Genomic Plasticity of the Human Fungal Pathogen Candida albicans

The genomic plasticity of Candida albicans, a commensal and common opportunistic fungal pathogen, continues to reveal unexpected surprises. Once thought to be asexual, we now know that the organism can generate genetic diversity through several mechanisms, including mating between cells of the opposite or of the same mating type and by a parasexual reduction in chromosome number that can be accompanied by recombination events (2, 12, 14, 53, 77, 115). In addition, dramatic genome changes can appear quite rapidly in mitotic cells propagated in vitro as well as in vivo. The detection of aneuploidy in other fungal pathogens isolated directly from patients (145) and from environmental samples (71) suggests that variations in chromosome organization and copy number are a common mechanism used by pathogenic fungi to rapidly generate diversity in response to stressful growth conditions, including, but not limited to, antifungal drug exposure. Since cancer cells often become polyploid and/or aneuploid, some of the lessons learned from studies of genome plasticity in C. albicans may provide important insights into how these processes occur in higher-eukaryotic cells exposed to stresses such as anticancer drugs.The purpose of this review is to describe the tools used to detect genome changes, to highlight recent advances in our understanding of large-scale chromosome changes that arise in Candida albicans, and to discuss the role of specific stresses in eliciting these genome changes. The types of genomic diversity that have been characterized suggest that C. albicans can undergo extreme genomic changes in order to survive stresses in the human host. We propose that C. albicans and other pathogens may have evolved mechanisms not only to tolerate but also to generate large-scale genetic variation as a means of adaptation.C. albicans is a polymorphic yeast with a 16-Mb (haploid) genome organized in 8 diploid chromosomes (140, 154, 203). The C. albicans genome displays a very high degree of plasticity. This plasticity includes the types of genomic changes frequently observed with cancer cells, including gross chromosomal rearrangements, aneuploidy, and loss of heterozygosity (reviewed in references 100, 117, and 157). Similar to somatic cancer cells, C. albicans reproduces primarily through asexual clonal division (65, 84). Nonetheless, it has retained much of the machinery needed for mating and meiosis (189), yet meiosis has never been observed (13, 120).C. albicans has two mating-type-like (MTL) alleles, MTLa and MTLα (76). The MTL locus is on the left arm of chromosome 5 (Chr5), approximately 80 kbp from the centromere. Most C. albicans isolates are heterozygous for the MTL locus, but approximately 3 to 10% of clinical isolates are naturally homozygous at MTL (104, 108). Mating can occur between strains carrying the opposite MTL locus, and most strains that were found to be naturally MTL homozygous are mating competent (104, 108). MTL-homozygous strains were also constructed from MTL-heterozygous strains by deletion of either the MTLa or MTLα locus (77) or by selection for Chr5 loss on sorbose (87, 115).Mating between these diploid strains of opposite mating type can occur both in vitro (115) and in vivo (77, 97). The products are tetraploid and do not undergo a conventional meiotic reduction in ploidy (12, 120). Rather, they undergo random loss of multiple chromosomes, a process termed “concerted chromosome loss,” until they reach a near-diploid genome content (2, 12, 53, 85). A subset of these cells also undergoes multiple gene conversion events reminiscent of meiotic recombination, and most remain trisomic for one to several chromosomes (53). While mating and concerted chromosome loss have been induced in the laboratory, the role of the parasexual cycle during the host-pathogen interaction and in the response to stresses, such as exposure to antifungal drugs, remains unclear. The prevailing model is that adaptive mutations (such as those that occur with the acquisition of drug resistance) evolve through somatic events, including point mutations, recombination, gene conversion, loss of heterozygosity, and/or aneuploidy (13).     

A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Yeast Cell Adhesion Molecules Have Functional Amyloid-Forming Sequences

The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776–782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The β-aggregation predictor TANGO found highly β-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were β-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High β-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.Protein amyloids are characteristic of pathological conditions, including neurodegenerative diseases (4, 11, 17, 38). These protein aggregates can also occur naturally in adhesive bacterial curli (3), melanosomes (14), condensed peptide hormone arrays (24), as regulatory prions in yeast (2, 5), and fungal hydrophobins, which are nonantigenic coats to some fungi (1, 33, 39). Nevertheless, such natural occurrences are relatively few, considering the negative free energy for amyloid formation (28).We have recently discovered that there are amyloid-forming sequences in the cell surface Als adhesins of Candida albicans. Cells that express these adhesins aggregate readily, and the aggregation has amyloid-like properties, including protein conformational shifting, surface birefringence, and ability to bind the amyloid-active dyes Congo red and amino-naphthalene sulfonic acid (ANS) (29). A five- to seven-residue sequence in Als1p, Als3p, and Als5p has extremely high potential for formation of β-aggregates, according to the protein state prediction program TANGO (13, 27, 31). Such β-aggregates include amyloids, which are ordered structures with paracrystalline regions of stacked parallel β-strands that are perpendicular to the long axis of micrometer-long fibrils. The strands are stabilized by interaction of identical sequences from many protein molecules (31, 32). Where TANGO analyses have shown that specific sequences have β-aggregate potentials greater than 20%, an insoluble β-aggregate state is likely to form. These β-aggregates nucleate formation of amyloids if the proteins can associate to form fibers (13, 27, 31). Sequences in the conserved 127-residue T region of Als1p, Als3p, and Als5p have β-aggregation potentials of >90% (27). An oligopeptide with this sequence, as well as 412- and 645-residue fragments of Als5p formed authentic amyloids, as determined by characteristic dye binding and fiber morphology. The amyloid-forming sequences were rich in the β-branched amino acids Thr, Val, and Ile. This amino acid composition is unusual among proteins in general, but is common in the Thr-rich mid-piece domains of yeast adhesins.Yeasts display many cell-wall-bound adhesins that mediate colonial and biofilm interactions as well as host-pathogen binding (9, 21, 41). Such adhesins have a common mosaic structure. In general, the adhesins have N-terminal globular binding domains (often immunoglobulin-like or lectin-like), Thr-rich mid-piece sequences including tandem repeats, and 300- to 800-residue heavily glycosylated Ser and Thr-rich “stalk” domains near the C-terminal domain that extend the active regions from the surface of the wall. The adhesins are covalently cross-linked to wall polysaccharides through modified glycosylphosphatidylinositol (GPI) anchors and/or glycosyl esters of glutamic acid (9, 18).Because the yeast adhesins share this common modular domain structure, we searched among known and putative yeast adhesins for sequences with high β-aggregation potential. We have found that many of these proteins share amyloid-forming sequences and amyloid-like behavior on activation.     

Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.Candida albicans is a major opportunistic fungal human pathogen that causes a wide variety of infections (9, 68). In healthy individuals C. albicans resides as a commensal within the oral cavity and gastrointestinal and urogenital tracts. However, in immunocompromised hosts, C. albicans causes infections ranging in severity from mucocutaneous infections to life-threatening disseminated diseases (9, 68). Research into the pathogenicity of C. albicans has revealed a complex mix of putative virulence factors (7, 60), perhaps reflecting the fine balance this species strikes between commensal colonization and opportunistic invasion of the human host.Melanins are biological pigments, typically dark brown or black, formed by the oxidative polymerization of phenolic compounds. They are negatively charged hydrophobic molecules with high molecular weights and are insoluble in both aqueous and organic solvents. Their insolubility makes melanins difficult to study, and no definitive structure has yet been found for them; they probably represent an amorphous mixture of polymers (35). There are various types of melanin in nature, including eumelanin and phaeomelanin (76). Two principal types of melanin are found in the fungal kingdom. The majority are 1.8-dihydroxynapthalene (DNH) melanins synthesized from acetyl-coenzyme A (CoA) via the polyketide pathway (5). DNH melanins have been found in a wide range of opportunistic fungal pathogens of humans, including dark (dematiaceous) molds, such as Cladosporium, Fonsecaea, Phialophora, and Wangiella species, and as conidial pigments in Aspergillus fumigatus and Aspergillus niger (41, 80, 87, 88). However, several other fungal pathogens, including Blastomyces dermatitidis, Coccidioides posadasii, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenckii, produce eumelanin (3,4-dihydroxyphenylalanine [DOPA]-melanin) through the activity of a polyphenol oxidase (laccase) and require an exogenous o-diphenolic or p-diphenolic substrate, such as l-DOPA (16, 30, 63,–65, 67, 79).The production of melanin in humans and other mammals is a function of specialized cells called melanocytes. Particles of melanin polymers, sometimes, including more than one melanin type, are built up within membrane-bound organelles called melanosomes (76), and these are actively transported along microtubules to the tips of dendritic outgrowths of melanocytes, from where they are transferred to neighboring cells (32, 81). The mechanism of intercellular transfer of melanosomes has not yet been established, but the export process probably involves the fusion of cell and vesicular membranes rather than secretion of naked melanin (82). In pathogenic fungi, melanins are often reported to be associated with or “in” the cell wall (35, 36, 50, 72, 79). However, there is variation between species: the melanin may be located external to the wall, e.g., in P. brasiliensis (79); within the wall itself (reviewed in reference 42); or as a layer internal to the wall and external to the cell membrane, e.g., in C. neoformans (22, 45, 85). However, mutants of C. neoformans bearing disruptions of three CDA genes involved in the biosynthesis of cell wall chitosan, or of CHS3, encoding a chitin synthase, or of CSR2, which probably regulates Chs3, all released melanin into the culture supernatant, suggesting a role for chitin or chitosan in retaining the pigment polymer in its normal intracellular location (3, 4). However, vesicles externalized from C. neoformans cells also show laccase activity (21), so the effect of chitin may be on vesicle externalization rather than on melanin itself. Internal structures compatible with mammalian melanosomes have been observed in Cladosporium carrionii (73) and in Fonsecaea pedrosoi (2, 26). Remarkably, F. pedrosoi also secretes melanin and locates the polymer within the cell wall (1, 2, 25, 27, 74).Melanization has been found to play an important role in the virulence of several human fungal pathogens, such as C. neoformans, A. fumigatus, P. brasiliensis, S. schenckii, H. capsulatum, B. dermatitidis, and C. posadasii (among recent reviews are references 29, 42, 62, 74, and 79). From these and earlier reviews of the extensive literature, melanin has been postulated to be involved in a range of virulence-associated properties, including interactions with host cells; protection against oxidative stresses, UV light, and hydrolytic enzymes; resistance to antifungal agents; iron-binding activities; and even the harnessing of ionizing radiation in contaminated soils (15). The most extensively studied fungal pathogen for the role of melanization is C. neoformans, which possesses two genes, LAC1 and LAC2, encoding melanin-synthesizing laccases (52, 69, 90). It has been known since early studies with naturally occurring albino variants of C. neoformans (39) that melanin-deficient strains are attenuated in mouse models of cryptococcosis. Deletion of both the LAC1 and LAC2 genes reduced survival of C. neoformans in macrophages (52), and a study based on otherwise isogenic LAC1⁺ and LAC1⁻ strains confirmed the importance of LAC1 in experimental virulence (66). Other genes in the regulatory pathway for LAC1 are similarly known to be essential to virulence (12, 84).C. albicans has been shown to produce melanin with DOPA as a substrate for production of the polymer (53). The cells could be treated with hot acids to produce typical melanin “ghosts,” and antibodies specific for melanin reacted with the fungal cells by immunohistochemistry with tissues from experimentally infected mice, demonstrating that C. albicans produces melanin in vivo (53). However, no candidate genes encoding laccases have yet been identified in the C. albicans genome (http://www.candidagenome.org/). In this study, we investigated the production of melanin by C. albicans and showed that its normal externalization from wild-type cells, including formation of melanosomes, can be altered to an intracellular and intrawall location by mutation of genes involved in chitin synthesis. C. albicans has four genes encoding chitin synthase enzymes. CHS1 is an essential gene under normal conditions (59), and its product is the main enzyme involved in septum formation (83). Chs3 forms the bulk of the chitin in the cell wall and the chitinous ring at sites of bud emergence (8, 51, 57), while Chs2 contributes to differential chitin levels found between yeast and hyphal forms of the fungus, and Chs8 influences the architecture of chitin microfibrils (43, 51, 55, 57, 58). We found that melanin externalization was unaffected in a chs8Δ mutant but was reduced or abrogated in chs2Δ and chs3Δ mutants. Expression profiles of melanin-producing cells grown in the presence of l-DOPA did not identify any potential laccase-synthesizing genes.     

Heterologous Expression of Candida albicans Cell Wall-Associated Adhesins in Saccharomyces cerevisiae Reveals Differential Specificities in Adherence and Biofilm Formation and in Binding Oral Streptococcus gordonii

Colonization and infection of the human host by opportunistic pathogen Candida albicans derive from an ability of this fungus to colonize mucosal tissues and prosthetic devices within the polymicrobial communities present. To determine the functions of C. albicans cell wall proteins in interactions with host or bacterial molecules, Saccharomyces cerevisiae was utilized as a surrogate host to express C. albicans cell wall proteins Als3p, Eap1p, Hwp1p, and Rbt1p. Salivary pellicle and fibrinogen were identified as novel substrata for Als3p and Hwp1p, while only Als3p mediated adherence of S. cerevisiae to basement membrane collagen type IV. Parental S. cerevisiae cells failed to form biofilms on salivary pellicle, polystyrene, or silicone, but cells expressing Als3p or Hwp1p exhibited significant attachment to each surface. Virulence factor Rbt1p also conferred lower-level binding to salivary pellicle and polystyrene. S. cerevisiae cells expressing Eap1p formed robust biofilms upon polystyrene surfaces but not salivary pellicle. Proteins Als3p and Eap1p, and to a lesser degree Hwp1p, conferred upon S. cerevisiae the ability to bind cells of the oral primary colonizing bacterium Streptococcus gordonii. These interactions, which occurred independently of amyloid aggregate formation, provide the first examples of specific C. albicans surface proteins serving as receptors for bacterial adhesins. Streptococcus gordonii did not bind parental S. cerevisiae or cells expressing Rbt1p. Taken collectively, these data suggest that a network of cell wall proteins comprising Als3p, Hwp1p, and Eap1p, with complementary adhesive functions, promotes interactions of C. albicans with host and bacterial molecules, thus leading to effective colonization within polymicrobial communities.Candida albicans is a pleiomorphic fungus found on mucosal surfaces of the gastrointestinal and genitourinary tracts, skin, and oral cavity (2). As an opportunistic pathogen, C. albicans can form potentially lethal fungal masses in the kidney, heart, and brain upon gaining access to the bloodstream (4), and invasive fungal infections are becoming increasingly problematic in the clinical setting (34). Candida species are now the third most common cause of nosocomial bloodstream infections. In the United States alone there are an estimated 70,000 cases per year of disseminated candidiasis (34), with an associated health care cost of $2 billion to $4 billion/year (44, 45). C. albicans is also responsible for >90% of oral fungal diseases derived from polymicrobial biofilms, and ≤90% of HIV-infected individuals suffer from oral candidiasis, which may progress to advanced esophageal candidiasis (10).C. albicans can colonize a wide variety of sites within the host in addition to mucosal tissues, such as catheters, stents, surgical implants, and dentures. This ability can be attributed, at least in part, to the large number of proteins expressed on the candidal cell surface, which mediate adhesion to a range of substrata. Cell wall proteins (CWPs) in C. albicans also play a critical role in biofilm formation. Within the host, Candida species are frequently found as part of polymicrobial biofilms, in which antagonistic, synergistic, and mutualistic interactions among microbes significantly influence composition of the community microflora (17). This is particularly pertinent for colonization of the oral cavity, where up to 100 different microbial species may be isolated from a single site at any given time. To successfully colonize the host and cause disease, C. albicans must therefore not only attach directly to host tissues or medical devices but also navigate interactions with a diverse microflora to ensure the availability of suitable binding sites, nutrients, and growth conditions.It has been shown that C. albicans coaggregates (coadheres) strongly with Streptococcus bacteria indigenous to the human oral cavity such as Streptococcus gordonii and Streptococcus sanguinis (13, 18). These bacteria are pioneer colonizers of oral cavity surfaces, and it is hypothesized that interactions with these streptococci may promote oral carriage and persistence of C. albicans, thereby supporting candidal reservoirs for opportunistic infections following disruption of the oral ecology. Previous work by Holmes et al. (13, 14) identified Streptococcus gordonii cell wall-associated polypeptides SspA, SspB, and CshA, together with linear cell wall phosphopolysaccharides, as potential targets for C. albicans binding streptococcal cells. However, the reciprocal receptors on the surface of C. albicans recognized by streptococci have yet to be identified.This work utilizes Saccharomyces cerevisiae, which does not bind streptococci, as a heterologous host for expression and identification of candidal surface proteins targeted by Streptococcus gordonii. Four surface proteins were selected that had been previously implicated in C. albicans colonization and pathogenesis: Als3p, Eap1p, Hwp1p, and Rbt1p. Als3p (comprehensively reviewed by Hoyer et al. [15]), Hwp1p (29, 40), and Eap1p (20, 22) are associated with mediating interactions of C. albicans with host epithelial cells and with biofilm formation in catheter models. Expression of Als3p or Hwp1p has been shown to be hypha specific, while Eap1p is expressed by each morphological form (16, 20, 41). Rbt1p shares 43% sequence identity with Hwp1p and has been associated with virulence in mouse and rabbit models of C. albicans infection (6). Using a recombinase-based Gateway cloning system (Invitrogen), each of the C. albicans proteins was expressed on the surface of S. cerevisiae. Their functional properties in adherence and biofilm formation were determined, and proteins Als3p and Eap1p were identified as potential Streptococcus gordonii receptors on the surface of C. albicans.