Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin.
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2010-03
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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.
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Kingsbury, Joanne M, and John H McCusker (2010). Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2{Delta}) mutants is influenced by the carbon source and rapamycin. Microbiology, 156(Pt 3). pp. 929–939. 10.1099/mic.0.034348-0 Retrieved from https://hdl.handle.net/10161/4165.
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Scholars@Duke
John Henry McCusker
My research uses whole genome analysis as well as standard genetic and molecular biological techniques in S. cerevisiae and, in particular, applies these techniques to the following areas:
The development of S. cerevisiae as a microbial model for quantitative genetics. Quantitative traits are extremely important and have been extensively studied in higher eukaryotes. Unfortunately, our understanding of quantitative traits is poor because of: the polygenic and additive nature of quantitative trait loci; the complexity of the structures and processes affected by quantitative traits in higher eukaryotes; and, the large genome size and genetic intractability (compared to microorganisms) of high eukaryotes. The simplicity and genetic tractability of S. cerevisiae will alllow us to define quantitative traits in precise genetic and molecular terms. This analysis will aid our understanding of quantitative traits in higher eukaryotes.
The development of S. cerevisiae as a model for the pathogenic fungi. Our knowledge of fungal pathogenesis has been hampered by the genetic intractability of the pathogenic fungi. I have discovered pathogenic variants of S. cerevisiae and have shown that these strains have properties typical of other pathogenic fungi, such as the ability to grow at very high temperatures and a novel phase variation switching system. S. cerevisiae is a close relative of many of the pathogenic fungi and, as we have shown, is an emerging opportunistic pathogen which is virulent in mouse model systems. Genetic analysis of S. cerevisiae pathogenesis and virulence will be applied to more common pathogenic fungi, such as Candida albicans.
The study of phase variation in s. cerevisiae as a model for phase variation (or colony morphology switching) in pathogenic fungi. Phase variants are genetically heritable gene expression states. S. cerevisiae phase variation is also a model for cellular differentiation and gene regulation in higher eukaryotes.
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