Identification of Drosophila gene products required for phagocytosis of Candida albicans.
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ABSTRACT:
SUBMITTER: Stroschein-Stevenson SL
PROVIDER: S-EPMC1310651 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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. DeltaDeltasfl2 cells failed to filament under a range of hypha-inducing conditions and exhibited greatly reduced RHE damage, reversed by reintroduction of SFL2 into the DeltaDeltasfl2 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, DeltaDeltasfl2 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.
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Project description:Candida albicans is an opportunistic fungal pathogen capable of life-threatening disseminated infections particularly in immunocompromised patients. Resistance to many clinically used antifungal agents has created a need to identify and develop a new generation of compounds for therapeutic use. A compound screen to identify potential antifungal natural products was undertaken, identifying 12 saponins, some of which have not been previously described. In the Caenorhabditis elegans model, some saponins conferred nematode survival comparable to that of amphotericin B. Of the 12 antifungal saponins identified, two were selected for further analysis. C. albicans isolates were inhibited by these compounds at relatively low concentrations (16 and 32 microg mL(-1)) including isolates resistant to clinically used antifungal agents. C. albicans hyphae and biofilm formation were also disrupted in the presence of these natural products, and studies demonstrate that fungal cells in the presence of saponins are more susceptible to salt-induced osmotic stress. Although saponins are known for their hemolytic activity, no hemolysis of erythrocytes was observed at three times the minimal inhibitory concentration for C. albicans, suggesting the saponins may have a preference for binding to fungal ergosterol when compared to cholesterol. Importantly, when used in combination with photosensitizer compounds, the fungus displayed increased susceptibility to photodynamic inactivation due to the ability of the saponins to increase cell permeability, thereby facilitating penetration of the photosensitizers. The large proportion of compounds identified as antifungal agents containing saponin structural features suggests it may be a suitable chemical scaffold for a new generation of antifungal compounds.
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Project description:BackgroundSystemic candidiasis is a major public health concern. In particular, in immunocompromised people, such as patients with neutropenia, patients with Acquired Immune Deficiency Syndrome (AIDS) and cancer who are undergoing antiballistic chemotherapy or bone marrow transplants, and people with diabetes. Since the clinical signs and symptoms are nonspecific, early diagnosis is often difficult. The 65-kDa mannoprotein (MP65) gene of Candida albicans is appropriate for detection and identification of systemic candidiasis. This gene encodes a putative b-glucanase mannoprotein of 65 kDa, which plays a major role in the host-fungus relationship, morphogenesis and pathogenicity.ObjectivesThe current study aimed to identify different species of Candida (C. albicans, C. glabrata and C. parapsilosis) using the Polymerase Chain Reaction (PCR) technique and also to evaluate C. albicans MP65 gene expression in BALB/C mice.Materials and methodsAll yeast isolates were identified on cornmeal agar supplemented with tween-80, germ tube formation in serum, and assimilation of carbon sources in the API 20 C AUX yeast identification system. Polymerase Chain Reaction was performed on all samples using species-specific primers for the MP65 65 kDa gene. After RNA extraction, cDNA synthesis was performed by the Maxime RT Pre Mix kit. Candida albicans MP65 gene expression was evaluated by quantitative Real-Time (q Real-Time) and Real-Time (RT) PCR techniques. The 2-ΔΔCT method was used to analyze relative changes in gene expression of MP65. For statistical analysis, nonparametric Wilcoxon test was applied using the SPSS version 16 software.ResultsUsing biochemical methods, one hundred, six and one isolates of clinical samples were determined as C. albicans, C. glabrata and C. parapsilosis, respectively. Species-specific primers for PCR experiments were applied to clinical specimens, and in all cases a single expected band for C. albicans, C. glabrata and C. parapsilosis was obtained (475, 361 and 124 base pairs, respectively). All species isolated by culture methods (100% positivity) were evaluated with PCR using species-specific primers to identify Candida species. Relative expression of Mp65 genes increased significantly after C. albicans injection into the mice (P < 0.05).ConclusionsThe results of the current study showed that the PCR method is reproducible for rapid identification of Candida species with specific primers. Mp65 gene expression of C. albicans after injection into the mice was 2.3 folds higher than before injection, with this difference being significant. These results indicated that increase of Mp65 gene expression might be an early stage of infection; however definitive conclusions require further studies.
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