John H. McCusker, PhD
Associate Professor
Department of Molecular Genetics and Microbiology

Saccharomyces cerevisiae — a model for the pathogenic fungi: S. cerevisiae, a close relative of the pathogenic Candida species, is observed clinically, in a variety of different body sites and patient types, and is an emerging opportunistic pathogen. Clinically derived strains of S. cerevisiae resemble more commonly observed pathogenic fungi in that they have traits associated with virulence, such as profuse pseudohyphal formation and high temperature growth. Also, clinically derived strains of S. cerevisiae proliferate and persist in immunocompetent mice and kill complement factor five deficient mice. The ease of genetically manipulating S. cerevisiae, relative to the more commonly observed pathogenic fungi makes S. cerevisiae a powerful, genetically tractable model system to identify pathways required for fungal survival in the mammalian host environment. We test S. cerevisiae mutants to find those that are severely deficient in their ability to survive in the host environment which provides insight into the basic processes of fungal pathogenesis. We then apply these results to other, less genetically tractable fungi.

S. cerevisiae — a model to study phenotypic variation/instability: Extensive phenotypic instability or variation exists in clonal populations of microorganisms, which is thought to play a role in adaptation to novel environments. This phenotypic variation or instability, which occurs by multiple mechanisms, may be a form of cellular differentiation and a stochastic means for modulating gene expression. This phenotypic instability or variation may also play a role in human genetic diseases. One case that we have dissected involves the high frequency formation of translational suppressors. All of these suppressors are mutants in the eight member tRNA-Tyr gene family that are dispersed throughout the genome. Most interestingly, there is a strong position effect on mutation frequency at different tRNA-Tyr loci. In addition to the tRNA-Tyr-dependent phenotypic variation, other phenotypic variation systems are under study which involve other genes and mechanisms.

S. cerevisiae — a microbial model for quantitative genetics: One observes a range of phenotypes (quantitative traits) in natural populations which is due to the complex interaction of multiple alleles of many different genes. Although these quantitative traits are very important, the genetic complexity of quantitative traits has made the identification of the genes underlying quantitative traits difficult. To better understand quantitative traits, we developed S. cerevisiae as a microbial model for quantitative genetics; we focus on the ability to grow at high temperatures, a virulence trait in pathogenic fungi. We combine genome-wide mapping and a new genetic technique named reciprocal hemizygosity analysis to dissect quantitative trait loci (QTL) in S. cerevisiae and find that QTL architecture is considerably more complex than anticipated. We also find a genetic explanation for heterosis (hybrid vigor), the increased fitness of the heterozygote compared to homozygotes. S. cerevisiae is a superb model for understanding quantitative genetics.

Using genetics to identify novel antifungal drug targets: The currently available antifungal drugs are few (compared to antibacterials) in number, are less effective than clinicians would like, and tend to have severe side effects. Therefore, there is a great need to develop new antifungals. The goal of these experiments is to identify Candida albicans and Cryptococcus neoformans mutants that are avirulent/non-pathogenic in experimental infections and thereby identify good potential drug targets. The primary focus is on a specific subset of amino acid and vitamin biosynthetic genes. The reason for focusing on these pathways is that many auxotrophs are avirulent. The choice of genes in these pathways is dictated by (i) their absence in humans and (ii) the highly deleterious in vitro phenotypes of specific mutants. We have identified a number of fungal mutants with severe defects. As targets are identified it will then be possible to focus attention on these gene products and use novel technologies, such as combinatorial chemistry, to develop small molecule inhibitors.