Project description:CaGAL102 is a sequence homolog of Rmlb. In Candida knock out of this gene causes abnormal hyphal morphogenesis and increased sensitivity to cell wall damaging agents. The knock out strain is also avirulent in mouse model of systemic infection. To get a larger insight into the function of the protein product of this gene we carried out global transcription analysis through micro array experiment. The gene is expressed under normal growth conditions and the knock out causes the cells to become hyphal under these conditions. Many of the cell wall proteins were upregulated recapitulating the cell morphology. Keywords: Candida albicans, Gene knockout, genome wide transcription profiling study
Project description:During infection the yeast Candida albicans undergoes a yeast-to-hyphal transition and secretes numerous proteins required for the invasion and damage of host tissues and the modulation of the host immune responses. Still little is known about the interplay of Candida albicans secreted proteins and the host adaptive immune system. For the characterization of the extracellular proteins of C. albicans and their ability to trigger a serological response in candidemia patients we applied 2D gel electrophoresis and LC-MS/MS-based approaches. These analyses identified the secretion of 101 different proteins from C. albicans yeast cells and 410 proteins from hyphal cells. Morphology-dependent changes of the secretome were mostly associated with proteins involved in cell wall organization, general stress response and the interplay with host immune cells. Our immunoproteomics workflow for the identification of secreted C. albicans protein antigens revealed a core set of 20 immunodominant specific anti- C. albicans protein antibodies. However, some secreted protein antigens showed cross-reactivity with sera from control groups without Candida infection. Glycoprotein staining of C. albicans hyphal secretome demonstrated that some secreted proteins are strongly glycosylated. Enzymatic deglycosylation of secreted proteins uncovered a significant contribution of glycan epitopes to the recognition of the secreted aspartyl protease Sap6 by IgG antibodies from patient sera.
Project description:Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.
Project description:CaGAL102 is a sequence homolog of Rmlb. In Candida knock out of this gene causes abnormal hyphal morphogenesis and increased sensitivity to cell wall damaging agents. The knock out strain is also avirulent in mouse model of systemic infection. To get a larger insight into the function of the protein product of this gene we carried out global transcription analysis through micro array experiment. The gene is expressed under normal growth conditions and the knock out causes the cells to become hyphal under these conditions. Many of the cell wall proteins were upregulated recapitulating the cell morphology. Keywords: Candida albicans, Gene knockout, genome wide transcription profiling study Wild type Sc5314 cells and the mutant CAS12 were grown in YPD till (OD 600nm) 1 following which total RNA was extracted using hot phenol method. The RNA was checked using Bioanalyser (Agilent). Single dye experiment was carried out using Cy3 labelled pooled wild type samples and two independent mutant RNA samples.
Project description:Cas5, a transcriptional regulator of Candida albicans, has profound effects on the biosynthesis of cell wall proteins through regulating Candida albicans cell wall remodeling
Project description:Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.
Project description:Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.
Project description:Whole cell lysates from yeast or hyphal Candida albicans cells were obtained and proteins were analysed by MS in order to have a global view of proteome differences between both fungal morphologies. This analysis constitute an interesting approach to decipher proteomic differences underlying morphological switch intimately related to Candida albicans virulence. A total of 811 different proteins were identified from hyphal whole cell lysates and 976 from yeast whole cell lysates. In terms of biological significance, we only took into consideration proteins that were identified in at least two biological replicates with at least two peptides and a q-value< 0.01.