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.

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. INTRODUCTIONAerobic life is associated with the production of reactive oxygen species (ROS) by various metabolic processes. ROS can modify lipids, proteins, and nucleic acids and can particularly cause mutations in DNA, which might contribute to tumor formation. Normally, ROS production is kept at bay by a variety of detoxifying enzymes, some of which derive their reducing power from glutathione (GSH) or thioredoxins (TRXs) (Hohmann and Mager, 1997;Jamieson and Storz, 1997;Grant et al., 1998). However, in certain pathological conditions caused by tissue damage or during treatment with certain pharmaceuticals, this protection fails, probably due to a compromised redox state: [NAD(P)H/ NAD(P)]. Although the mitochondrial respiratory chain is an important source of ROS, peroxisomal metabolism is another contributor in this respect. For instance, in rodents, application of hypolipidemic drugs resulted in enlargement of the peroxisome compartment, and long-term treatment even caused cancer (Lock et al., 1989;Reddy and Mannaerts, 1994).Peroxisomes house a number of oxidative enzymes producing ROS, such as H 2 O 2 , which is formed during the ␤-oxidation of fatty acids (Beevers, 1969;Van den Bosch et al., 1992). In the classic view, the raison d'etre of the organelle is to provide a boundary to keep ROS confined within a compartment where they can be quickly detoxified. Several considerations indicate that this concept may be too simple (Tabak et al., 1999). H 2 O 2 can easily permeate through membranes and loss of peroxisomal catalase remains without symptoms. Is this due to the fact that other detoxifying enzymes come to the rescue? There are indeed suggestions that peroxisomes harbor additional GSH or thioredoxin-dependent detoxifying enzymes (Jeong et al., 1999;Lee et al., 1999b), but it may also be that cytosolic enzymes are recruited. An opportunity to study the role of perox...

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.

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.