Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the alpha1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the alpha1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1delta null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence.     

Methionine is required for cAMP‐PKA‐mediated morphogenesis and virulence of Candida albicans

Candida albicans is a major human fungal pathogen, causing superficial, as well as life‐threatening invasive infections. Therefore, it has to adequately sense and respond to the host defense by expressing appropriate virulence attributes. The most important virulence factor of C. albicans is the yeast‐to‐hyphae morphogenetic switch, which can be induced by numerous environmental cues, including the amino acid methionine. Here, we show an essential role for methionine permease Mup1 in methionine‐induced morphogenesis, biofilm formation, survival inside macrophages and virulence. Furthermore, we demonstrate that this process requires conversion of methionine into S‐adenosyl methionine (SAM) and its decarboxylation by Spe2. The resulting amino‐propyl group is then used for biosynthesis of polyamines, which have been shown to activate adenylate cyclase. Inhibition of the SPE2 SAM decarboxylase gene strongly impairs methionine‐induced morphogenesis on specific media and significantly delays virulence in the mouse systemic infection model system. Further proof of the connection between methionine uptake and initial metabolism and the cAMP‐PKA pathway was obtained by showing that both Mup1 and Spe2 are required for cAMP production in response to methionine. Our results suggest that amino acid transport and further metabolism are interesting therapeutic targets as inhibitors of this may prevent the morphogenetic switch, thereby preventing virulence.     

Candida albicans SRR1, a putative two-component response regulator gene, is required for stress adaptation, morphogenesis, and virulence

We report here the identification and characterization of a previously uncharacterized, two-component response regulator gene (orf19.5843) from Candida albicans. Because of its apparent functions in stress adaptation, we have named this gene SRR1 (stress response regulator 1). Disruption of SRR1 causes defects in hyphal development, reduced resistance to stress, and severe virulence attenuation in the mouse model of disseminated candidiasis.     

Role of CaECM25 in cell morphogenesis, cell growth and virulence in Candida albicans

Candida albicans is the most prominent opportunistic fungal pathogen in humans. Multiple factors are associated with the virulence of C. albicans, including morphogenesis, cell wall organization and growth rate. Here, we describe the identification and functional characterization of CaECM25, a gene that has not been reported before. We constructed Caecm25?/? mutants and investigated the role of the gene in morphogenesis, cell wall organization and virulence. CaECM25 deletion resulted in defects in cell separation, a slower growth rate, reduced filamentous growth and attenuated adherence to plastic surfaces. The Caecm25?/? mutant was also significantly less virulent than wild type when tested for systemic infection in mice. Therefore, CaECM25 plays important roles in morphogenesis, cell wall organization and virulence.     

PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis.

Candida albicans, like many fungi, exhibits morphological plasticity, a property which may be related to its biological capacity as an opportunistic pathogen of humans. Morphogenesis and alterations in cell shape require integration of many cellular functions and occur in response to environmental signals, most notably pH and temperature in the case of C. albicans. In the course of our studies of differential gene expression associated with dimorphism of C. albicans, we have isolated a gene, designated PHR1, which is regulated in response to the pH of the culture medium. PHR1 expression was repressed at pH values below 5.5 and induced at more alkaline pH. The predicted amino acid sequence of the PHR1 protein was 56% identical to that of the Saccharomyces cerevisiae Ggp1/Gas1 protein, a highly glycosylated cell surface protein attached to the membrane via glycosylphosphatidylinositol. A homozygous null mutant of PHR1 was constructed and found to exhibit a pH-conditional morphological defect. At alkaline pH, the mutant, unlike the parental type, was unable to conduct apical growth of either yeast or hyphal growth forms. This morphological aberration was not associated with defective cytoskeletal polarization or secretion. The results suggest that PHR1 defines a novel function required for apical cell growth and morphogenesis.     

Mucosal damage and neutropenia are required for Candida albicans dissemination

Candida albicans fungemia in cancer patients is thought to develop from initial gastrointestinal (GI) colonization with subsequent translocation into the bloodstream after administration of chemotherapy. It is unclear what components of the innate immune system are necessary for preventing C. albicans dissemination from the GI tract, but we have hypothesized that both neutropenia and GI mucosal damage are critical for allowing widespread invasive C. albicans disease. We investigated these parameters in a mouse model of C. albicans GI colonization that led to systemic spread after administration of immunosuppression and mucosal damage. After depleting resident GI intestinal flora with antibiotic treatment and achieving stable GI colonization levels of C. albicans, it was determined that systemic chemotherapy with cyclophosphamide led to 100% mortality, whereas selective neutrophil depletion, macrophage depletion, lymphopenia or GI mucosal disruption alone resulted in no mortality. Selective neutrophil depletion combined with GI mucosal disruption led to disseminated fungal infection and 100% mortality ensued. GI translocation and dissemination by C. albicans was also dependent on the organism's ability to transform from the yeast to the hyphal form. This mouse model of GI colonization and fungemia is useful for studying factors of innate host immunity needed to prevent invasive C. albicans disease as well as identifying virulence factors that are necessary for fungal GI colonization and dissemination. The model may also prove valuable for evaluating therapies to control C. albicans infections.     

Calcineurin A of Candida albicans: involvement in antifungal tolerance,cell morphogenesis and virulence

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.     

Asc1, a WD-repeat protein, is required for hyphal development and virulence in Candida albicans

Candida albicans is a human pathogenic fungus which can undergo a morphological transition from yeast to hyphae in response to a variety of environmental stimuli. We analyzed a C. albicans Asc1 (Absence of growth Suppressor of Cyp1) protein which is entirely composed of seven repeats of the WD domain, and is conserved from fungi to metazoan. Deleting the ASC1 in C. albicans led to a profound defect in hyphal development under hypha-inducing conditions examined. Furthermore, deletion of the ASC1 attenuated virulence of C. albicans in a mouse model of systemic infection. These data strongly suggested that the conserved WD-repeat protein Asc1 is required for morphogenesis and pathogenesis of C. albicans.     

Pga26 mediates filamentation and biofilm formation and is required for virulence in Candida albicans

The Candida albicans gene PGA26 encodes a small cell wall protein and is upregulated during de novo wall synthesis in protoplasts. Disruption of PGA26 caused hypersensitivity to cell wall-perturbing compounds (Calcofluor white and Congo red) and to zymolyase, which degrades the cell wall β-1,3-glucan network. However, susceptibility to caspofungin, an inhibitor of β-1,3-glucan synthesis, was decreased. In addition, pga26Δ mutants show increased susceptibility to antifungals (fluconazol, posaconazol or amphotericin B) that target the plasma membrane and have altered sensitivities to environmental (heat, osmotic and oxidative) stresses. Except for a threefold increase in β-1,6-glucan and a slightly widened outer mannoprotein layer, the cell wall composition and structure was largely unaltered. Therefore, Pga26 is important for proper cell wall integrity, but does not seem to be directly involved in the synthesis of cell wall components. Deletion of PGA26 further leads to hyperfilamentation, increased biofilm formation and reduced virulence in a mouse model of disseminated candidiasis. We propose that deletion of PGA26 may cause an imbalance in the morphological switching ability of Candida, leading to attenuated dissemination and infection.     

Comparison of the epidemiology, drug resistance mechanisms, and virulence of Candida dubliniensis and Candida albicans

Candida dubliniensis is a pathogenic yeast species that was first identified as a distinct taxon in 1995. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and that it is primarily associated with oral carriage and oropharyngeal infections in human immunodeficiency virus (HIV)-infected and acquired immune deficiency syndrome (AIDS) patients. However, unlike Candida albicans, C. dubliniensis is rarely found in the oral microflora of normal healthy individuals and is responsible for as few as 2% of cases of candidemia (compared to approximately 65% for C. albicans). The vast majority of C. dubliniensis isolates identified to date are susceptible to all of the commonly used antifungal agents, however, reduced susceptibility to azole drugs has been observed in clinical isolates and can be readily induced in vitro. The primary mechanism of fluconazole resistance in C. dubliniensis has been shown to be overexpression of the major facilitator efflux pump Mdr1p. It has also been observed that a large number of C. dubliniensis strains express a non-functional truncated form of Cdr1p, and it has been demonstrated that this protein does not play a significant role in fluconazole resistance in the majority of strains examined to date. Data from a limited number of infection models reflect findings from epidemiological studies and suggest that C. dubliniensis is less pathogenic than C. albicans. The reasons for the reduced virulence of C. dubliniensis are not clear as it has been shown that the two species express a similar range of virulence factors. However, although C. dubliniensis produces hyphae, it appears that the conditions and dynamics of induction may differ from those in C. albicans. In addition, C. dubliniensis is less tolerant of environmental stresses such as elevated temperature and NaCl and H(2)O(2) concentration, suggesting that C. albicans may have a competitive advantage when colonising and causing infection in the human body. It is our hypothesis that a genomic comparison between these two closely-related species will help to identify virulence factors responsible for the far greater virulence of C. albicans and possibly identify factors that are specifically implicated in either superficial or systemic candidal infections.     

Dimorphism and virulence in Candida albicans

Two regulatory pathways govern filamentation in the pathogenic fungus Candida albicans. Recent virulence studies of filamentation regulatory mutants argue that both yeast and filamentous forms have roles in infection. Filamentation control pathways seem closely related in C. albicans and in Saccharomyces cerevisiae, thus permitting speculation about C. albicans filamentation genes not yet discovered.     

Growth, morphogenesis, and virulence of Candida albicans after oral inoculation in the germ-free and conventional chick

Balish, Edward (Syracuse University, Syracuse, N.Y.), and A. W. Phillips. Growth, morphogenesis, and virulence of Candida albicans after oral inoculation in the germ-free and conventional chick. J. Bacteriol. 91:1736-1743. 1966.-The effects of intestinal bacteria on the multiplication, morphogenesis, and infectivity of Candida albicans in the alimentary tract were investigated by comparing results obtained in germ-free and conventional chicks after oral inoculation. This challenge resulted in the establishment of large numbers of the pathogen in the alimentary tract of each group of chicks; these numbers were increased in crop contents from challenged bacteria-free chicks wherein hyphae predominated over the yeast form. These animals also had lesions of the crop epithelium containing numerous hyphae and few yeast-like forms. In contrast, challenged conventional chicks receiving an adequate diet displayed no evidence of infection. Their alimentary tract contained the yeast form of C. albicans; no hyphae were seen. Although we found bacterial inhibition of C. albicans multiplication in the alimentary tract, this in itself did not seem to explain the resistance to intestinal candidiasis in our conventional chicks. We argued that this resistance to infection was due chiefly to the prevention of hyphal development in C. albicans by intestinal bacteria. C. albicans in the gut of our conventional chicks resulted in some increase in numbers of enterococci in contents from the crop. Increased pH values in contents from the gut of germ-free chicks were not clearly related to infection after challenge. The E(h) of the above crop contents were only slightly decreased in the germ-free crop. Thus the E(h) did not appear to be involved in susceptibility to infection. Invasion of the blood stream and kidneys of conventional chicks by the yeast form of C. albicans occurred in challenged animals receiving a purified diet which had been radiation-sterilized and stored for 6 months at room temperature (25 C). Their growth rate decreased and they became moribund; no hyphae were observed in tissues or intestine of these animals. Challenged bacteria-free chicks receiving the same diet were resistant to the above invasion, although they had crop lesions containing hyphae as described. The resistance of these chicks to systemic invasion was attributed to absence of intestinal bacteria competing for low levels of vitamins in the stored diet. Germ-free chicks had decreased levels of serum gamma-globulin which increased after challenge, whereas this value was unchanged in conventional birds after challenge.     

Microtubule motor protein Kar3 is required for normal mitotic division and morphogenesis in Candida albicans

The kinesin-related protein Kar3 is a minus end-directed molecular motor that plays a multifunctional role in microtubule-directed nuclear movement. Previously, it was shown that Candida albicans Kar3p is critical for nuclear fusion during mating as kar3 mutants were defective in karyogamy. In this study, we confirm that Kar3p is required for nuclear congression in mating but that neither Kar3p nor the dynein motor protein Dyn1p is required for nuclear migration in the mating projection prior to cell fusion. In addition, we show that C. albicans Kar3p plays an important role in the cell and colony morphology of mitotically dividing cells, as evidenced by diminished filamentation of kar3 cells on Spider medium and an increased tendency of mutant cells to form pseudohyphal cells in liquid culture. Loss of Kar3p also led to defects in nuclear division, causing cells to grow slowly and exhibit reduced viability compared to wild-type cells. Slow growth was due, at least in part, to delayed cell cycle progression, as cells lacking Kar3p accumulated in anaphase of the cell cycle. Consistent with a role in mitotic division, Kar3 protein was shown to localize to the spindle pole bodies. Finally, kar3 cells exhibited unstable or aberrant mitotic spindles, a finding that accounts for the delay in cell cycle progression and decreased viability of these cells. We suggest that the altered morphology of kar3 cells is a direct consequence of the delay in anaphase, and this leads to increased polarized growth and pseudohypha formation.     

Candida albicans GRX2, encoding a putative glutaredoxin, is required for virulence in a murine model

Resistance of Candida albicans to reactive oxygen species is thought to enhance its virulence in mammalian hosts. Genes such as SOD1, which encodes the anti-oxidant, superoxide dismutase, are known virulence factors. We disrupted the gene GRX2, which encodes a putative glutathione reductase (glutaredoxin) in C. albicans, and we compared the mutant with an sod1Deltamutant. In vitro, the grx2Deltastrain, but not the sod1Delta strain, was defective in hypha formation. The grx2Deltastrain, but not sod1Delta, was significantly more susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, both mutants were susceptible to 1 mM menadione, but grx2Deltanull alone was resistant to diamide. Both mutants were attenuated in a murine intravenous challenge model, and a GRX2 reintegrant regained partial virulence. Emphasis on the putative function of products of genes such as SOD1 and GRX2 in resistance to oxidative stress may oversimplify their functions in the virulence process, since the grx2Deltastrain also gave defective hypha formation. Both mutants were sensitive to menadione and were slow to form germ tubes, though growth rates matched controls once the lag phase was passed.     

GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to beta1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029-1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Deltagwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Deltagwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25-32), is due to the properties of Gwt1p itself and not to other membrane components.     

Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was addressed through the characterization of C. albicans strains without a functional HOG1 gene. Analysis of the phenotype of hog1 mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions, hog1 mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the hog1 mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in C. albicans. We show that C. albicans hog1 mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that HOG1 overexpression in Saccharomyces cerevisiae represses the pseudodimorphic transition. Most interestingly, deletion of HOG1 resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.     

Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans

Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTL(alpha) strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mf(alpha), prevented mating in MTL(alpha) but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTL(alpha) cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.