Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin

Abstract

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Delta mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Delta mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Delta mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Delta starvation-cidal defects in either species, the cidal phenotype was not due to alpha-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Delta starvation viability, it increased Ca. albicans ilv1Delta and ilv2Delta viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Delta mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Delta and ilv2 Delta viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

Fig. 1.

Isoleucine, valine and leucine biosynthetic pathways.

Fig. 2.

Virulence phenotypes of Ca. albicans isoleucine and valine biosynthetic mutants. Strains included SC5314 (wild-type), CJK27 (ilv2Δ/ilv2Δ), CJK133 (ILV2/ilv2Δ, ILV2-complemented strain), CJK140 (ilv1Δ/ilv1Δ), CJK142 (ILV1/ilv1Δ, ILV1-complemented strain) and CJK132 (ilv1Δ/ilv1Δ ilv2Δ/ilv2Δ).

Fig. 3.

(a) Recovery on YPD following starvation of S. cerevisiae (and Ca. albicans met2Δ) auxotrophs in SD. (b) Recovery on YPD and YPEG following starvation in SD or SEG±amino acid supplementation, as indicated. (c) Recovery of S. cerevisiae ilv2Δ mutant on YPD following starvation in SD or SD+rapamycin (SD+rap). Strains included S. cerevisiae YJK2486 (ilv2Δ), YJK2487 (ilv1Δ ilv2Δ), YJK2484 (ilv1Δ), S3782 (leu1Δ), S3807 (leu2Δ), S3783 (leu4Δ leu9Δ), YJK564 (met2Δ) and Ca. albicans CJK103 (ca met2Δ).

Fig. 4.

Influence of genotype, amino acid addition, carbon source present during recovery and starvation, and rapamycin on the viability of Ca. albicans mutants following starvation. Strains included CJK27 (ilv2Δ), CJK132 (ilv1Δ ilv2Δ) and CJK140 (ilv1Δ).

Fig. 5.

Effect of starvation on S. cerevisiae ilv2Δ colony size. Cells were starved in SD medium and streaked onto YPD plates at the times indicated.