The interaction between Candida albicans and cells of the innate immune system is a key determinant of disease progression. Transcriptional profiling has revealed that C. albicans has a complex response to phagocytosis, much of which is similar to carbon starvation. This suggests that nutrient limitation is a significant stress in vivo, and we have shown that glyoxylate cycle mutants are less virulent in mice. To examine whether other aspects of carbon metabolism are important in vivo during an infection, we have constructed strains lacking FOX2 and FBP1, which encode key components of fatty acid ␤-oxidation and gluconeogenesis, respectively. As expected, fox2⌬ mutants failed to utilize several fatty acids as carbon sources. Surprisingly, however, these mutants also failed to grow in the presence of several other carbon sources, whose assimilation is independent of ␤-oxidation, including ethanol and citric acid. Mutants lacking the glyoxylate enzyme ICL1 also had more severe carbon utilization phenotypes than were expected. These results suggest that the regulation of alternative carbon metabolism in C. albicans is significantly different from that in other fungi. In vivo, fox2⌬ mutants show a moderate but significant reduction in virulence in a mouse model of disseminated candidiasis, while disruption of the glyoxylate cycle or gluconeogenesis confers a severe attenuation in this model. These data indicate that C. albicans often encounters carbon-poor conditions during growth in the host and that the ability to efficiently utilize multiple nonfermentable carbon sources is a virulence determinant. Consistent with this in vivo requirement, C. albicans uniquely regulates carbon metabolism in a more integrated manner than in Saccharomyces cerevisiae, such that defects in one part of the machinery have wider impacts than expected. These aspects of alternative carbon metabolism may then be useful as targets for therapeutic intervention.

Carbon starvation is one of the many stresses to which microbial pathogens are subjected while in the host. Pathways necessary for the utilization of alternative carbon sources, such as gluconeogenesis, the glyoxylate cycle, and ␤-oxidation of fatty acids, have been shown to be required for full virulence in several systems, including the fungal pathogen Candida albicans. We have investigated the regulatory network governing alternative carbon metabolism in this organism through characterization of transcriptional regulators identified based on the model fungi, Saccharomyces cerevisiae and Aspergillus nidulans. C. albicans has homologs of the ScCAT8/AnFacB and ScADR1/AnAmdX transcription factors that regulate induction of genes encoding the proteins of gluconeogenesis, the glyoxylate cycle, and ethanol utilization. Surprisingly, C. albicans mutants lacking CAT8 or ADR1 have no apparent phenotypes and do not regulate genes for key enzymes of these pathways. Fatty acid degradation and peroxisomal biogenesis are controlled by nonhomologous regulators, OAF1/PIP2 in S. cerevisiae and FarA/FarB in A. nidulans; C. albicans is missing OAF1 and PIP2 and, instead, has a single homolog of the Far proteins, CTF1. We have shown that CTF1 is required for growth on lipids and for expression of genes necessary for ␤-oxidation, such as FOX2. ctf1⌬/ctf1⌬ (ctf1⌬/⌬) strains do not, however, show the pleiotropic phenotypes observed for fox2⌬/⌬ mutants. The ctf1⌬/⌬ mutant confers a mild attenuation in virulence, like the fox2⌬/⌬ mutant. Thus, phenotypic and genotypic observations highlight important differences in the regulatory network for alternative carbon metabolism in C. albicans compared to the paradigms developed in other model fungi.Candida albicans is both a ubiquitous commensal of the human microbial flora and the most important fungal pathogen of humans (9, 43). While C. albicans can infect nearly any site in the body, the most serious manifestation, disseminated bloodstream infections, particularly affects immunocompromised individuals and is fatal in about 40% of cases (67). Candida species are responsible for ϳ9% of cases of hospitalacquired sepsis and up to 12% of central line-associated bloodstream infections, with C. albicans causing about half of these infections (23,67). Studying the biology of C. albicans in its natural niche, the mammalian host, provides insights into how this intriguing species has adapted to become such a successful pathogen and, as a result, is crucial to the development of new drug targets and treatment strategies.An increasing body of literature indicates that some host niches are carbon limited and that mutations that abrogate utilization of nonfermentable carbon sources are compromised in virulence models for many (but not all) fungal pathogens of both plants and animals, including C. albicans, Magnaporthe grisea, Leptosphaeria maculans, Stagonospora nodorum, and Colletotrichum lagenarium (2,5,27,33,46,48,59,66). In particular, studies have focused on the pathways of gluconeogenesis, the g...

To investigate the role of the prevacuolar secretion pathway in biofilm formation and virulence in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae prevacuolar trafficking gene PEP12. C. albicans PEP12 encodes a deduced t-SNARE that is 28% identical to S. cerevisiae Pep12p, and plasmids bearing C. albicans PEP12 complemented the abnormal vacuolar morphology and temperaturesensitive growth of an S. cerevisiae pep12 null mutant. The C. albicans pep12⌬ null mutant was defective in endocytosis and vacuolar acidification and accumulated 40-to 60-nm cytoplasmic vesicles near the plasma membrane. Secretory defects included increased extracellular proteolytic activity and absent lipolytic activity. The pep12⌬ null mutant was more sensitive to cell wall stresses and antifungal agents than the isogenic complemented strain or the control strain DAY185. Notably, the biofilm formed by the pep12⌬ mutant was reduced in overall mass and fragmented completely upon the slightest disturbance. The pep12⌬ mutant was markedly reduced in virulence in an in vitro macrophage infection model and an in vivo mouse model of disseminated candidiasis. These results suggest that C. albicans PEP12 plays a key role in biofilm integrity and in vivo virulence.

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