Reintroduction of the PLB1 gene into Candida albicans restores virulence in vivo.

Phospholipases have been proposed to contribute to the virulence of Candida albicans. Recently, a candidal strain deleted for PLB1, the gene encoding the predominant phospholipase B (Plb1) secreted by C. albicans, was constructed and its virulence in an intravenous murine model of disseminated candidiasis was evaluated. In the present study, the PLB1 gene was reintroduced back into the plb1 null mutant to generate the revertant strain, which showed similar growth and morphology to its isogenic parent strain. Virulence of the revertant strain was found to be comparable to that of the parent strain in an intravenous murine model of disseminated candidiasis. To compare the abilities of the plb1 null mutant, the revertant and the isogenic parent strains to cross the gastrointestinal (GI) tract and cause systemic infection, an oral-intragastric infant mouse model of candidiasis was used. Histological examinations and analysis of c.f.u. of the pathogen in liver homogenates revealed that the parental and revertant strains were able to invade and traverse the GI mucosa to a significantly greater extent than the plb1 null mutant. Immunofluorescence and immunoelectron microscopic studies of infected host tissue using anti-Plb1 antibody showed that Plb1 is secreted during invasion of the gastric mucosa by the parental and revertant strains. In contrast, little or no labelling was observed in the null mutant strain. The results indicate that the Plb1 secreted by C. albicans enhances the ability of this organism to cross the GI tract and disseminate haematogenously. These studies provide unequivocal evidence supporting a role for Plb1 during the course of infection by C. albicans.

Antifungal resistance of candidal biofilms formed on denture acrylic in vitro.

Denture biofilms represent a protective reservoir for oral microbes. The study of the biology of Candida in these biofilms requires a reliable model. A reproducible model of C. albicans denture biofilm was developed and used to determine the susceptibility of two clinically relevant C. albicans isolates against 4 antifungals. C. albicans, growing as a biofilm, exhibited resistance to amphotericin B, nystatin, chlorhexidine, and fluconazole, with 50% reduction in metabolic activity (50% RMA) at concentrations of 8, 16, 128, and > 64 microg/mL, respectively. In contrast, planktonically cultured C. albicans were susceptible (50% RMA for the same antifungals was obtained at 0.25, 1.0, 4.0, and 0.5 microg/mL, respectively). In conclusion, results obtained by means of our biofilm model show that biofilm-associated C. albicans cells, compared with cells grown in planktonic form, are resistant to antifungals used to treat denture stomatitis.

Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans.

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.

Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans.

The Candida albicans PLB1 gene was cloned using a polymerase chain reaction-based approach relying on degenerate oligonucleotide primers designed according to the amino acid sequences of two peptide fragments obtained from a purified candidal enzyme displaying phospholipase activity (Mirbod, F., Banno, Y., Ghannoum, M. A., Ibrahim, A. S., Nakashima, S., Yasuo, K., Cole, G. T., and Nozawa, Y. (1995) Biochim. Biophys. Acta 1257, 181-188). Sequence analysis of a 6.7-kilobase pair EcoRI-ClaI genomic clone revealed a single open reading frame of 1818 base pairs that predicts for a pre-protein of 605 residues. Comparison of the putative candidal phospholipase with those of other proteins in data base revealed significant homology to known fungal phospholipase Bs from Saccharomyces cerevisiae (45%), Penicillium notatum (42%), Torulaspora delbrueckii (48%), and Schizosaccharomyces pombe (38%). Thus, we have cloned the gene encoding a C. albicans phospholipase B homolog. This gene, designated caPLB1, was mapped to chromosome 6. Disruption experiments revealed that the caplb1 null mutant is viable and displays no obvious phenotype. However, the virulence of strains deleted for caPLB1, as assessed in a murine model for hematogenously disseminated candidiasis, was significantly attenuated compared with the isogenic wild-type parental strain. Although deletion of caPLB1 did not produce any detectable effects on candidal adherence to human endothelial or epithelial cells, the ability of the caplb1 null mutant to penetrate host cells was dramatically reduced. Thus, phospholipase B may well contribute to the pathogenicity of C. albicans by abetting the fungus in damaging and traversing host cell membranes, processes which likely increase the rapidity of disseminated infection.

Gpi1, a Saccharomyces cerevisiae protein that participates in the first step in glycosylphosphatidylinositol anchor synthesis.

The temperature-sensitive Saccharomyces cerevisiae gpi1 mutant is blocked in [3H]inositol incorporation into protein and defective in the synthesis of N-acetylglucosaminylphosphatidylinositol, the first step in glycosylphosphatidylinositol (GPI) anchor assembly (Leidich, S. D., Drapp, D. A., and Orlean, P. (1994) J. Biol. Chem. 269, 10193-10196). The GPI1 gene, which encodes a 609-amino acid membrane protein, was cloned by complementation of the temperature sensitivity of gpi1 and corrects the mutant's [3H]inositol labeling and enzymatic defects. Disruption of GPI1 yields viable haploid cells that are temperature-sensitive for growth, for [3H]inositol incorporation into protein, and for GPI anchor-dependent processing of the Gas1/Ggp1 protein and that lack in vitro N-acetylglucosaminylphosphatidylinositol synthetic activity. The Gpi1 protein thus participates in GPI synthesis and is required for growth at 37 degrees C. When grown at a semipermissive temperature of 30 degrees C, gpi1 cells and gpi1::URA3 disruptants form large, round, multiply budded cells with a separation defect. Homozygous gpi1/gpi1, gpi1::URA3/gpi1::URA3, gpi2/gpi2, and gpi3/gpi3 diploids undergo meiosis, but are defective in ascospore wall maturation for they fail to give the fluorescence due to the dityrosine-containing layer in the ascospore wall. These findings indicate that GPIs have key roles in the morphogenesis and development of S. cerevisiae.

Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene.

To identify genes required for the synthesis of glycosyl phosphatidylinositol (GPI) membrane anchors in yeast, we devised a way to isolate GPI anchoring mutants in which colonies are screened for defects in [3H]-inositol incorporation into protein. The gpi1 mutant, identified in this way, is temperature sensitive for growth and defective in vitro in the synthesis of GlcNAc-phosphatidylinositol, the first intermediate in GPI biosynthesis (Leidich, S. D., Drapp, D. A., and Orlean, P. (1994) J. Biol. Chem. 269, 10193-10196). We report the isolation of two more conditionally lethal mutants, gpi2 and gpi3, which, like gpi1, have a temperature-sensitive defect in the incorporation of [3H]inositol into protein and which lack in vitro GlcNAc-phosphatidylinositol synthetic activity. Haploid Saccharomyces cerevisiae strains containing any pairwise combination of the gpi1, gpi2, and gpi3 mutations are inviable. The GPI2 gene, cloned by complementation of the gpi2 mutant's temperature sensitivity, encodes a hydrophobic 269-amino acid protein that resembles no other gene product known to participate in GPI assembly. Gene disruption experiments show that GPI2 is required for vegetative growth. Overexpression of the GPI2 gene causes partial suppression of the gpi1 mutant's temperature sensitivity, a result that suggests that the Gpi1 and Gpi2 proteins interact with one another in vivo. The gpi3 mutant is defective in the SPT14 gene, which encodes a yeast protein similar to the product of the mammalian PIG-A gene, which complements a GlcNAc-phosphatidylinositol synthesis-defective human cell line. In yeast, at least three gene products are required for the first step in GPI synthesis, as is the case in mammalian cells, and utilization of several different proteins at this stage is therefore likely to be a general characteristic of the GPI synthetic pathway.

A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis.

Glycosyl phosphatidylinositols (GPIs) anchor many proteins to the surface of eukaryotic cells and may also serve as sorting signals on proteins and participate in signal transduction. We have isolated a Saccharomyces cerevisiae GPI anchoring mutant, gpi1, using a colony screen for cells blocked in [3H]inositol incorporation into protein. The gpi1 mutant is defective in vitro in the synthesis of N-acetylglucosaminyl phosphatidylinositol, the first intermediate in GPI synthesis, and is also temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for growth of the unicellular eukaryote S. cerevisiae. GPI synthesis could therefore be exploited as a target for antifungal or antiparasitic agents.

Isolation of temperature-sensitive yeast GPI-anchoring mutants.

We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI). We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate [3H]inositol into protein. Among our isolates are strains blocked in mannosylation of the GPI-anchorprecursor, and strains defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). We have characterized one mutant, gpi1, further. This strain is defective in GlcNAc-PI synthesis and is temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S. cerevisiae. We have isolated plasmids that complement the gpi1 mutation from S. cerevisiae genomic DNA-and fission yeast cDNA libraries.

Isolation of temperature-sensitive yeast GPI-anchoring mutants

We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI). We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate [3H]inositol into protein. Among our isolates are strains blocked in mannosylation of the GPI-anchor precursor, and strains defective in the synthesis of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have characterized one mutant, gpil, further. This strain is defective in GlcNAC-PI synthesis and is temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S. cerevisiae. We have isolated plasmids that complement the gpil mutation from S. cerevisiae genomic DNA- and fission yeast cDNA libraries