Specific recognition of Candida albicans by macrophages requires galectin-3 to discriminate Saccharomyces cerevisiae and needs association with TLR2 for signaling

Stimulation of cells of the macrophage lineage is a crucial step in the sensing of yeasts by the immune system. Glycans present in both Candida albicans and Saccharomyces cerevisiae cell walls have been shown to act as ligands for different receptors leading to different stimulating pathways, some of which need receptor co-involvement. However, among these ligand-receptor couples, none has been shown to discriminate the pathogenic yeast C. albicans. We explored the role of galectin-3, which binds C. albicans beta-1,2 mannosides. These glycans are specifically and prominently expressed at the surface of C. albicans but not on S. cerevisiae. Using a mouse cell line and galectin-3-deleted cells from knockout mice, we demonstrated a specific enhancement of the cellular response to C. albicans compared with S. cerevisiae, which depended on galectin-3 expression. However, galectin-3 was not required for recognition and endocytosis of yeasts. In contrast, using PMA-induced differentiated THP-1, we observed that the presence of TLR2 was required for efficient uptake and endocytosis of both C. albicans and S. cerevisiae. TLR2 and galectin-3, which are expressed at the level of phagosomes containing C. albicans, were shown to be associated in differentiated macrophages after incubation with this sole species. These data suggest that macrophages differently sense C. albicans and S. cerevisiae through a mechanism involving TLR2 and galectin-3, which probably associate for binding of ligands expressing beta-1,2 mannosides specific to the C. albicans cell wall surface.     

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).     

Inhibitory effect of glucose and adenosine 3',5'-monophosphate on the synthesis of inducible N-acetylglucosamine catabolic enzymes in yeast

Glucose can block the utilization of N-acetylglucosamine in Saccharomyces cerevisiae, a facultative aerobe, but not in Candida albicans, an obligatory aerobe. Furthermore, glucose represses the synthesis of the enzymes of the N-acetylglucosamine catabolic pathway in S. cerevisiae, but not in C. albicans. The results suggest that catabolite repression is present in S. cerevisiae, but not in C. albicans. Cyclic AMP added to S. cerevisiae cells maintained in a glucose medium cannot bring about their release from catabolite repression. On the contrary, the synthesis of inducible enzymes of N-acetylglucosamine pathway was inhibited by cyclic AMP in both the yeasts. This seems to indicate that cyclic AMP can penetrate into the yeast cells. Furthermore, cyclic AMP inhibits protein synthesis, suggesting that protein synthesis in yeast is under cyclic AMP control.     

Effect of phosphatidylserine enrichment on amino acid transport in yeast

A 1.5- to 3.5-fold accumulation of phosphatidylserine was observed when Candida albicans and Saccharomyces cerevisiae cells were grown in the presence of hydroxylamine, a known inhibitor of phosphatidylserine decarboxylase. However, as compared to S. cerevisiae cells, the levels of phosphatidylcholine and phosphatidylethanolamine were much lower in C. albicans cells. The enrichment of phosphatidylserine selectively affected the transport of several amino acids.     

Inactivation of CaMIT1 inhibits Candida albicans phospholipomannan beta-mannosylation, reduces virulence, and alters cell wall protein beta-mannosylation

Studies on Candida albicans phospholipomannan have suggested a novel biosynthetic pathway for yeast glycosphingolipids. This pathway is thought to diverge from the usual pathway at the mannose-inositol-phospho-ceramide (MIPC) step. To confirm this hypothesis, a C. albicans gene homologue for the Saccharomyces cerevisiae SUR1 gene was identified and named MIT1 as it coded for GDP-mannose:inositol-phospho-ceramide mannose transferase. Two copies of this gene were disrupted. Western blots of cell extracts revealed that strain mit1Delta contained no PLM. Thin layer chromatography and mass spectrometry confirmed that mit1Delta did not synthesize MIPC, demonstrating a role of MIT1 in the mannosylation of C. albicans IPCs. As MIT1 disruption prevented downstream beta-1,2 mannosylation, mit1Delta represents a new C. albicans mutant affected in the expression of these specific virulence attributes, which act as adhesins/immunomodulators. mit1Delta was less virulent during both the acute and chronic phases of systemic infection in mice (75 and 50% reduction in mortality, respectively). In vitro, mit1Delta was not able to escape macrophage lysis through down-regulation of the ERK1/2 phosphorylation pathway previously shown to be triggered by PLM. Phenotypic analysis also revealed pleiotropic effects of MIT1 disruption. The most striking observation was a reduced beta-mannosylation of phosphopeptidomannan. Increased beta-mannosylation of mannoproteins was observed under growth conditions that prevented the association of beta-oligomannosides with phosphopeptidomannan, but not with PLM. This suggests that C. albicans has strong regulatory mechanisms associating beta-oligomannoses with different cell wall carrier molecules. These mechanisms and the impact of the different presentations of beta-oligomannoses on the host response need to be defined.     

Doxorubicin-efflux pump in Haloferax voleanii is energized by ATP

Abstract Treatment of Candida albicans with low concentrations of either hydrogen peroxide or menadione (a Superoxide generating agent) induces an adaptive response which protects cells from the lethal effects of a subsequent challenge with higher concentrations of these oxidants. Pre-treatment with either menadione or hydrogen peroxide is protective against cell killing by either oxidant. This suggests that the pathogenic yeast C. albicans (unlike the budding yeast Saccharomyces cerevisiae which has separate responses) possesses an adaptive response that responds to both these oxidants. In addition, we found that C. albicans showed a greater level of resistance to oxidants, both H₂O₂ and redox-cycling agents, compared to that observed with S. cerevisiae . In an attempt to characterise the oxidative stress response in more detail we have analysed the effect of oxidants on the activities of a number of enzymes with known antioxidant activity.     

The mating projections of Saccharomyces cerevisiae and Candida albicans show key characteristics of hyphal growth

Fungi can grow in a variety of growth forms: yeast, pseudohyphae and hyphae. The human fungal pathogen Candida albicans can grow in all three of these forms. In this fungus, hyphal growth is distinguished by the presence of a Spitzenk?rper-like structure at the hyphal tip and a band of septin bars around the base of newly evaginated germ tubes. The budding yeast Saccharomyces cerevisiae grows as yeast and pseudohyphae, but is not normally considered to show hyphal growth. We show here that in mating projections of both C. albicans and S. cerevisiae a Spitzenk?rper-like structure is present at the growing tip and a band of septin bars is present at the base. Furthermore, in S. cerevisiae mating projections, Spa2 and Bni1 form a cap to the 3-dimensional ball of FM4-64 staining, exactly as previously observed in C. albicans hyphae, suggesting that the putative Spitzenk?rper may be a distinct structure from the polarisome. Taken together this work shows that mating projections of both S. cerevisiae and C. albicans show the key characteristics of hyphal growth.     

Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p

The Candida albicans INT1 gene is important for hyphal morphogenesis, adherence, and virulence (C. Gale, C. Bendel, M. McClellan, M. Hauser, J. M. Becker, J. Berman, and M. Hostetter, Science 279:1355-1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, an S. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required for INT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes to INT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute to INT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2Delta mutants for growth at 37 degrees C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis of INT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene, SLA2, that is required for hyphal growth in C. albicans.     

The Candida albicans myristoyl-CoA:protein N-myristoyltransferase gene. Isolation and expression in Saccharomyces cerevisiae and Escherichia coli.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) has recently been identified as a target for antiviral and antifungal therapy. Candida albicans is a dimorphic, asexual yeast that is a major cause of systemic fungal infections in immunosuppressed humans. Metabolic labeling studies indicate that C. albicans synthesizes one principal 20-kDa N-myristoyl-protein. The single copy C. albicans NMT gene (ca-NMT1) was isolated and encodes a 451-amino acid protein that has 55% identity with Saccharomyces cerevisiae NMT. C. albicans NMT1 is able to complement the lethal phenotype of S. cerevisiae nmt1 null mutants by directing efficient acylation of the approximately 12 endogenous N-myristoylproteins produced by S. cerevisiae. C. albicans NMT was produced in Escherichia coli, a prokaryote with no endogenous NMT activity. In vitro studies of purified E. coli-derived S. cerevisiae and C. albicans NMTs revealed species-specific differences in the kinetic properties of synthetic octapeptide substrates derived from known N-myristoylproteins. Together these data indicate that C. albicans and S. cerevisiae NMTs have similar yet distinct substrate specificities which may be of therapeutic significance.     

Candida albicans CHT3 encodes the functional homolog of the Cts1 chitinase of Saccharomyces cerevisiae

Chitin synthesis and chitin degradation play an important role in cellular morphogenesis and influence the cell shape of fungal organisms. The Candida albicans genome contains four chitinase genes, CHT1, CHT2, and CHT3, which are homologous to the Saccharomyces cerevisiae CTS1 gene and C. albicans CHT4, which is homologous to S. cerevisiae CTS2. To determine which of the C. albicans CHT genes represents the functional homolog of the S. cerevisiae CTS1 gene we constructed mutants of these genes and characterized the resulting phenotypes using morphological assays such as in vivo time lapse microscopy and enzymatic assays to determine the chitinase activity. Deletion of CaCHT1 and CaCHT2 provided no phenotypic alterations in liquid culture but resulted in increased hyphal growth on solid media. Deletion of CaCHT3 generated chains of unseparated cells in the yeast growth phase strongly resembling the cts1 deletion phenotype of S. cerevisiae cells. Expression of CHT3 under control of the regulatable MAL2-promoter in C. albicans resulted in the reversion of the cell separation defect when cells were grown in maltose. Cht3, but not Cht2 when expressed in S. cerevisiae was also able to reverse the cell separation defect of the S. cerevisiae c ts1 deletion strain. Measurements of chitinase activity from yeast cells of C. albicans showed that Cht2 is bound to cells, consistent with it being GPI-anchored while Cht3 is secreted into growth medium; Cht3 is also the principal, observed activity.     

Over-expression of Saccharomyces cerevisiae hsp90 enhances the virulence of this yeast in mice

Abstract Saccharomyces cerevisiae , a yeast of low pathogenic potential, is a rare but well-documented cause of invasive infections in humans. The yeast Candida albicans is a much commoner cause of significant and life-threatening infections. In such infections the heat shock protein hsp90 is an immunodominant antigen associated with protective humoral immunity. In this study it was shown that over-expression of S. cerevisiae hsp90, the amino acid sequence of which shows 84% identity to C. albicans hsp90, significantly increased the virulence of a laboratory strain of S. cerevisiae in mice, both in terms of colony counts in the kidney, liver and spleen, and in terms of mortality. This is the first direct evidence that hsp90 is a virulence factor.     

Elongation factor 3 (EF-3) from Candida albicans shows both structural and functional similarity to EF-3 from Saccharomyces cerevisiae

As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino-acid stretch. That the cloned C. albicans sequence did indeed encode EF-3 was confirmed by demonstrating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional similarity to EF-3 from S. cerevisiae.     

Efficient translation of synthetic and natural mRNAs in an mRNA-dependent cell-free system from the dimorphic fungus Candida albicans.

An mRNA-dependent cell-free translation system has been developed from the human pathogenic fungus Candida albicans using either S30 or S100 lysates prepared from glass-bead-disrupted whole cells. Translation of the synthetic template poly(U) in this system is highly efficient at temperatures up to 37 degrees C and is ATP-dependent. Studies using a range of elongation-specific inhibitors suggest that the mechanism of translational elongation in C. albicans is similar to that of another yeast, Saccharomyces cerevisiae. A micrococcal-nuclease-treated C. albicans S100 lysate was able to translate exogenously-supplied homologous mRNAs, and a range of heterologous natural mRNAs, using an initiation mechanism that is inhibited by the antibiotic edeine and the 5' cap analogue 7-methylguanosine 5'-monophosphate (m7GMP). As with cell-free lysates prepared from S. cerevisiae, the C. albicans lysate is unable to initiate translation upon natural mRNAs at temperatures above 20 degrees C.     

Candida albicans, a distinctive fungal model for cellular aging study

The unicellular eukaryotic organisms represent the popular model systems to understand aging in eukaryotes. Candida albicans, a polymorphic fungus, appears to be another distinctive unicellular aging model in addition to the budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe. The two types of Candida cells, yeast (blastospore) form and hyphal (filamentous) form, have similar replicative lifespan. Taking the advantage of morphologic changes, we are able to obtain cells of different ages. Old Candida cells tend to accumulate glycogen and oxidatively damaged proteins. Deletion of the SIR2 gene causes a decrease of lifespan, while insertion of an extra copy of SIR2 extends lifespan, indicating that like in S. cerevisiae, Sir2 regulates cellular aging in C. albicans. Interestingly, Sir2 deletion does not result in the accumulation of extra-chromosomal rDNA molecules, but influences the retention of oxidized proteins in mother cells, suggesting that the extra-chromosomal rDNA molecules may not be associated with cellular aging in C. albicans. This novel aging model, which allows efficient large-scale isolation of old cells, may facilitate biochemical characterizations and genomics/proteomics studies of cellular aging, and help to verify the aging pathways observed in other organisms including S. cerevisiae.     

The GRR1 gene of Candida albicans is involved in the negative control of pseudohyphal morphogenesis

The opportunistic fungal pathogen Candida albicans can grow as yeast, pseudohyphae or true hyphae. C. albicans can switch between these morphologies in response to various environmental stimuli and this ability to switch is thought to be an important virulence trait. In Saccharomyces cerevisiae, the Grr1 protein is the substrate recognition component of an SCF ubiquitin ligase that regulates cell cycle progression, cell polarity and nutrient signaling. In this study, we have characterized the GRR1 gene of C. albicans. Deletion of GRR1 from the C. albicans genome results in a highly filamentous, pseudohyphal morphology under conditions that normally promote the yeast form of growth. Under hypha-inducing conditions, most cells lacking GRR1 retain a pseudohyphal morphology, but some cells appear to switch to hyphal-like growth and express the hypha-specific genes HWP1 and ECE1. The C. albicans GRR1 gene also complements the elongated cell morphology phenotype of an S. cerevisiae grr1Delta mutant, indicating that C. albicans GRR1 encodes a true orthologue of S. cerevisaie Grr1. These results support the hypothesis that the Grr1 protein of C. albicans, presumably as the F-box subunit of an SCF ubiquitin ligase, has an essential role in preventing the switch from the yeast cell morphology to a pseudohyphal morphology.     

Inhibition of endothelium-dependent relaxation by Candida albicans

This study examines the effects of Candida albicans on acethylcholine-induced, endothelium-dependent relaxation of thoracic aorta of rabbits, precontracted by phenylephrine (10(-7) M). Isolated vessel rings were incubated with C. albicans, Saccharomyces cerevisiae, or their mannans, and endothelium-dependent relaxation was measured by the induction of acethylcholine. Endothelium-dependent relaxation remained unaffected after 3 hours by either C. albicans or S. cerevisiae, or their mannans. After 24 hours, however, incubation with C. albicans had completely abolished relaxation, whereas relaxation was decreased by mannan of C. albicans and continued unaffected by S. cerevisiae. In contrast, no change was registered with a 24 hours incubation of C. Albicans in a sodium nitroprusside-induced, endothelium-independent, vascular smooth muscle relaxation. Microscopical investigation of the morphological structure of vessel walls revealed penetration of C. albicans on the intimal surface after 3 hours incubation and infiltration of the yeast through the vessel wall after 24 hours. No changes in vessel morphology occurred after 3 or 24 hours with S. cerevisiae or the mannan of C. albicans. These results show the ability of C. albicans to inhibit endothelium-dependent, but not endothelium-independent, relaxation of vascular smooth muscle and may have important implications for functional damage to endothelial cells and the regulation of vessel tone and blood flow.