Signal Mediated Defense Mechanisms of Yeast

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The yeast Saccharomyces cerevisiae has been a powerful model system for the study of many fundamental processes in cell biology. One important unsolved cell biology problem is how cells sense and defend themselves against chemical attack by other cells. To directly study this problem, this research will develop a yeast-mouse macrophage co-culture system to uncover and analyze the signaling pathways and genomic responses of yeast to specific noxious chemicals, e.g., nitric oxide and superoxide anion, releasedby macrophages. In previous results, it was demonstrated that the osmostress sensing MAP kinase pathway called the HOG pathway also functions to sense and respond to oxidants.The goals of this research are: 1.) a combination of two different approaches will be used to identify the yeast signaling pathways activated by macrophage-produced superoxide, nitric oxide, and non-oxidant attacking chemicals, respectively. First, genome-wide analysis of mRNA expression using hybridization to DNA microarrays will be used to define the different sets of yeast genes that are specifically induced by macrophages producing nitric oxide and not superoxide or producing superoxide and not nitric oxide, etc. Second, yeast strains with deletions of putative defense signaling pathway genes will be analyzed and compared to a wild type strain for changes in gene induction and survival in response to macrophage-produced superoxide, nitric oxide, and non-oxidant attacking chemicals, respectively. 2.) the transcription factor target of the HOG pathway involved in the oxidant response will be identified by three complementary approaches. The promoter sequences necessary for HOG pathway-dependent, oxidant-induced expression of the thioredoxin gene TRX2 will be identified by mutational analysis of a TRX2-lacZ reporter gene. The HOG pathway dependent, oxidant-induced set of yeast genes will be identified using hybridization to DNA microarrays. Finally, yeast strains with deletions of putative HOG pathway transcription factors will be analyzed and compared to a wild type strain for changes in oxidant-induced TRX2-lacZ expression and genome-wide changes in gene expression.