Identification of Candida albicans ALS2 and ALS4 and Localization of Als Proteins to the Fungal Cell Surface

Additional genes in the growing ALS family of Candida albicans were isolated by PCR screening of a genomic fosmid library with primers designed from the consensus tandem-repeat sequence of ALS1. This procedure yielded fosmids encoding ALS2 and ALS4. ALS2 and ALS4 conformed to the three-domain structure of ALS genes, which consists of a central domain of tandemly repeated copies of a 108-bp motif, an upstream domain of highly conserved sequences, and a domain of divergent sequences 3′ of the tandem repeats. Alignment of five predicted Als protein sequences indicated conservation of N- and C-terminal hydrophobic regions which have the hallmarks of secretory signal sequences and glycosylphosphatidylinositol addition sites, respectively. Heterologous expression of an N-terminal fragment of Als1p in Saccharomyces cerevisiae demonstrated function of the putative signal sequence with cleavage following Ala17. This signal sequence cleavage site was conserved in the four other Als proteins analyzed, suggesting identical processing of each protein. Primary-structure features of the five Als proteins suggested a cell-surface localization, which was confirmed by indirect immunofluorescence with an anti-Als antiserum. Staining was observed on mother yeasts and germ tubes, although the intensity of staining on the mother yeast decreased with elongation of the germ tube. Similar to other ALS genes, ALS2 and ALS4 were differentially regulated. ALS4 expression was correlated with the growth phase of the culture; ALS2 expression was not observed under many different in vitro growth conditions. The data presented here demonstrate that ALS genes encode cell-surface proteins and support the conclusion that the size and number of Als proteins on the C. albicans cell surface vary with strain and growth conditions.     

Candida albicans Als3, a multifunctional adhesin and invasin

Candida albicans is part of the normal human flora, and it grows on mucosal surfaces in healthy individuals. In susceptible hosts, this organism can cause both mucosal and hematogenously disseminated disease. For C. albicans to persist in the host and induce disease, it must be able to adhere to biotic and abiotic surfaces, invade host cells, and obtain iron. The C. albicans hypha-specific surface protein Als3 is a member of the agglutinin-like sequence (Als) family of proteins and is important in all of these processes. Functioning as an adhesin, Als3 mediates attachment to epithelial cells, endothelial cells, and extracellular matrix proteins. It also plays an important role in biofilm formation on prosthetic surfaces, both alone and in mixed infection with Streptococcus gordonii. Als3 is one of two known C. albicans invasins. It binds to host cell receptors such as E-cadherin and N-cadherin and thereby induces host cells to endocytose the organism. Als3 also binds to host cell ferritin and enables C. albicans to utilize this protein as a source of iron. Because of its multiple functions and its high expression level in vivo, Als3 is a promising target for vaccines that induce protective cell-mediated and antibody responses. This review will summarize the multiple functions of this interesting and multifunctional protein.     

Recognition of Candida albicans Als3 by the germ tube-specific monoclonal antibody 3D9.3

Monoclonal antibody 3D9.3 (MAb 3D9.3) reacts with the surface of Candida albicans germ tubes and recognizes a protein epitope. We used a two-step chromatography procedure to purify and identify the antigen (3D9) from C. albicans strain 66396 germ tubes. MAb 3D9.3 recognized two intense protein bands at 140 and 180 kDa. A comparative analysis between theoretical and experimental mass spectrum peaks showed that both bands corresponded to Als3. This conclusion was supported by lack of reactivity between MAb 3D9.3 and an als3 Δ /als3 Δ mutant strain, and the fact that an immunoglobulin preparation enriched for Als3 specificity recognized the purified 3D9 antigen. PCR demonstrated that C. albicans strain 66396 has two different-sized ALS3 alleles that correspond to the two purified protein bands. Strain- and species-specificity of the 3D9 epitope were studied with various C. albicans strains and Candida species, such as closely related Candida dubliniensis . The 3D9 epitope was detected only in C. albicans , demonstrating the utility of MAb 3D9.3 for differentiation between C. albicans and C. dubliniensis . Adhesion assays demonstrated that MAb 3D9.3 blocks adhesion of C. albicans germ tubes to human buccal epithelial cells and vascular endothelial cells.     

Functional and structural diversity in the Als protein family of Candida albicans

The human fungal pathogen Candida albicans colonizes and invades a wide range of host tissues. Adherence to host constituents plays an important role in this process. Two members of the C. albicans Als protein family (Als1p and Als5p) have been found to mediate adherence; however, the functions of other members of this family are unknown. In this study, members of the ALS gene family were cloned and expressed in Saccharomyces cerevisiae to characterize their individual functions. Distinct Als proteins conferred distinct adherence profiles to diverse host substrates. Using chimeric Als5p-Als6p constructs, the regions mediating substrate-specific adherence were localized to the N-terminal domains in Als proteins. Interestingly, a subset of Als proteins also mediated endothelial cell invasion, a previously unknown function of this family. Consistent with these results, homology modeling revealed that Als members contain anti-parallel beta-sheet motifs interposed by extended regions, homologous to adhesins or invasins of the immunoglobulin superfamily. This finding was confirmed using circular dichroism and Fourier transform infrared spectrometric analysis of the N-terminal domain of Als1p. Specific regions of amino acid hypervariability were found among the N-terminal domains of Als proteins, and energy-based models predicted similarities and differences in the N-terminal domains that probably govern the diverse function of Als family members. Collectively, these results indicate that the structural and functional diversity within the Als family provides C. albicans with an array of cell wall proteins capable of recognizing and interacting with a wide range of host constituents during infection.     

The N-terminal part of Als1 protein from Candida albicans specifically binds fucose-containing glycans

The opportunistic pathogen Candida albicans expresses on its surface Als (Agglutinin like sequence) proteins, which play an important role in the adhesion to host cells and in the development of candidiasis. The binding specificity of these proteins is broad, as they can bind to various mammalian proteins, such as extracellular matrix proteins, and N- and E-cadherins. The N-terminal part of Als proteins constitutes the substrate-specific binding domain and is responsible for attachment to epithelial and endothelial cells. We have used glycan array screening to identify possible glycan receptors for the binding domain of Als1p-N. Under those conditions, Als1p-N binds specifically to fucose-containing glycans, which adds a lectin function to the functional diversity of the Als1 protein. The binding between Als1p-N and BSA-fucose glycoconjugate was quantitatively characterized using surface plasmon resonance, which demonstrated a weak millimolar affinity between Als1p-N and fucose. Furthermore, we have also quantified the affinity of Als1p-N to the extracellular matrix proteins proteins fibronectin and laminin, which is situated in the micromolar range. Surface plasmon resonance characterization of Als1p-N-Als1p-N interaction was in the micromolar affinity range.     

Induction of N-acetyl-D-glucosamine catabolic enzymes and germinative response in Candida albicans

The regulation of N-acetylglucosamine catabolic enzymes was studied in both yeast and germ tube forms of the dimorphic fungus Candida albicans. The induction pattern of these enzymes was the same for yeast cells incubated at 28 degrees C and in cells incubated at 37 degrees C which formed germ tubes. However, the level of activity of these enzymes in germ tube stage is lower as compared to yeast phase cells. A strain of C. albicans that did not form germ tubes was endowed with a pronounced ability for induction of N-acetylglucosamine catabolic enzymes. This result suggests that germ tube formation and N-acetylglucosamine metabolism are mutually exclusive events.     

Evaluation of the role of Candida albicans agglutinin-like sequence (Als) proteins in human oral epithelial cell interactions

The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.     

Differential expression of cytoplasmic proteins during yeast bud and germ tube formation in Candida albicans

Changes in the identity and quantity of proteins synthesized during morphogenesis may result from alterations in gene expression in the dimorphic yeast Candida albicans. Stationary phase yeast cells, upon resuming growth at 25 degrees C, form budding yeast and at 37 degrees C form germ tubes. In order to identify proteins associated with morphogenesis, we compared cytoplasmic proteins synthesized during germ tube and bud formation. Proteins synthesized during this period were labeled at four intervals with either [3H]leucine or [35S]methionine and separated by two-dimensional polyacrylamide gel electrophoresis. This study shows that, of the 230 proteins resolved on each gel, 5 were specific to the yeast morphology and 2 proteins showed reduction in net synthesis in the mycelial phase. There were, however, no mycelium-specific proteins at any labeling period. The majority of proteins were common to both morphologies and showed no major shift in number during resumption of growth. The observations reported here suggest that differential gene expression occurs during morphogenesis of C. albicans.     

Involvement of calcium, calmodulin and protein phosphorylation in morphogenesis of Candida albicans

N-Acetyl-D-glucosamine-induced germ tube formation in Candida albicans at 37 degrees C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 degrees C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2(+)-induced shift in electrophoretic mobility.     

Septin function in Candida albicans morphogenesis

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 and CDC12 septins are essential for viability. In contrast, the C. albicans cdc10Delta and cdc11Delta mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. The cdc11Delta mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.     

Inhibition of Candida albicans adhesion by recombinant human antibody single-chain variable fragment specific for Als3p

The Candida albicans adhesin, Als3p, was identified as a potential cognate antigen for previously described human antibody fragments [single-chain variable fragment (scFv)] based on similarity of the binding pattern of the scFv to the distribution of this protein on the hyphal surface. Although all scFv bound avidly to wild type, scFv3 showed no detectable binding via immunofluorescence assay to strain 1843, containing a homozygous deletion of ALS3. Binding to the ALS3 reintegrant strain, 2322, was preserved, and scFv3 also bound to Saccharomyces cerevisiae expressing ALS3. Other scFv retained binding to 1843, but with a markedly altered pattern. To determine if scFv3 could interfere with Als3p function, adhesion assays were conducted using human epithelial or endothelial cells as target. Treatment of wild-type C. albicans with scFv3 reduced adhesion of the fungus to both cell types to levels comparable to the als3Delta/als3Delta mutant. These experiments confirm that phage display is a viable method to isolate human scFv specific to an antigen implicated in C. albicans virulence, and that the scFv interfere with adhesion to human cells. The altered pattern of immunostaining with other scFv that retain binding to the als3Delta/als3Delta mutant suggest that Als3p may also have a role in structural organization of the C. albicans cell surface.     

Ethanol-induced germ tube formation in Candida albicans

Ethanol is the first reported compound which can induce germ tube formation in Candida albicans without the addition of any nitrogen-containing nutrients. Conditions controlling induction of germ tubes in C. albicans by ethanol were investigated. Ethanol (17.1 mM) in buffered salts solution containing sodium bicarbonate induced 70 to 80% of yeast phase cells of C. albicans to form germ tubes. Germ tubes could be induced by ethanol (0.08 to 340 mM) at temperatures ranging from 29 to 41 degrees C (optimum 37 degrees C) and at pH values ranging from 3.0 to 8.0 (optimum 5.75). The germ tubes averaged 11 micron in length after 6 h at 37 degrees C. The percentage of cells forming germ tubes decreased as the concentration of cells in the induction solution was increased above 4 X 10(5) cells ml-1. Germ tubes first appeared 45 to 60 min after continuous exposure to ethanol at 37 degrees C and all cells which formed germ tubes did so by 2 h. Germ tube length decreased as the pH was increased but was independent of the concentration of ethanol. Oxygen was required for germ tube formation. In addition to ethanol, 1-propanol, 2-propanol, 1-butanol and acetic acid could induce germ tube formation, whereas methanol could not. These results indicate that the cells must mobilize their endogenous nitrogen and probably carbohydrate reserves in order to initiate formation of germ tubes. The evidence is inconclusive as to whether ethanol itself must be metabolized for germ tube induction to occur, although it is not thought to act by a nonspecific interaction with the cell membrane.     

Candida albicans internalization by host cells is mediated by a clathrin-dependent mechanism

Candida albicans is a major cause of oropharyngeal, vulvovaginal and haematogenously disseminated candidiasis. Endocytosis of C. albicans hyphae by host cells is a prerequisite for tissue invasion. This internalization involves interactions between the fungal invasin Als3 and host E- or N-cadherin. Als3 shares some structural similarity with InlA, a major invasion protein of the bacterium Listeria monocytogenes . InlA mediates entry of L. monocytogenes into host cells through binding to E-cadherin. A role in internalization, for a non-classical stimulation of the clathrin-dependent endocytosis machinery, was recently highlighted. Based on the similarities between the C. albicans and L. monocytogenes invasion proteins, we studied the role of clathrin in the internalization of C. albicans . Using live-cell imaging and indirect immunofluorescence of epithelial cells infected with C. albicans , we observed that host E-cadherin, clathrin, dynamin and cortactin accumulated at sites of C. albicans internalization. Similarly, in endothelial cells, host N-cadherin, clathrin and cortactin accumulated at sites of fungal endocytosis. Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans internalization by epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner. These data indicate that C. albicans , like L. monocytogenes, hijacks the clathrin-dependent endocytic machinery to invade host cells.     

Characterization of agglutinin-like sequence genes from non-albicans Candida and phylogenetic analysis of the ALS family

The ALS (agglutinin-like sequence) gene family of Candida albicans encodes cell-surface glycoproteins implicated in adhesion of the organism to host surfaces. Southern blot analysis with ALS-specific probes suggested the presence of ALS gene families in C. dubliniensis and C. tropicalis; three partial ALS genes were isolated from each organism. Northern blot analysis demonstrated that mechanisms governing expression of ALS genes in C. albicans and C. dubliniensis are different. Western blots with an anti-Als serum showed that cross-reactive proteins are linked by beta 1,6-glucan in the cell wall of each non-albicans Candida, suggesting similar cell wall architecture and conserved processing of Als proteins in these organisms. Although an ALS family is present in each organism, phylogenetic analysis of the C. albicans, C. dubliniensis, and C. tropicalis ALS genes indicated that, within each species, sequence diversification is extensive and unique ALS sequences have arisen. Phylogenetic analysis of the ALS and SAP (secreted aspartyl proteinase) families show that the ALS family is younger than the SAP family. ALS genes in C. albicans, C. dubliniensis, and C. tropicalis tend to be located on chromosomes that also encode genes from the SAP family, yet the two families have unexpectedly different evolutionary histories. Homologous recombination between the tandem repeat sequences present in ALS genes could explain the different histories for co-localized genes in a predominantly clonal organism like C. albicans.     

Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells

Candida albicans is the most common cause of hematogenously disseminated and oropharyngeal candidiasis. Both of these diseases are characterized by fungal invasion of host cells. Previously, we have found that C. albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the fungal surface protein and host cell receptors that mediate this process. We found that the C. albicans Als3 is required for the organism to be endocytosed by human umbilical vein endothelial cells and two different human oral epithelial lines. Affinity purification experiments with wild-type and an als3Δ/als3Δ mutant strain of C. albicans demonstrated that Als3 was required for C. albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. Furthermore, latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not. Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin–cadherin binding. Therefore, Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. These results uncover the first known fungal invasin and provide evidence that C. albicans Als3 is a molecular mimic of human cadherins.     

Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity

The opportunistic pathogenic yeast Candida albicans exhibits growth phase-dependent changes in cell surface hydrophobicity, which has been correlated with adhesion to host tissues. Cell wall proteins that might contribute to the cell surface hydrophobicity phenotype were released by limited glucanase digestion. These proteins were initially characterized by their rates of retention during hydrophobic interaction chromatography--high-performance liquid chromatography and used as immunogens for monoclonal antibody production. The present work describes the cloning and functional analysis of a C. albicans gene encoding a 38-kDa protein recognized by the monoclonal antibody 6C5-H4CA. The 6C5-H4CA antigen was resolved by two-dimensional electrophoresis, and a partial protein sequence was determined by mass spectrometry analysis of tryptic fragments. The obtained peptides were used to identify the gene sequence from the unannotated C. albicans DNA database. The antibody epitope was provisionally mapped by peptide display panning, and a peptide sequence matching the epitope was identified in the gene sequence. The gene sequence encodes a novel open reading frame (ORF) of unknown function that is highly similar to several other C. albicans ORFs and to a single Saccharomyces cerevisiae ORF. Knockout of the gene resulted in a decrease in measurable cell surface hydrophobicity and in adhesion of C. albicans to fibronectin. The results suggest that the 38-kDa protein is a hydrophobic surface protein that meditates binding to host target proteins.