2007 Meritorious Research Travel Award Recipients

Abstract:

Multilocus Genotyping Method (MLST) that allows unambiguous genotyping of strains of C. neoformans
Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause infections in immunocompromised and immunocompetent individuals. Many laboratories study molecular epidemiology of these important pathogens. However, current methods of molecular genotyping preclude exchange of genotypic information among the researchers. In 2006, we developed a Multilocus Genotyping Method (MLST) that allows unambiguous genotyping of strains of C. neoformans and simplifies sharing genotypic information among the laboratories.  Currently, in collaboration with Dr. Matthew C. Fisher at the Imperial College in London, we are establishing a database of MLST protocols and markers on the Internet that will be universally available to any scientist wanting to genotype isolates of C. neoformans and C. gattii. At the third meeting of Trends of Medical Mycology in Turin, Italy (TIMM3), I will be meeting with the group of researchers that represent all major laboratories interested in molecular genotyping of Cryptococcus species complex. The purpose of this meeting will be (i) to reach consensus about key MLST markers, (ii) to designate common reference strains, (iii) to establish recommended protocols, and (iv) to review the MLST web site.

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Abstract:

Extreme serum sensitivity of yeast thr1 mutants due to homoserine accumulation
The threonine pathway is interesting from the perspective of antifungal drug targets since it does not exist in humans, is conserved throughout fungi, and various threonine biosynthetic mutants have deleterious phenotypes in addition to threonine auxotrophy. We found aspartate kinase (hom3), homoserine kinase (thr1) and threonine synthase (thr4) mutants of a clinically derived S. cerevisiae strain unable to survive in mice, and in particular, thr1 and thr4 mutants were severely depleted after only 4 hours in vivo. Consistent with this, these mutants were extremely sensitive to incubation in serum, a phenotype that was conserved for Candida albicans thr1 mutants, while Cryptococcus neoformans thr1 mutation was lethal. Serum sensitivity results from accumulation of the pathway intermediate homoserine since we find homoserine to be toxic to thr1 and thr4 mutants, and mutation of genes required for homoserine production (HOM3 and HOM6) block serum sensitivity. Increasing levels of threonine overcomes the serum and homoserine sensitivity of thr1 mutants, therefore the toxic effects of homoserine may be due to homoserine acting as a threonine analog. Homoserine and serum toxicity is also blocked by cycloheximide, consistent with a role for protein synthesis in the sensitivity. We therefore hypothesize that homoserine replaces threonine in proteins, resulting in aberrant proteins. In low threonine environments such as serum, a higher homoserine:threonine ratio would result in increased homoserine misincorporation, thus a higher degree of aberrant proteins and a higher level of toxicity.
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Abstract:

Resistance to hydrogen peroxide in Saccharomyces cerevisiae is a quantitative trait and prevalent in clinical isolates
Hydrogen peroxide (H₂O₂) is used by animals and plants alike to prevent the growth and proliferation of microbial intruders. Research on fungal pathogens including C. albicans, A. fumigatus and C. neoformans has shown the importance of resistance to reactive oxygen species (ROS). However, it appears that the catalase enzymes and genes are not primarily responsible for survival in the presence of elevated H₂O₂ levels.

While characterizing the response of S. cerevisiae to H₂O₂, we observed that a clinical isolate exhibited a higher resistance than S288c. Both strains have been crossed and 1,272 segregants have been assayed. Resistance to H₂O₂ is a dominant quantitative trait with 3-4 segregating loci. None of the genes encoding the ROS deactivating enzymes, i.e. superoxide dismutases (SOD1+SOD2), catalases (CTT1, CTA1), and peroxidase (CCP1) could be linked to the trait. Both parental strains have similar expression levels of CTA1 and catalase activities. We genotyped resistant segregants using custom designed microarrays and the S98 array and identified three candidate quantitative loci.

Furthermore, we have compared the H₂O₂ phenotypes of strains from clinical, environmental, and industrial backgrounds. Clinical isolates were more resistant than soil or brewery isolates. Industrial isolates covered a wide range of resistance phenotypes with a high degree of strain-to-strain variation. Environmental strains from the soil were most sensitive and had almost identical phenotypes.