Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which β(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp–YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis

The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans

Open Access via PMC2649417Peer reviewedPublisher PD

Niche-specific regulation of central metabolic pathways in a fungal pathogen

To establish an infection, the pathogen Candida albicans must assimilate carbon and grow in its mammalian host. This fungus assimilates six-carbon compounds via the glycolytic pathway, and two-carbon compounds via the glyoxylate cycle and gluconeogenesis. We address a paradox regarding the roles of these central metabolic pathways in C. albicans pathogenesis: the glyoxylate cycle is apparently required for virulence although glyoxylate cycle genes are repressed by glucose at concentrations present in the bloodstream. Using GFP fusions, we confirm that glyoxylate cycle and gluconeogenic genes in C. albicans are repressed by physiologically relevant concentrations of glucose, and show that these genes are inactive in the majority of fungal cells infecting the mouse kidney. However, these pathways are induced following phagocytosis by macrophages or neutrophils. In contrast, glycolytic genes are not induced following phagocytosis and are expressed in infected kidney. Mutations in all three pathways attenuate the virulence of this fungus, highlighting the importance of central carbon metabolism for the establishment of C. albicans infections. We conclude that C. albicans displays a metabolic program whereby the glyoxylate cycle and gluconeogenesis are activated early, when the pathogen is phagocytosed by host cells, while the subsequent progression of systemic disease is dependent upon glycolysis

Spontaneous Second-site Suppressors of the Filamentation Defect of prr1Delta Mutants Define a Critical Domain of Rim101p in Candida Albicans

In response to changes in ambient pH the opportunistic pathogen Candida albicans differentially expresses a number of genes. The response to pH affects morphological differentiation and virulence. The pathway controlling the pH response terminates in the zinc-finger containing transcription factor encoded by RIM101/PRR2. By analogy to the pH response pathway of Aspergillus nidulans, PRR1 of C. albicans encodes a protein that is presumably required to convert Rim101p from an inactive to an active form by proteolytic removal of a C-terminal peptide. A prr1Delta mutant is compromised in its ability to differentiate into the filamentous form. Spontaneous phenotypic revertants of a prr1Delta mutant were selected by their ability to form filamentous colonies. These mutants were also found to be defective in pH-dependent gene expression. Each of the eight mutants examined contained a heterozygous dominant mutation at the RIM101 locus. This was demonstrated genetically in all of the mutants, and directly by sequence determination of both alleles in two of the mutants. The mutant alleles conferred the ability to filament to a prr1Delta mutant, thus demonstrating that they were directly responsible for suppressing the filamentation defect. Seven of the mutant alleles contained a 1-bp substitution and one contained two substitutions at adjacent positions. The mutations were clustered within a 90-bp region near the 3'-end of the gene. In all cases the mutation generated a nonsense codon that resulted in premature termination of Rim101p; the mutant proteins were truncated by 75-104 amino acids. The results define a critical region in the C-terminal region of Rim101p and are consistent with the proposed proteolytic activation of Rim101p

Cell wall glucan remodeling is required for Candida albicans adhesion and invasion

The fungal cell wall plays a crucial role in host-pathogen interactions. Its formation is the result of the coordinated activity of several extracellular enzymes which assemble the constituents, remodel and hydrolyze them in the extracellular space. Candida albicans Phr1 and Phr2 proteins belong to Family GH72 of \uf062(1,3)-glucanosyltransferases and play a crucial role in cell wall assembly. PHR1 and PHR2 are differently regulated by extracellular pH. PHR1 is expressed when ambient pH is 5.5 or higher whereas PHR2 has the reverse expression pattern. Their deletion causes a pH-conditional defect in morphogenesis and virulence. In this work we explored whether PHR1 deletion affects C. albicans potential to invade human epithelia. We exploited a reconstituted human epithelium (RHE) as a model system. After 24 h from the exposure of RHE to the control cells (CAI-10) or to a PHR1 null mutant (CAS-10) the effects on invasion were scored. Control cells penetrated the entire epithelium layer very efficiently and invaded the underlying collagen matrix whereas the incubation with the mutant cells did not result in penetration of the epithelium and consequently no invasion of the matrix was detectable. The lack of cells in proximity of the epithelium layer suggested that adhesion might also be affected. Thus we studied the behavior of delta-phr1 cells in different adhesion assays. The mutant cells exhibited a marked reduction in the adhesion to abiotic surfaces. About 80% of the control cells were adhered within 30 min from transfer to adhesion conditions increasing to about 95% by 2h. The extent of adherence of PHR1 null mutant cells was greatly reduced since only 20% adhered by 30 min increasing to a maximum of about 30% by 2h. Next we tested the ability of PHR1 null mutant to adhere to monolayers of human epithelial cells. A similar defect in adhesion was detected. Transcription profiling of selected hyphal-specific and adhesin-encoding genes during adhesion indicates that in the PHR1 null mutant HWP1 and ECE1 transcript levels are markedly reduced. Our results suggest that expression of PHR1 strongly contributes to adhesion and invasion, two processes that promote the establishment of C. albicans infections and progression

Phr1p, a glycosylphosphatidylinsitol-anchored \u3b2(1,3)-glucanosyltransferase critical for hyphal wall formation, localizes to the apical growth sites and septa in Candida albicans

Cell wall biogenesis is a dynamic process relying on the coordinated activity of several extracellular enzymes. PHR1 is a pH-regulated gene of Candida albicans encoding a glycosylphosphatidylinositol-anchored \u3b2(1,3)-glucanosyltransferase of family GH72 which acts as a cell wall remodelling enzyme and is crucial for morphogenesis and virulence. In order to explore the function of Phr1p, we obtained a green fluorescent protein (GFP) fusion to determine its localization. During induction of vegetative growth, Phr1p-GFP was concentrated in the plasma membrane of the growing bud, in the mother-bud neck, and in the septum. Phr1p-GFP was recovered in the detergent-resistant membranes indicating its association with the lipid rafts as the wild type Phr1p. Upon induction of hyphal growth, Phr1p-GFP highly concentrated at the apex of the germ tubes and progressively distributed along the lateral sides of the hyphae. Phr1p-GFP also labelled the hyphal septa, where it colocalized with chitin. Localization to the hyphal septa was perturbed in nocodazole-treated cells, whereas inhibition of actin polymerization hindered the apical localization. Electron Microscopy analysis of the hyphal wall ultrastructure of a PHR1 null mutant showed loss of compactness and irregular organization of the surface layer. These observations indicate that Phr1p plays a crucial role in hyphal wall formation, a highly regulated process on which morphogenesis and virulence rely