additionally, our YPS3 clones and the genomic sequence of YPS3 revealed a shorter open reading frame than originally published (20). Specifically, there is an omission of a guanosine in the published sequence. The guanosine occurs after position 1687 of the YPS3 found in GenBank (accession number L16844). The published sequence predicts a 184-amino-acid protein, including a putative glycosyl-phosphatidylinositol (GPI) processing site near the C terminus (20). Our corrected protein sequence predicts a 137-amino-acid protein, truncated by 47 residues, including the putative GPI processing site.



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  		FIG. 1. Yps3p protein of Histoplasma capsulatum strain G217B and alignment with Blastomyces dermatitidis Bad1p. The predicted Yps3p protein product bears homology to the N and C termini of Blastomyces dermatitidis Bad1p. The white box indicates the internal 1,070-amino -acid tandem repeat region that constitutes the majority of Bad1p, which we have omitted for this alignment. The shaded boxes indicate six conserved cysteine residues thought to be important in disulfide bonding of EGF-like domains. The middle line indicates the GenBank Yps3p sequence (accession no. AAA33384), which misreports the length of the open reading frame.

 
Yps3p is surface exposed. Although Yps3p has been described as cell wall associated (35), it had not been determined whether this cell wall-associated fraction is surface localized. Flow cytometric analysis of representatives from each of the three RFLP classes revealed that Yps3p was surface localized on class 2 strains G217B (Fig. 2) and G222B (data not shown). As expected, Yps3p could not be detected on the surface of the class 1 strains UCLA 531S (Fig. 2) and Downs (data not shown). We also detected no surface-exposed Yps3p on class 3 strains G184AS (Fig. 2), G186AS, and G184AR (data not shown) that had been grown continuously as yeasts.



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  		FIG. 2. Flow cytometric determination of Yps3p surface localization. The top left histogram is a representative class 1 strain, UCLA 531S, which never expresses YPS3. The top right histogram is a representative class 2 strain, G217B, which both secretes and surface localizes Yps3p. The bottom left histogram is a class 3 strain, G184AS, which has turned off its transient Yps3p expression during continuous culture in the yeast phase. On all histograms, preimmune serum was included to demonstrate a lack of any autofluorescence of yeasts or cross-reactivity of antibody. Yps3p is only expressed in class 2 strains, where it is also surface localized.

 
Interestingly, in some instances strain G217B yeasts appeared to have two different populations of Yps3p binding cells, detectable as a bimodal peak in flow cytometry (Fig. 3A). The cells that stained with a lower fluorescence were also the smallest and least granular cells on the flow cytometric scatter plots, so we hypothesize that this low portion of the bimodal population represents yeasts which have recently budded and therefore have a smaller surface area to coat with Yps3p. We also detected surface-localized protein on class 2 strains via immunofluorescence microscopy (Fig. 3B, top panel). These differences in strain expression and localization to the cell wall fraction of expressing strains were confirmed by Western immunoblotting (data not shown).



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  		FIG. 3. Yps3p secreted from the class 3 strain G217B binds to the surface of the class 1 strain UCLA 531S. Flow cytometry (A) and immunofluorescence microscopy (B) revealed that incubation of UCLA 531S with filtered, concentrated G217B supernatant resulted in accumulation of Yps3p on the yeast surface. The bottom half of the UCLA 531S immunofluorescence panel is data captured from a 10-fold-longer exposure, revealing the very low level of background fluorescence on the yeasts in the field. This interaction did not require live UCLA 531S cells (C) and happened within minutes of exposure (D).

 
Yps3p released into the culture supernatant binds to the yeast cell surface. As Yps3p is also secreted from the cell and, except for the putative signal sequence, has no obvious transmembrane or GPI anchor domain, we examined whether the secreted protein could adhere to the surface of Histoplasma capsulatum yeasts, a mechanism of localization employed by Bad1p (4). The RFLP class 1 strain UCLA 531S never expresses Yps3p. After exposure to filtered concentrated supernatant from the class 2 strain G217B, which contains Yps3p, UCLA 531S yeasts bound Yps3p to their surface, as detected by flow cytometry (Fig. 3A). Immunofluorescence microscopy revealed this bound Yps3p to be uniformly distributed, similar to the surface-localized Yps3p of class 2 strains (Fig. 3B). The viability of "recipient" yeasts was not necessary for binding of Yps3p (Fig. 3C). Binding occurred rapidly after exposure (Fig. 3D) and was saturable (data not shown). Yps3p from filtered concentrated G217B supernatant bound to all other class 1 and 3 strains tested, including Downs, G184AS, and G184AR (Fig. 4A). We also tested whether the Yps3p could bind to the mycelial form of H. capsulatum, which does not express the YPS3 gene (21). When strain G217B was grown under conditions in which both yeast and mycelial cells were present, we found Yps3p coating both forms (Fig. 4B), although yeasts showed more immunofluorescence than the mycelial filaments. When we examined chitin exposure in a mixed culture via fluorescence microscopy with FITC-labeled wheat germ agglutinin, we found a similar distribution of chitin (data not shown).



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  		FIG. 4. A. Yps3p from G217B binds to class 1 and class 3 strains. Strains Downs and UCLA 531S are representatives of RFLP class 1. Strains G184AS and G184AR are representatives of RFLP class 3. All strains tested showed the ability to bind Yps3p on their surface, as detected by flow cytometry. B. Yps3p in a culture of mycelial and yeast cells binds to both morphotypes, as detected by immunofluorescence microscopy.

 
Chitinase digestion reduces Yps3p surface binding. To identify a ligand for Yps3p on the surface of H. capsulatum, we performed enzymatic digestions of yeast cell surface components prior to exposure to Yps3p. Proteinase, lipase, and PIPLC treatment of heat-killed UCLA 531S cells prior to filtered concentrated G217B supernatant exposure did not inhibit Yps3p binding (data not shown). Chitinase treatment degrades the cell wall polysaccharide chitin, which is surface exposed in H. capsulatum, as shown by fluorescently labeled wheat germ agglutinin (WGA) binding (Fig. 5A). Chitinase treatment removed Yps3p from the surface of G217B cells as well as reduced binding of Yps3p from G217B to the surface of UCLA 531S cells (Fig. 5B). As expected, chitinase treatment reduced the binding of FITC-WGA, but enzymatic digestion did not completely abolish FITC-WGA binding (data not shown).



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  		FIG. 5. Enzymatic digestion of Histoplasma capsulatum cell surface. A. FITC-WGA binding to surface-localized chitin in H. capsulatum strains G217B (left panel) and UCLA 531S (right panel). B. Heat-killed H. capsulatum cells were treated with chitinase prior to exposure to filtered concentrated supernatant. From left to right: heat-killed UCLA 531S plus supernatant; heat-killed, chitinase-digested UCLA 531S plus supernatant; heat-killed G217B; and heat-killed, chitinase-digested G217B. Chitinase treatment reduced Yps3p surface binding.

 
Wheat germ agglutinin incubation reduces Yps3p binding. We incubated UCLA 531S cells with the chitin binding lectin WGA prior to incubation with filtered concentrated G217B supernatant. After an hour of incubation with WGA, we added identical amounts of G217B supernatant to WGA-treated and untreated cells. Yps3p binding was reduced by more than 50% in cells that had been incubated with wheat germ agglutinin, as assessed by flow cytometry (Fig. 6).



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  		FIG. 6. Preincubation with wheat germ agglutinin, a lectin that binds chitin, decreased Yps3p surface binding in UCLA 531S by more than 50%, as detected by flow cytometry. Dotted lines, UCLA 531S cells (mean fluorescence intensity, 6.84). Solid lines, UCLA 531S cells after incubation with G217B supernatant (mean fluorescence intensity, 255). Dashed lines, UCLA 531S cells incubated with wheat germ agglutinin prior to exposure to G217B supernatant (mean fluorescence intensity, 102).

 
Yps3p from G217B supernatant binds to purified chitin. We incubated filtered concentrated G217B supernatant with either crab shell chitin or cellulose as a polysaccharide control for 1 h and then separated the insoluble polysaccharide fraction from the remaining supernatant. We then refiltered the supernatant, extensively washed the polysaccharide, and examined the fractions by Western immunoblotting. The polysaccharide chitin bound and extracted all of the Yps3p from the supernatant (Fig. 7, top panel). To assay the specificity of this binding, we then stripped the membrane and reblotted with an antibody raised against total G217B supernatant (11) (Fig. 7, bottom panel). Among the supernatant components detected by this technique, chitin specifically bound Yps3p. Although a few higher-molecular-weight bands were visible, these also appeared in the cellulose control column and presumably represent nonspecific polysaccharide-binding proteins. When polysaccharide was incubated with PBS alone, neither chitin nor cellulose demonstrated any components or contaminants cross-reactive with these antibodies (Fig. 7).



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  		FIG. 7. Chitin but not cellulose binds and extracts Yps3p from G217B supernatant. Lane 1, concentrated G217B supernatant; 20 mg of chitin flakes after incubation with PBS (lane 2) or G217B supernatant (lane 3); 20 mg of fibrous cellulose powder after incubation with PBS (lane 4) or G217B supernatant (lane 5). Supernatant from chitin flakes after incubation with PBS (lane 6) or G217B supernatant (lane 7). Supernatant from fibrous cellulose after incubation with PBS (lane 8) or G217B supernatant (lane 9). Chitin binds to and extracts Yps3p from culture supernatants.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated that cell wall-localized Yps3p is surface exposed and characterized H. capsulatum strains of three RFLP classes for natural surface localization and the ability for surface localization in expressing and nonexpressing strains. We demonstrated that secreted Yps3p has the ability to bind to the surface of Histoplasma capsulatum. This surface binding occurs quickly upon exposure to the protein. Enzymatic digestions and in vitro polysaccharide binding assays indicate that Yps3p binds to chitin, a component of fungal cell walls.

The binding of Yps3p to chitin is unusual. Saturable and specific binding often suggests a protein, but proteinase treatment did not decrease binding. Our results demonstrate binding to chitin both in its purified form and in yeast cell walls. The residual Yps3p binding after chitinase digestion could be due to incomplete chitin digestion or to some contribution from other cell wall constituents resistant to the degradative enzymes used in this study. We also cannot completely exclude the possibility that Yps3p interacts with another H. capsulatum supernatant component and this interaction contributes to surface binding, but two lines of evidence are inconsistent with this. First, although we mainly used supernatant from strain G217B for binding experiments, recombinant Yps3p purified from E. coli does bind to yeast cell surfaces and purified chitin (data not shown). Second, chitin affinity purification from strain G217B supernatant revealed Yps3p as the only specific product (Fig. 7). Strain G184AR, which possess the cell wall polysaccharide {alpha}-(1,3)-glucan, bound Yps3p severalfold less well than G184AS, the related variant strain that lacks {alpha}-(1,3)-glucan (22, 23). This polymer has been reported to make up the outermost layer of the Histoplasma cell wall (19), and it may shield chitin and therefore limit Yps3p binding. Immunofluorescence microscopy with FITC-WGA supported this hypothesis; we detected severalfold less surface-exposed chitin in strain G184AR than in strain G184AS (data not shown).

Although a function of Yps3p has not been determined, the release and surface attachment mechanism that we have demonstrated are features shared with other important microbial products that play important roles in pathogenesis. Secretion followed by surface localization has been described for surface entities from a range of microbes, both prokaryotes and eukaryotes (3, 4, 12). Interestingly, some secreted and reattached proteins, like Blastomyces dermatitidis Bad1p and MIC3 of Toxoplasma gondii, contain EGF-like or chitin binding domains (5, 12). This mechanism of localization has also been described for nonproteinaceous surface components. The extensive capsule of the pathogenic fungus Cryptococcus neoformans is attached to the cell wall polysaccharide {alpha}-1,3-glucan in a similar manner (29). In some cases, the receptor for surface binding has been identified, as is the case for chitin for Bad1p and Yps3p, but even in many of the cases where the receptor has been identified, detailed characterization of surface molecules involved in binding has not been accomplished. Intermolecular interactions and structural features involved in binding have also generally not been fully described.

It is not yet clear whether the secreted form, the surface-localized form, either, or both are important during Histoplasma capsulatum infection. Based on homology with Bad1, we are tempted to speculate that Yps3p could function as an adhesin, mediating interaction between Histoplasma cells and the mammalian host. The identification of Hsp60 as the ligand on H. capsulatum that binds to CD11 and CD18 receptors on macrophages (24) as well as the fact that Yps3p expression is limited to class 2 strains suggest it may not function in an adhesin capacity, however.

Another potential role for Yps3p could involve the modulation of cytokines. Research done in Cryptococcus neoformans and Blastomyces dermatitis has demonstrated that fungal pathogens can modulate host cytokines via secreted or cell surface-localized proteins (8, 27). Tumor necrosis factor alpha suppression by secreted and surface-localized Bad1 is the attribute of the protein that makes it a Blastomyces virulence factor (9).

Any function of the protein would obviously be restricted to strains that express it, when they are expressing it. We are currently exploring differential expression and regulation among strains and under different environmental conditions in vitro or during host infection. Interestingly, YPS3 was originally identified because of its yeast phase-specific expression, yet another feature shared with Blastomyces dermatitidis BAD1. Transcriptional regulation and promoter sequences important for yeast expression have been reported for BAD1 and YPS3 (30), providing evidence for some conserved mechanism(s).

We are also interested in exploring the regions of Yps3p necessary for surface binding. A truncation mutant of Bad1p which lacks the EGF-like domain can no longer bind to the Blastomyces cell surface (5). The EGF-like domain is the predominant feature of the much smaller Yps3p, so we are currently using site-directed mutagenesis of recombinant protein to examine the features of Yps3p involved in surface localization. Examination of the similarities and differences in structure, function, and regulatory mechanisms of these genes and encoded proteins in two related but different microbes will provide insights into fungal pathogenesis.


   ACKNOWLEDGMENTS
 
We thank Elizabeth Heinecke, Thanh Hoang, John Luecke, and Parul Trivedi for preliminary observations, James Bangs for use of a fluorescence microscope, Robert Zarnowski for critical reading of the manuscript, and Tristan Brandhorst, George Deepe, and Bruce Klein for helpful discussions.

This work was supported by NIH R01s HL55949 and AI52303 (to J.P.W.), NIH R37 AI42747 (to George Deepe), and a traineeship on NIH T32 AI055397 [GenBank] (M.L.B.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, 420 SMI, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706-1532. Phone: (608) 265-6292. Fax: (608) 265-6132. E-mail: jpwoods{at}wisc.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Berliner, M. D. 1968. Primary subcultures of Histoplasma capsulatum. I. Macro and micro-morphology of the mycelial phase. Sabouraudia 6:111-118.[Medline]
  2. Berliner, M. D., and N. Biundo, Jr. 1973. Effects of continuous light and total darkness on cultures of Histoplasma capsulatum. Sabouraudia 11:48-51.[Medline]
  3. Boot, H. J., and P. H. Pouwels. 1996. Expression, secretion and antigenic variation of bacterial S-layer proteins. Mol. Microbiol. 21:1117-1123.[CrossRef][Medline]
  4. Brandhorst, T., and B. Klein. 2000. Cell wall biogenesis of Blastomyces dermatitidis. Evidence for a novel mechanism of cell surface localization of a virulence-associated adhesin via extracellular release and reassociation with cell wall chitin. J. Biol. Chem. 275:7925-7934.[Abstract/Free Full Text]
  5. Brandhorst, T., M. Wuthrich, B. Finkel-Jimenez, and B. Klein. 2003. A C-terminal EGF-like domain governs BAD1 localization to the yeast surface and fungal adherence to phagocytes, but is dispensable in immune modulation and pathogenicity of Blastomyces dermatitidis. Mol. Microbiol. 48:53-65.[CrossRef][Medline]
  6. Brandhorst, T. T., M. Wuthrich, T. Warner, and B. Klein. 1999. Targeted gene disruption reveals an adhesin indispensable for pathogenicity of Blastomyces dermatitidis. J. Exp. Med. 189:1207-1216.[Abstract/Free Full Text]
  7. Di Lallo, G., S. Gargano, and B. Maresca. 1994. The Histoplasma capsulatum cdc2 gene is transcriptionally regulated during the morphologic transition. Gene 140:51-57.[CrossRef][Medline]
  8. Finkel-Jimenez, B., M. Wuthrich, T. Brandhorst, and B. S. Klein. 2001. The WI-1 adhesin blocks phagocyte TNF-alpha production, imparting pathogenicity on Blastomyces dermatitidis. J. Immunol. 166:2665-2673.[Abstract/Free Full Text]
  9. Finkel-Jimenez, B., M. Wuthrich, and B. S. Klein. 2002. BAD1, an essential virulence factor of Blastomyces dermatitidis, suppresses host TNF-alpha production through TGF-beta-dependent and -independent mechanisms. J. Immunol. 168:5746-5755.[Abstract/Free Full Text]
  10. Fisher, K. L., G. S. Deepe, Jr., and J. P. Woods. 1999. Histoplasma capsulatum strain variation in both H antigen production and beta-glucosidase activity and overexpression of HAG1 from a telomeric linear plasmid. Infect. Immun. 67:3312-3316.[Abstract/Free Full Text]
  11. Fisher, K. L., and J. P. Woods. 2000. Determination of beta-glucosidase enzymatic function of the Histoplasma capsulatum H antigen using a native expression system. Gene 247:191-197.[CrossRef][Medline]
  12. Garcia-Reguet, N., M. Lebrun, M. N. Fourmaux, O. Mercereau-Puijalon, T. Mann, C. J. Beckers, B. Samyn, J. Van Beeumen, D. Bout, and J. F. Dubremetz. 2000. The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite. Cell Microbiol. 2:353-364.[CrossRef][Medline]
  13. Gargano, S., G. Di Lallo, G. S. Kobayashi, and B. Maresca. 1995. A temperature-sensitive strain of Histoplasma capsulatum has an altered delta 9-fatty acid desaturase gene. Lipids 30:899-906.[Medline]
  14. Gass, M., and G. S. Kobayashi. 1969. Histoplasmosis. An illustrative case with unusual vaginal and joint involvement. Arch. Dermatol. 100:724-727.[CrossRef][Medline]
  15. Harris, G. S., E. J. Keath, and J. Medoff. 1989. Characterization of alpha and beta tubulin genes in the dimorphic fungus Histoplasma capsulatum. J. Gen. Microbiol. 135:1817-1832.[Medline]
  16. Harris, G. S., E. J. Keath, and J. Medoff. 1989. Expression of alpha- and beta-tubulin genes during dimorphic-phase transitions of Histoplasma capsulatum. Mol. Cell. Biol. 9:2042-2049.[Abstract/Free Full Text]
  17. Hwang, L., D. Hocking-Murray, A. K. Bahrami, M. Andersson, J. Rine, and A. Sil. 2003. Identifying phase-specific genes in the fungal pathogen Histoplasma capsulatum using a genomic shotgun microarray. Mol. Biol. Cell 14:2314-2326.[Abstract/Free Full Text]
  18. Johnson, C. H., M. G. Klotz, J. L. York, V. Kruft, and J. E. McEwen. 2002. Redundancy, phylogeny and differential expression of Histoplasma capsulatum catalases. Microbiology 148:1129-1142.[Abstract/Free Full Text]
  19. Kanetsuna, F., L. M. Carbonell, F. Gil, and I. Azuma. 1974. Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma capsulatum. Mycopathol. Mycol. Appl. 54:1-13.[CrossRef][Medline]
  20. Keath, E. J., and F. E. Abidi. 1994. Molecular cloning and sequence analysis of yps-3, a yeast-phase-specific gene in the dimorphic fungal pathogen Histoplasma capsulatum. Microbiology 140:759-767.[Abstract]
  21. Keath, E. J., A. A. Painter, G. S. Kobayashi, and G. Medoff. 1989. Variable expression of a yeast-phase-specific gene in Histoplasma capsulatum strains differing in thermotolerance and virulence. Infect. Immun. 57:1384-1390.[Abstract/Free Full Text]
  22. Klimpel, K. R., and W. E. Goldman. 1988. Cell walls from avirulent variants of Histoplasma capsulatum lack alpha-(1,3)-glucan. Infect. Immun. 56:2997-3000.[Abstract/Free Full Text]
  23. Klimpel, K. R., and W. E. Goldman. 1987. Isolation and characterization of spontaneous avirulent variants of Histoplasma capsulatum. Infect. Immun. 55:528-533.[Abstract/Free Full Text]
  24. Long, K. H., F. J. Gomez, R. E. Morris, and S. L. Newman. 2003. Identification of heat shock protein 60 as the ligand on Histoplasma capsulatum that mediates binding to CD18 receptors on human macrophages. J. Immunol. 170:487-494.[Abstract/Free Full Text]
  25. Medoff, G., B. Maresca, A. M. Lambowitz, G. Kobayashi, A. Painter, M. Sacco, and L. Carratu. 1986. Correlation between pathogenicity and temperature sensitivity in different strains of Histoplasma capsulatum. J. Clin. Investig. 78:1638-1647.
  26. Medoff, G., M. Sacco, B. Maresca, D. Schlessinger, A. Painter, G. S. Kobayashi, and L. Carratu. 1986. Irreversible block of the mycelial-to-yeast phase transition of Histoplasma capsulatum. Science 231:476-479.[Abstract/Free Full Text]
  27. Mozaffarian, N., A. Casadevall, and J. W. Berman. 2000. Inhibition of human endothelial cell chemokine production by the opportunistic fungal pathogen Cryptococcus neoformans. J. Immunol. 165:1541-1547.[Abstract/Free Full Text]
  28. Patel, J. B., J. W. Batanghari, and W. E. Goldman. 1998. Probing the yeast phase-specific expression of the CBP1 gene in Histoplasma capsulatum. J. Bacteriol. 180:1786-1792.[Abstract/Free Full Text]
  29. Reese, A. J., and T. L. Doering. 2003. Cell wall alpha-1,3-glucan is required to anchor the Cryptococcus neoformans capsule. Mol. Microbiol. 50:1401-1409.[CrossRef][Medline]
  30. Rooney, P. J., and B. S. Klein. 2004. Sequence elements necessary for transcriptional activation of BAD1 in the yeast phase of Blastomyces dermatitidis. Eukaryot. Cell. 3:785-794.[Abstract/Free Full Text]
  31. Spitzer, E. D., E. J. Keath, S. J. Travis, A. A. Painter, G. S. Kobayashi, and G. Medoff. 1990. Temperature-sensitive variants of Histoplasma capsulatum isolated from patients with acquired immunodeficiency syndrome. J. Infect. Dis. 162:258-261.[Medline]
  32. Tian, X., and G. Shearer, Jr. 2001. Cloning and analysis of mold-specific genes in the dimorphic fungus Histoplasma capsulatum. Gene 275:107-114.[CrossRef][Medline]
  33. Tian, X., and G. Shearer, Jr. 2002. The mold-specific MS8 gene is required for normal hypha formation in the dimorphic pathogenic fungus Histoplasma capsulatum. Eukaryot. Cell 1:249-256.[Abstract/Free Full Text]
  34. Vincent, R. D., R. Goewert, W. E. Goldman, G. S. Kobayashi, A. M. Lambowitz, and G. Medoff. 1986. Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms. J. Bacteriol. 165:813-818.[Abstract/Free Full Text]
  35. Weaver, C. H., K. C. Sheehan, and E. J. Keath. 1996. Localization of a yeast-phase-specific gene product to the cell wall in Histoplasma capsulatum. Infect. Immun. 64:3048-3054.[Abstract]
  36. Woods, J. P. 2002. Histoplasma capsulatum molecular genetics, pathogenesis, and responsiveness to its environment. Fungal Genet. Biol. 35:81-97.[CrossRef][Medline]
  37. Woods, J. P., and W. E. Goldman. 1992. In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum. Mol. Microbiol. 6:3603-3610.[Medline]
  38. Woods, J. P., E. L. Heinecke, and W. E. Goldman. 1998. Electrotransformation and expression of bacterial genes encoding hygromycin phosphotransferase and beta-galactosidase in the pathogenic fungus Histoplasma capsulatum. Infect. Immun. 66:1697-1707.[Abstract/Free Full Text]

Eukaryotic Cell, April 2005, p. 685-693, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.685-693.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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