Project description:Candida albicans is an opportunistic pathogenic fungus that is able to assume several morphologies, including yeast and hyphal growth forms. The hyphal morphology can be induced by various environmental stimuli and is accompanied by expression of a large set of hyphal-specific genes (HSGs). Cell cycle and morphogenetic programs are interconnected: notably, inhibition of cell cycle progression often causes a switch to filamentous growth. Here we identify and characterize CaNrm1, a C. albicans homolog of the S. cerevisiae Whi5 and Nrm1 transcription inhibitors that, analogous to mammalian Rb, regulate the cell cycle transcription program in the G1 phase and at the G1/S transition. CaNRM1 is able to complement the phenotypes of both whi5 and nrm1 mutants in S. cerevisiae. Deletion of CaNRM1 causes a reduction in cell size and results in increased resistance to hydroxyurea (HU), an inhibitor of DNA replication; analysis of the expression of ribonucleotide reductase, the target of HU, suggests that its transcriptional induction in response to HU is mainly dependent upon CaNrm1. Genetic epistasis analysis suggests that CaNrm1 interacts with the SBF and MBF transcription factors in S. cerevisiae and with the MBF functional homolog in C. albicans. At the transcription level, deletion of CaNRM1 causes an induction of many G1 and G1/S-specific genes. Induction of the HSGs is dampened under certain conditions in the Canrm1-/- mutant, suggesting that the cell cycle transcription program influences the morphogenetic transcription program of C. albicans.
Project description:Candida albicans is an opportunistic pathogenic fungus that is able to assume several morphologies, including yeast and hyphal growth forms. The hyphal morphology can be induced by various environmental stimuli and is accompanied by expression of a large set of hyphal-specific genes (HSGs). Cell cycle and morphogenetic programs are interconnected: notably, inhibition of cell cycle progression often causes a switch to filamentous growth. Here we identify and characterize CaNrm1, a C. albicans homolog of the S. cerevisiae Whi5 and Nrm1 transcription inhibitors that, analogous to mammalian Rb, regulate the cell cycle transcription program in the G1 phase and at the G1/S transition. CaNRM1 is able to complement the phenotypes of both whi5 and nrm1 mutants in S. cerevisiae. Deletion of CaNRM1 causes a reduction in cell size and results in increased resistance to hydroxyurea (HU), an inhibitor of DNA replication; analysis of the expression of ribonucleotide reductase, the target of HU, suggests that its transcriptional induction in response to HU is mainly dependent upon CaNrm1. Genetic epistasis analysis suggests that CaNrm1 interacts with the SBF and MBF transcription factors in S. cerevisiae and with the MBF functional homolog in C. albicans. At the transcription level, deletion of CaNRM1 causes an induction of many G1 and G1/S-specific genes. Induction of the HSGs is dampened under certain conditions in the Canrm1-/- mutant, suggesting that the cell cycle transcription program influences the morphogenetic transcription program of C. albicans. One aspect of the characterization of the C. albicans Whi5/Nrm1 gene was to examine how this molecule influences global gene expression. To that end, we isolated total RNA from log-phase nrm1-/nrm1- (as well as parent/background strain) cells in two independent replicate experiments. These samples were processed, labeled, and subsequently used for hybridization of custom C. albicans Affymetrix arrays.
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:We perform microarray analysis of HUVECs upon stimulation with virulent wildtype C. albicans strain SC5314 or its efg1/efg1 cph1/cph1 hyphal-deficient derivative strain CAN34 to compare the gene expression profiles elicited from HUVECs in response to these strains. In addition, these responses are compared to that of TNF-alpha induced responses to determine which responses are Candida-specific. Keywords: comparison of host response to different Candida albicans morphologies
Project description:Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. ΔΔsfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the ΔΔsfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔsfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.
Project description:Candida albicans and Candida dubliniensis are closely related species displaying differences in virulence and genome content, therefore providing potential opportunities to identify novel C. albicans virulence genes. C. albicans gene arrays were used for comparative analysis of global gene expression in the two species in reconstituted human oral epithelium (RHE). C. albicans (SC5314) showed upregulation of hypha-specific and virulence genes within 30 min postinoculation, coinciding with rapid induction of filamentation and increased RHE damage. C. dubliniensis (CD36) showed no detectable upregulation of hypha-specific genes, grew as yeast, and caused limited RHE damage. Several genes absent or highly divergent in C. dubliniensis were upregulated in C. albicans. One such gene, SFL2 (orf19.3969), encoding a putative heat shock factor, was deleted in C. albicans. ΔΔsfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the ΔΔsfl2 strain. Moreover, SFL2 overexpression in C. albicans triggered hyphal morphogenesis. Although SFL2 deletion had no apparent effect on host survival in the murine model of systemic infection, ΔΔsfl2 strain-infected kidney tissues contained only yeast cells. These results suggest a role for SFL2 in morphogenesis and an indirect role in C. albicans pathogenesis in epithelial tissues.
Project description:Candida albicans demonstrates three main growth morphologies yeast, pseudohyphal and true hyphal forms. Cell separation is distinct in these morphological forms and the process of separation is closely linked to the completion of mitosis and cytokinesis. In Saccharomyces cerevisiae the small GTPase Tem1 is known to initiate the mitotic exit network, a signalling pathway involved in signalling the end of mitosis and initiating cytokinesis and cell separation. We have characterised the role of Tem1 in C. albicans, and demonstrate that it is essential for mitotic exit and cytokinesis, and that this essential function is signalled through the kinase Cdc15. Consistent with its role in activating the mitotic exit network Tem1 localised to spindle pole bodies in a cell cycle dependent manner. Cells depleted of Tem1 displayed highly polarised growth but ultimately fail to complete cytokinesis and re-enter the cell cycle following nuclear division. At the transcriptional level genes downregulated following the depletion of Tem1 where significantly enriched for genes whose expression peaks early in the cell cycle and for those associated with glycolysis.