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.     

HIV-1 and its transmembrane protein gp41 bind to different Candida species modulating adhesion

Oral candidiasis in HIV-1-infected individuals is widely believed to be triggered by the acquired T-lymphocyte immunodeficiency. Recently, binding of the HIV-1 envelope protein gp160 and its subunit gp41, and also of the whole virus itself, to Candida albicans has been shown. The present study shows that, in addition to C. albicans, HIV-1 gp41 also binds to yeast and hyphal forms of Candida dubliniensis, a species which is closely related to C. albicans, and to Candida tropicalis but not to Candida krusei, Candida glabrata or Saccharomyces cerevisiae. The previous finding that gp41 binding to C. albicans augments fungal virulence in vitro is supported by the observation that the yeast showed an enhanced adhesion to HIV-infected H9 cells in comparison to uninfected cells. In line with these results soluble gp41 itself reduced binding of C. albicans to both endothelial and epithelial cell lines, confirming a dominant role of the gp41 binding moiety on the surface of Candida for adhesion. Surface-associated secreted aspartic proteinases (Saps) play an important role in candidial adhesion, but are not likely to be involved in the interaction as gp41 binding to the C. albicans parental wild-type strain was comparable to that of three different isogenic Sap deletion mutants. Furthermore, gp41 binding to the yeast killer toxin-susceptible C. albicans strain 10S was not inhibitable by an anti-YKT receptor antibody. In conclusion, HIV-1 interacts with different clinically important Candida spp., and may thereby affect the outcome of the respective fungal infection.     

The DNA binding protein Rfg1 is a repressor of filamentation in Candida albicans

We have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor.     

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.     

The genes of two G-proteins involved in protein transport in Pichia pastoris

Members of the Rab protein family play essential roles in vesicle fusion during protein secretion and represent highly conserved GTP binding proteins. The Saccharomyces cerevisiae Sec4p and Ypt1p, promoting vesicle fusion at the plasma membrane and in ER-Golgi transport, respectively, are among the best characterised yeast members. We have here cloned the Pichia pastoris SEC4 homologue using a S. cerevisiae SEC4 probe. In addition we isolated a crosshybridising clone encoding another Rab-/Ypt-like protein. The deduced full-length PpSec4p comprises 204 amino acid residues with an over all identity of 64% to the Sec4p from S. cerevisiae and 72% to the Candida albicans Sec4p. The YPT-like gene encodes a 216 amino acid residue protein showing highest similarity to the S. cerevisiae Ypt10p and Ypt53p. Both PpSec4p and the Ypt-like protein carry a -Cys-Cys C-terminus, indicating that these proteins are targets for geranyl-geranylation by a type II prenyltransferase.     

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.     

Analysis and expression of STE13ca gene encoding a putative X-prolyl dipeptidyl aminopeptidase from Candida albicans

Candida albicans STE13ca gene was identified by its homology to the Saccharomyces cerevisiae STE13 gene that encodes for the dipeptidyl aminopeptidase A (DAP A) involved in the maturation of alpha-factor mating pheromone. Our study revealed that C. albicans ATCC 10231 depicts dipeptidyl aminopeptidase activity. We also analyzed the expression of the STE13ca gene homologue from this pathogenic yeast. This gene of 2793 pb is homozygotic and encodes for a predicted protein of 930 amino acids with a molecular weight of 107,035 Da. The predicted protein displays significant sequence similarity to S. cerevisiae Ste13p. This C. albicans gene is located in chromosome R. STE13ca gene increases its levels of expression in conditions of nutritional stress (proline as nitrogen source) and during formation of the germinal tube, suggesting a basic biological function for the STE13ca in this yeast.     

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.     

Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.     

The expression of Candida albicans enolase is not heat shock inducible

Abstract An isoprotein of enolase from the yeast Saccharomyces cerevisiae was reported to be a heat shock protein. The possible role of the C. albicans enolase as a heat shock protein was therefore investigated. The de novo synthesis of C. albicans enolase protein and mRNA did not increase during heat stress, but remained constitutively expressed. Amino acid similarity to the heat shock proteins suggests that although the C. albicans enolase is not a classical heat shock protein, it may be a memberof a group of constitutively expressed, structurally related proteins, the heat shock cognate proteins.     

Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.

We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.     

Role of CaBud6p in the polarized growth of Candida albicans

Bud6p is a component of a polarisome that controls cell polarity in Saccharomyces cerevisiae. In this study, we investigated the role of the Candida albicans Bud6 protein (CaBud6p) in cell polarity and hyphal development. CaBud6p, which consists of 703 amino acids, had 37% amino-acid sequence identity with the Bud6 protein of S. cerevisiae. The homozygous knock-out of CaBUD6 resulted in several abnormal phenotypes, such as a round and enlarged cells, widened bud necks, and a random budding pattern. In hypha-inducing media, the mutant cells had markedly swollen tips and a reduced ability to switch from yeast to hypha. In addition, a yeast two-hybrid analysis showed a physical interaction between CaBud6p and CaAct1p, which suggests that CaBud6p may be involved in actin cable organization, like Bud6p in S. cerevisiae. Taken together, these results indicate that CaBud6 plays an important role in the polarized growth of C. albicans.     

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.     

The MAP kinase-activated protein kinase Rck2p plays a role in rapamycin sensitivity in Saccharomyces cerevisiae and Candida albicans

Rck2p is a Hog1p-MAP kinase-activated protein kinase and regulates osmotic and oxidative stresses in budding yeast. In this study, we have demonstrated in both Saccharomyces cerevisiae and, the most medically important human fungal pathogen, Candida albicans that deletion of RCK2 renders cells sensitive to rapamycin, the inhibitor of target of rapamycin protein kinase controlling cell growth. The kinase activity of Rck2p does not seem to be required for this rapamycin sensitivity function in both eukaryotic microorganisms. Interestingly, the HOG pathway is not directly involved in cell sensitivity to rapamycin in S. cerevisiae, whereas disruption of CaHOG1 renders cells sensitive to rapamycin in C. albicans. In addition, we have shown that CaRck2p and its kinase activity are required for cell growth in C. albicans.     

Deletion analysis of yeast Sec65p reveals a central domain that is sufficient for function in vivo

The Saccharomyces cerevisiae SEC65 gene encodes a 32 kDa subunit of yeast signal recognition particle that is homologous to human SRP19. Sequence comparisons suggest that the yeast protein comprises three distinct domains. The central domain (residues 98–171) exhibits substantial sequence similarity to the 144 residue SRP19. In contrast, the N-terminal and C-terminal domains (residues 1–97 and 172–273 respectively) share no similarity to SRP19, with the exception of a cluster of positively charged residues at the extreme C-terminus of both proteins. Here, we report the cloning of a Sec65p homologue from the yeast Candida albicans that shares the same extended domain structure as its S. cerevisiae counterpart. This conservation of sequence is reflected at the functional level, as the C. albicans gene can complement the conditional lethal sec65-1 mutation in S. cerevisiae . In order to examine the role of the N- and C- terminal domains in Sec65p function, we have engineered truncation mutants of S. cerevisiae SEC65 and tested these for complementing activity in vivo and for SRP integrity in vitro . These studies indicate that a minimal Sec65p comprising residues 76–209, which includes the entire central SRP19-like domain, is sufficient for SRP function in yeast.