Contributions of carnitine acetyltransferases to intracellular acetyl unit transport in Candida albicans.

Transport of acetyl-CoA between intracellular compartments is mediated by carnitine acetyltransferases (Cats) that reversibly link acetyl units to the carrier molecule carnitine. The genome of the opportunistic pathogenic yeast Candida albicans encodes several (putative) Cats: the peroxisomal and mitochondrial Cat2 isoenzymes encoded by a single gene and the carnitine acetyltransferase homologs Yat1 and Yat2. To determine the contributions of the individual Cats, various carnitine acetyltransferase mutant strains were constructed and subjected to phenotypic and biochemical analyses on different carbon sources. We show that mitochondrial Cat2 is required for the intramitochondrial conversion of acetylcarnitine to acetyl-CoA, which is essential for a functional tricarboxylic acid cycle during growth on oleate, acetate, ethanol, and citrate. Yat1 is cytosolic and contributes to acetyl-CoA transport from the cytosol during growth on ethanol or acetate, but its activity is not required for growth on oleate. Yat2 is also cytosolic, but we were unable to attribute any function to this enzyme. Surprisingly, peroxisomal Cat2 is essential neither for export of acetyl units during growth on oleate nor for the import of acetyl units during growth on acetate or ethanol. Oxidation of fatty acids still takes place in the absence of peroxisomal Cat2, but biomass formation is absent, and the strain displays a growth delay on acetate and ethanol that can be partially rescued by the addition of carnitine. Based on our results, we present a model for the intracellular flow of acetyl units under various growth conditions and the roles of each of the Cats in this process.

Identification and characterization of a complete carnitine biosynthesis pathway in Candida albicans.

Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl units. Here we show that the yeast Candida albicans can synthesize carnitine de novo, and we identify the 4 genes of the pathway. Null mutants of orf19.4316 (trimethyllysine dioxygenase), orf19.6306 (trimethylaminobutyraldehyde dehydrogenase), and orf19.7131 (butyrobetaine dioxygenase) lacked their respective enzymatic activities and were unable to utilize fatty acids, acetate, or ethanol as a sole carbon source, in accordance with the strict requirement for carnitine-mediated transport under these growth conditions. The second enzyme of carnitine biosynthesis, hydroxytrimethyllysine aldolase, is encoded by orf19.6305, a member of the threonine aldolase (TA) family in C. albicans. A strain lacking orf19.6305 showed strongly reduced growth on fatty acids and was unable to utilize either acetate or ethanol, but TA activity was unaffected. Growth of the null mutants on nonfermentable carbon sources is restored only by carnitine biosynthesis intermediates after the predicted enzymatic block in the pathway, which provides independent evidence for a specific defect in carnitine biosynthesis for each of the mutants. In conclusion, we have genetically characterized a complete carnitine biosynthesis pathway in C. albicans and show that a TA family member is mainly involved in the aldolytic cleavage of hydroxytrimethyllysine in vivo.

The activity of the glyoxylate cycle in peroxisomes of Candida albicans depends on a functional beta-oxidation pathway: evidence for reduced metabolite transport across the peroxisomal membrane.

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 pex5Delta/Delta 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 fox2Delta/Delta strain that is completely deficient in fatty acid beta-oxidation, 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 fox2Delta/Delta 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 fox2Delta/Delta 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 fox2Delta/Delta mutant.

Peroxisomal fatty acid beta-oxidation is not essential for virulence of Candida albicans.

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 beta-oxidation pathway. Here, we asked whether fatty acid beta-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5Delta/pex5Delta mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2Delta/fox2Delta mutant, which lacks the second enzyme of the beta-oxidation pathway. Both mutant strains had strongly reduced beta-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2Delta/fox2Delta mutant, and not the pex5Delta/pex5Delta 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 fox2Delta/fox2Delta mutant was comparable to that of the icl1Delta/icl1Delta mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid beta-oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2Delta/fox2Delta mutant is largely due to a dysfunctional glyoxylate cycle.

Dissection of transient oxidative stress response in Saccharomyces cerevisiae by using DNA microarrays.

Yeast cells were grown in glucose-limited chemostat cultures and forced to switch to a new carbon source, the fatty acid oleate. Alterations in gene expression were monitored using DNA microarrays combined with bioinformatics tools, among which was included the recently developed algorithm REDUCE. Immediately after the switch to oleate, a transient and very specific stress response was observed, followed by the up-regulation of genes encoding peroxisomal enzymes required for fatty acid metabolism. The stress response included up-regulation of genes coding for enzymes to keep thioredoxin and glutathione reduced, as well as enzymes required for the detoxification of reactive oxygen species. Among the genes coding for various isoenzymes involved in these processes, only a specific subset was expressed. Not the general stress transcription factors Msn2 and Msn4, but rather the specific factor Yap1p seemed to be the main regulator of the stress response. We ascribe the initiation of the oxidative stress response to a combination of poor redox flux and fatty acid-induced uncoupling of the respiratory chain during the metabolic reprogramming phase.