Virulence factors of Candida albicans.

Candidiasis is a common infection of the skin, oral cavity and esophagus, gastrointestinal tract, vagina and vascular system of humans. Although most infections occur in patients who are immunocompromised or debilitated in some other way, the organism most often responsible for disease, Candida albicans, expresses several virulence factors that contribute to pathogenesis. These factors include host recognition biomolecules (adhesins), morphogenesis (the reversible transition between unicellular yeast cells and filamentous, growth forms), secreted aspartyl proteases and phospholipases. Additionally, 'phenotypic switching' is accompanied by changes in antigen expression, colony morphology and tissue affinities in C. albicans and several other Candida spp. Switching might provide cells with a flexibility that results in the adaptation of the organism to the hostile conditions imposed not only by the host but also by the physician treating the infection.

Isolation and characterization of the Candida albicans MOT2 gene.

A putative Candida albicans homologue of Saccharomyces cerevisiae MOT2 (modulator of transcription) has been cloned and analyzed. A cDNA fragment corresponding to a portion of S. cerevisiae MOT2 was used to isolate a similar C. albicans gene (CaMOT2). CaMOT2 is comprised of two exons of 50 bp and 1,714 bp, respectively, with a single 82 bp intron located near the 5' end of the gene. The gene encodes a protein (CaMot2p) with an estimated mass of 67 kDa. The 5' region of the gene shows sequence homology with S. cerevisiae MOT2, whereas no significant similarity was observed in the 3' region. Similarly, the N-terminal portion of C. albicans Mot2p exhibits approximately 80% homology with S. cerevisiae Mot2p, while no significant homology to any known protein was observed in the carboxy-terminal half of the C. albicans protein. The N-terminal portion of CaMot2p contains a cysteine-rich domain (amino acids 18-62). The distribution of the cysteine residues identifies CaMot2p as a zinc-finger protein. The data suggest two potential Zn-binding sites, similar to the arrangement found in S. cerevisiae. Reverse-transcriptase polymerase chain reaction was used to compare the level of CaMOT2 expression between C. albicans grown in vitro and growth during in vivo infection in the rat model of oral candidiasis. The results showed CaMOT2 is down-regulated during growth in the rat oral cavity compared to in vitro culture. Although the function of C. albicans MOT2 has not been determined, comparison to S. cerevisiae MOT2 suggests the gene product may act as a general negative regulator.

Identification of a putative response regulator two-component phosphorelay gene ( CaSSK1) from Candida albicans.

We have identified and analysed a putative response regulator two-component gene (CaSSK1) from Candida albicans and its encoding protein (CaSsk1p). CaSSK1 has an open reading frame of 2022 bp. In the promotor region of CaSSK1 a short sequence is found that matches the consensus sequence of the stress response elements (STRE) from Saccharomyces cerevisiae. CaSSK1 is located on chromosome 1 and is expressed in either yeast or mycelial phases of C. albicans. CaSSK1 encodes a 674 amino acid protein (CaSsk1p) with an estimated molecular mass of 73.5 kDa and a basic isoelectric point (pI 9.5). It has a tripeptide (NKA) located in its C-terminus, which resembles the peroxisomal signalling target type 1 sequence (PST1) of most of the peroxisomal matrix proteins. A homology search of CaSsk1p with other proteins in databases showed that the C-terminus of CaSsk1p exhibits the greatest similarity with the C-terminus of Ssk1p and Mcs4 from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The response regulator domain of CaSsk1p contains the motifs that are characteristic of all response regulators, including the conserved aspartate and lysine residues as well as the putative aspartate, which is phosphorylated by a phosphohistidine residue. Finally, in spite of the structural similarities among CaSsk1p, Ssk1p and Mcs4, CaSsk1p does not seem to exhibit functional homology with these proteins.

Identification of a putative histidine kinase two-component phosphorelay gene (CaHK1) in Candida albicans.

We have cloned and analysed the sequence of a putative histidine kinase, two-component gene (CaHK1) from Candida albicans. This gene encodes a 2471 amino acid protein (Cahk1p) with an estimated molecular mass of 281.8 kDa. A homology search of Cahk1p with other proteins in the databases showed that Cahk1p exhibits the greatest homology at its C-terminus with both the sensor and regulator components of prokaryotic and eukaryotic two-component histidine kinases. A further analysis of this homology showed that the Cahk1p possessed both sensor and regulator domains in the same polypeptide. Also, Cahk1p is likely to be a soluble protein. The sensor kinase domain of Cahk1p contains conserved motifs that are characteristic of all histidine kinase proteins, including the putative histidine which is believed to be autophosphorylated during activation, ATP binding motifs and others (F- and N-motifs), with unknown function. The Cahk1p regulator domain also contains conserved aspartate and lysine residues and the putative aspartate, which is secondarily phosphorylated by the autophosphorylated histidine. Finally, according to the codon usage frequency of the CaHK1 gene in comparison with other genes from C. albicans, there would appear to be a low level of expression of the gene.

Expression of the complement-binding protein (MP60) of Candida albicans in experimental vaginitis.

The expression of the Candida albicans complement-binding C3d protein (MP60) was investigated both in vitro and in vivo by immunogold labelling and electron microscopy. In vivo expression was determined in a rat vaginitis model. Reactivity of in vitro-grown cells to an anti-MP60 rabbit serum was associated with both cytoplasmic and cell wall sites. Immunostaining in the cell wall of both yeast and hyphae was most concentrated in the inner, electron-lucid layer. Immunogold stained preparations of C. albicans from vaginal smears of infected animals also showed intense localization of the MP60 in the inner cell wall, plasma membrane. However, immunogold label was also intense at the cell surface in these samples, mostly in the area of close adherence with the keratinocytes of the vaginal epithelia. These observations indicate that MP60 is expressed both in vitro and in vivo, but to a different degree in the different cell wall layers.

Isolation of phosphoribosylpyrophosphate synthetase (PRS1) gene from Candida albicans.

We have isolated a 3.7 kb EcoR1 fragment from a genomic library of Candida albicans which displayed a 65% level of identity with the PRS gene family (PRS) of Saccharomyces cerevisiae. The PRS gene encodes a phosphoribosylpyrophosphate (PRPP) synthetase of S. cerevisiae, which catalyses the synthesis of purines, pyrimidines, and amino acids such as histidine and tryptophan. By Northern analyses, we observed that the entire 3.7 kb EcoR1 fragment as well as 1.1 kb KpnI-SacI internal fragment of the 3.7 kb EcoR1 fragment hybridized to the same 1.4 kb transcript. An internal 2.6 kb KpnI fragment was subcloned and sequenced. A deduced sequence of 321 amino acids representing a polypeptide of 35.2 kDa was determined. A FASTA search indicated that the C. albicans PRS (Ca PRS1) had an overall homology at the amino acid level of 91% with the S. cerevisiae PRS3. Putative transcriptional start and termination sequences as well as a cation-binding, PRPP synthetase signature sequence were identified. Ca PRS1 was localized to chromosome 2 of the C. albicans genome. Low stringency hybridizations indicates that the organism may possess multiple PRS genes. The function of these genes in nitrogen signaling is discussed.

Isolation of avirulent clones of Candida albicans with reduced ability to recognize the CR2 ligand C3d.

Four clones of the yeast Candida albicans, isolated on the basis of their tolerance to clotrimazole, were compared with their parental strains in terms of growth, morphology, virulence, and cell surface complement receptor activity. In a newly described synthetic medium, these clones, designated C1, C2, N, and P, produced germ tubes or pseudohyphae, but no true hyphae, in a pattern which was specific for each strain. The growth of each clone at 37 degrees C, under conditions which favor the filamentous growth form of the organism, was equal to that of the parental strain (H12). The pathogenicity of each clone was tested in an intravenous mouse model. None of the mice infected with the tolerant clones but all of the mice infected with H12 developed severe renal candidiasis after infection with 1.4 x 10(6) to 2.0 x 10(6) CFU/ml. The number of CFU of each clone from the mouse kidney was reduced about 3 or 4 orders of magnitude in comparison with the wild type. As a correlate, we measured the complement receptor activity (CR2 and CR3) of each clone. The C3 ligands, iC3b and C3d, were conjugated to sheep erythrocytes (E) sensitized with antibody (A) to the erythrocytes (EA). We found that all tolerant clones showed reduced recognition of C3d-bearing sheep erythrocytes (EAC3d) in rosetting assays. Clone P showed more than an 80% reduction in rosetting of EAC3d in comparison with H12 cells. In contrast, recognition of iC3b (EAiC3b) by each of the clones was similar to that by H12 cells. When dithiothreitol extracts of clone P and H12 were compared by immunoblot, both quantitative and qualitative differences in reactivities were observed with antibodies specific for the Candida C3d receptor and with antiserum from a patient with chronic mucocutaneous candidiasis.

Recognition between Candida albicans and host cells.

The cell surface of Candida albicans is a complex mosaic of polysaccharide and protein, of which mannoproteins constitute the major antigens and host cell recognition molecules. One group of mannoproteins is known as 'adhesins' and has properties similar to lectins and integrins. The adhesins recognize either host cell fucosyl glycosides or peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD peptides).

Molecular interactions at the interface of Candida albicans and host cells.

The interaction of C. albicans with host cells has been shown to be quite complex. At least three recognition systems have been described, and, probably additional ones exist. The recognition systems are classified by the type of host cell, the growth form of the organism and the nature of the interaction at the molecular level, i.e., protein-protein or protein-oligosaccharide. It would appear that C. albicans has at least two distinct mannoprotein adhesins. One resembles a lectin in that it recognizes host cell glycosides containing fucose, and with some strains of C. albicans, N-acetylglucosamine. The second adhesin has properties similar to the integrin or transmembrane glycoproteins such as the CR3 (complement receptor 3). Still to be resolved is the characterization of the lectin-like adhesin as well as the relationship of the CR2-like activity to the CR3 of Candida. A third adhesin which is a mannan polysaccharide has been suggested as a recognition molecule in the adherence of the organism to buccal epithelial cells.

Cell extracts of Candida albicans block adherence of the organisms to endothelial cells.

Cell extracts of Candida albicans were fractionated by concanavalin A affinity chromatography. Eluted mannosylated proteins (fraction II) and nonbinding, nonmannosylated proteins (fraction I) were collected and assayed directly for inhibition of adherence of C. albicans to endothelium. Fraction II blocked blastospore adherence to endothelial cells. Fraction I blocked both blastospore and germ tube adherence to endothelial cells. Monoclonal antibody OKM-1 (anti-CR3) and an anti-C. albicans monoclonal antibody, CA-A (anti-CR2), reacted in Western blots with proteins from fraction I, suggesting the presence of the CR2- and CR3-like proteins that have been previously identified on C. albicans germ tubes.

Evidence for expression of the C3d receptor of Candida albicans in vitro and in vivo obtained by immunofluorescence and immunoelectron microscopy.

The complement conversion product C3d binds to a receptor on the cell surface of Candida albicans. While the function of this receptor is still uncertain, we investigated whether it is expressed during a murine infection. Rabbit antiserum raised against purified receptor was used in conjunction with immunofluorescence microscopy and immunocolloidal gold electron microscopy to examine kidney tissue and peritoneal lavages from infected mice for receptor expression by C. albicans in vivo. Specificity of the antiserum was indicated by reactivity with purified receptor (55 to 60 kDa) and with a protein of similar molecular mass from whole hyphal extracts in Western blots (immunoblots). In vitro analysis by immunofluorescence microscopy showed that the antiserum reacted with both yeast and pseudohyphal forms of the organism, but reactivity was strongest with pseudohyphae. Immunocolloidal gold electron microscopy of fungal cells from peritoneal lavages revealed intense staining of mother cells of germinative forms, germ tubes, and pseudohyphae. Staining of the mother cells was heaviest at the innermost layers of the cell wall but only scant on the cell surface. In contrast, staining was observed throughout the cell walls of germ tubes and pseudohyphae. In kidney, expression of the C3d receptor was found primarily on the cell walls of hyphae and pseudohyphae, although some staining was observed in the cytoplasm. These data support that the C3d receptor of C. albicans is expressed in vivo.

Adherence of cell surface mutants of Candida albicans to buccal epithelial cells and analyses of the cell surface proteins of the mutants.

Strains of Candida albicans, selected on the basis of their reduced agglutination with a polyclonal anti-Candida antiserum, were tested for their adherence to human buccal epithelial cells (BEC). Of four strains, one (A9V2) had reduced binding to BEC in vitro. Adherence of wild type (wt) yeast cells (A9), as measured by the percentage of BEC with adhering Candida cells, was 73.4% +/- 3.8% compared with 49.3% +/- 3.1% for A9V2 (P less than 0.01). From yeast cells of A9 and A9V2, whole-cell extracts and dithiothreitol-, Zymolyase-, or beta-mercaptoethanol-solubilized cell extracts were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). From dithiothreitol-solubilized cell extracts, proteins with molecular masses of 55 to 60, 80 to 84, 115, and 165 kDa were observed from wt (A9) cells but were highly reduced in amount or absent from A9V2 cells. Western blot profiles of Zymolyase-solubilized extracts from both A9 and A9V2 were similar in appearance, while 55-, 80- to 84-, 115-, and 165-kDa proteins were observed only in A9 cells extracted with beta-mercaptoethanol. Strain A9V4, also selected by reduced agglutination but which adhered as well as strain A9, lacked the 80- to 84-kDa and 115-kDa proteins but otherwise was similar to strain A9. These results indicate that the 55- to 60- and 165-kDa proteins may be related to an adhesin function in C. albicans. The differences observed in the protein profiles of the wt adhering strain and its derived nonadhering mutant are similar to those described for another matched pair of C. albicans strains.

Adherence and receptor relationships of Candida albicans.

The cell surface of Candida albicans is composed of a variety of polysaccharides such as glucan, chitin, and mannan. The first two components primarily provide structure, while the mannan, often covalently linked to protein, constitutes the major antigen of the organism. Mannoproteins also have enzymatic activity (acid protease) and ligand-receptor functions. The complement receptors of C. albicans appear to be mannoproteins that are required for the adherence of the organism to endothelial cells. This is certainly true of the CR3-like protein of C. albicans. Proof that the CR3 is the Candida receptor for endothelial cells is derived from two observations. First, mutants lacking CR3 activity are less adherent in vitro and, in fact, less virulent. Second, the ligand recognized by the CR3 receptor (C3bi) as well as anti-CR3 antibodies blocks adherence of the organism to endothelial cells. The CR2 of C. albicans appears to promote the adherence of the organism to plastic substrates. Unlike the CR2 of mammalian cells, the Candida CR2 recognizes ligands containing the RGD sequence of amino acids in addition to the C3d ligand, which does not contain the RGD sequence. There is uncertainty as to whether the Candida CR2 and CR3 are, in fact, different proteins. A mannoprotein has also been described as the adhesin for epithelial cells. In this case, the receptor has a lectinlike activity and recognizes fucose- or glucosamine-containing glycoproteins of epithelial cells, depending on the strain of C. albicans. The oligosaccharide component of the receptor is probably not involved in ligand recognition and may serve to stabilize the receptor. However, the oligosaccharide factor 6 epitope of mannan may also provide adhesin activity in the recognition of epithelial cells. Mannoproteins can be extracted from cells by a number of reagents. Zymolyase, for instance, tends to remove structural mannoproteins, which contain relatively little protein and are linked to glucan. Reagents such as dithiothreitol, on the other hand, tend to extract mannoproteins containing higher amounts of protein that appear to have receptor function. The mannoproteins of C. albicans are dynamically expressed and may be growth phase and growth form specific.

Granulocyte-macrophage colony-stimulating factor augments human monocyte fungicidal activity for Candida albicans.

The ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to augment the fungicidal activity of human monocytes for Candida albicans was evaluated. Purified human monocytes cultured with [3H]leucine-labeled C. albicans caused a dose-dependent release of the [3H]leucine. The amount of [3H]leucine released correlated with a decrease in the number of viable yeast colonies. Monocyte cytotoxicity for C. albicans was reduced by superoxide dismutase and catalase and by inhibitors of myeloperoxidase and scavengers of hydroxyl radical and single oxygen, consistent with monocyte candidacidal activity being partly dependent upon products of oxidative metabolism. Monocytes incubated with rhGM-CSF produced more superoxide anion (O2-) spontaneously and after stimulation than control monocytes (P less than .05). Enhanced O2- production was dose-dependent and specific for rhGM-CSF and could be inhibited by antibody to rhGM-CSF. In association with rhGM-CSF-induced production of O2-, the cytokine enhanced cytotoxic activity for C. albicans. These findings indicate that rhGM-CSF stimulates human monocyte fungicidal activity for C. albicans.

Reduced expression of the functionally active complement receptor for iC3b but not for C3d on an avirulent mutant of Candida albicans.

Pseudohyphae of Candida albicans bear surface receptors for iC3b and C3d. In order to determine a possible role for these receptors in the pathogenesis of candidiasis, a spontaneous C. albicans mutant, m-10, which exhibits reduced ability to adhere in vitro to fibrin platelet clots and epithelial cells or to cause endocarditis in a rabbit model, and its parent wild-type (wt) strain were compared for receptor expression in rosetting assays with sheep erythrocytes carrying iC3b (EAC1423bi) or C3d (EAC1423d). An equally high attachment to wt and m-10 was seen with EAC1423d, whereas rosetting with EAC1423bi was reduced by 53% in m-10 compared with wt. In inhibition studies, rosetting of wt with EAC1423bi was markedly inhibited by culture filtrate, hyphal-cell extract, and DEAE-fractionated material prepared from wt (54, 87, and 70% decreases in rosetting, respectively), thus suggesting the presence of the soluble, functionally active iC3b receptor of C. albicans in each of these preparations. Minimal inhibition of iC3b rosetting, however, was seen with the identical materials from m-10 (21, 5, and 12%, respectively). All of the preparations from the two strains were equally effective in their inhibitory activities against rosetting of C3d. A human serum specimen obtained from a patient with chronic mucocutaneous candidiasis blocked iC3b rosetting of the wt strain almost completely. When used in an immunoblot, this serum recognized proteins of 68 to 71, 55, and 50 kilodaltons (kDa) in hyphal-cell extracts of the wt. With the same preparation of the avirulent mutant, only weak reactions with the 68- to 71-kDa and 55-kDa proteins occurred, while the 50-kDa protein was not detectable. Taken together, these results indicate that the expression of the functionally active iC3b receptor on C. albicans may be involved in the virulence of the organism, possibly by mediating adherence to mammalian cells.

A monoclonal antibody that defines a surface antigen on Candida albicans hyphae cross-reacts with yeast cell protoplasts.

Female BALB/c mice were immunized with a whole-hyphal-cell extract obtained from Candida albicans wild-type strain 4918 grown in Lee medium. Monoclonal antibody (MAb)-producing hybridomas were prepared by fusing immune splenocytes with NS-1 myeloma cells. One of the hybrid cell clones (1.183) secreted an immunoglobulin G1 antibody that reacted with C. albicans hyphae in an indirect immunofluorescence assay but not with yeast cells and pseudohyphal segments directly originating from parent blastoconidia. In the same assay eight of nine recent clinical C. albicans isolates and Candida stellatoidea tested positive for hyphal cell-specific reactivity with MAb 1.183. The recognized antigen on hyphal cells was sensitive to heat treatment, beta-mercaptoethanol reduction, and proteolysis with pronase, trypsin, and subtilisin. Western blot (immunoblot) analysis of hyphal whole-cell and dithiothreitol extracts with MAb 1.183 revealed two major proteins with approximate molecular masses of 55 and 60 kilodaltons (kDa) under reducing conditions. Endo-alpha-N-acetylgalactosaminidase (O-glycanase) treatment reduced the molecular mass of the 60-kDa protein slightly but did not affect recognition by MAb 1.183, whereas peptide:N-glycosidase F (N-glycanase) had no effect on either protein. When exponentially growing yeast cells were treated sequentially with EDTA, beta-mercaptoethanol, and Zymolase to form protoplasts, a specific immunofluorescence signal was obtained with MAb 1.183. In a Western blot, MAb 1.183 showed reactivity with a 20-kDa protein in the sodium dodecyl sulfate extract from protoplasts, whereas no reactivity was found with cell wall material obtained from yeast cells. In summary, these experiments indicated that specific cell surface components from C. albicans hyphae are related to antigens which are present in yeast cells but are not detectable on the surface of the latter.

Host-parasite relationships in candidosis.

Candida albicans has several properties which allow it to colonize and invade host tissues, often resisting eradication. Two of these properties, adherence and acid proteinase production, seem to be genuine factors. Phenotypic switching and molecular mimicry may also provide the organism with an arsenal of mechanisms to evade host defenses.

Characterization of mannoproteins from a virulent Candida albicans strain and its derived, avirulent strain.

Antigenic differences between a wild-type virulent strain of Candida albicans 4918 (wt) and its spontaneous avirulent mutant (m-10) were found with crossed-immunoelectrophoresis and western blotting. Mannoprotein fractions from yeast and mycelial forms were obtained by affinity chromatography on concanavalin A and analyzed with use of patient sera or sera from rabbits infected with live wt cells and boosted with whole extracts or rabbits immunized with purified wt cell wall preparations. A spike-formed precipitate was detected only with the wt yeast cells with crossed-electrophoresis and patient and rabbit sera. Otherwise serum from infected rabbits precipitated about equally the wt and m-10 cell wall mannoprotein antigens. The nature of this precipitate, with extremely slow electromobility in the first dimension and the possibility of its relation to some special immunodeterminant of the wt mannan molecule, is discussed. Likewise, when mycelial mannoproteins were analyzed by crossed-immunoelectrophoresis, a precipitate specific for wt cells was observed with rabbit anti-Candida sera. Also, a mannoprotein of approximately 47 kilodaltons was observed by western blotting only for wt mycelial cells. The observed antigenic differences between the virulent and the avirulent derived strain might be related to the greater adherence and infectivity of the wt strain.