Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans. Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid -oxidation pathway. Here, we asked whether fatty acid -oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5⌬/pex5⌬ mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2⌬/ fox2⌬ mutant, which lacks the second enzyme of the -oxidation pathway. Both mutant strains had strongly reduced -oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2⌬/fox2⌬ mutant, and not the pex5⌬/pex5⌬ mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2⌬/fox2⌬ mutant was comparable to that of the icl1⌬/icl1⌬ mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid -oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2⌬/fox2⌬ mutant is largely due to a dysfunctional glyoxylate cycle.
SummaryPreviously, we have shown that PDE2 is required for hyphal development and cell wall integrity in Candida albicans. In the present study, we have investigated the effects of its deletion by genome-wide transcriptome profiling. Changes in expression levels of genes involved in metabolism, transcription, protein and nucleic acids synthesis, as well as stress responses, cell wall and membrane biogenesis, adherence and virulence have been observed. By comparing these changes with previously reported transcriptome profiles of pde2D mutants of Saccharomyces cerevisiae, as well as cdc35D, ras1D and efg1D mutants of C. albicans, conserved and speciesspecific cAMP-regulated genes have been identified. The genes whose transcription is altered upon deletion of PDE2 in C. albicans has also allowed us to predict that the pde2D mutant would have a defective ability to adhere to, and invade host cells, and an impaired virulence as well as response to different stresses. Using appropriate assays, we have tested these predictions and compared the roles of the highand low-affinity cAMP phosphodiesterases, Pde2p and Pde1p in stress, adhesion and virulence. We suggest that phosphodiesterases, and in particular the high-affinity cAMP phosphodiesterase encoded by PDE2, have real potential as targets for antifungal chemotherapy.
The glyoxylate cycle, a metabolic pathway required for generating C 4 units from C 2 compounds, is an important factor in virulence, in both animal and plant pathogens. Here, we report the localization of the key enzymes of this cycle, isocitrate lyase (Icl1; EC 4.1.3.1) and malate synthase (Mls1; EC 2.3.3.9), in the human fungal pathogen Candida albicans. Immunocytochemistry in combination with subcellular fractionation showed that both Icl1 and Mls1 are localized to peroxisomes, independent of the carbon source used. Although Icl1 and Mls1 lack a consensus type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on the PTS1 receptor Pex5p, suggesting the presence of non-canonical targeting signals in both proteins. Peroxisomal compartmentalization of the glyoxylate cycle is not essential for proper functioning of this metabolic pathway because a pex5D/D strain, in which Icl1 and Mls1 were localized to the cytosol, grew equally as well as the wild-type strain on acetate and ethanol. Previously, we reported that a fox2D/D strain that is completely deficient in fatty acid boxidation, but has no peroxisomal protein import defect, displayed strongly reduced growth on non-fermentable carbon sources such as acetate and ethanol. Here, we show that growth of the fox2D/D strain on these carbon compounds can be restored when Icl1 and Mls1 are relocated to the cytosol by deleting the PEX5 gene. We hypothesize that the fox2D/D strain is disturbed in the transport of glyoxylate cycle products and/or acetyl-CoA across the peroxisomal membrane and discuss the possible relationship between such a transport defect and the presence of giant peroxisomes in the fox2D/D mutant.
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