The pathogenic yersiniae inject a number of virulence determinants, or effectors, directly into the host cell cytoplasm by the type III secretion system. This proposal focuses on uncovering the cellular activity of one of these type III effectors, the Yersinia protein kinase A (YpkA). We have observed that ypkA strains of Yersinia pseudotuberculosis are handicapped in proliferation- and antibiotic protection-based cell culture infection assays compared to the wild-type strain. YpkA is composed of a eukaryotic-like ser/thr kinase and GTPase-binding domains within its amino and carboxyl termini, respectively. An enzymatically inactive YpkA is attenuated in cell and animal infection assays but is fully competent in blocking phagocytosis. The latter activity appears to involve YpkA's GTPase-binding domain that has been previously linked to YpkA's cytoskeletal-disrupting activity. We believe that YpkA's proliferation-promoting and antiphagocytic activities are based on its ability modulate host cell functions that normally serve to limit microbial proliferation. Our working hypothesis is that YpkA's cellular activity is dependent on it physically contacting specific host proteins following its injection into the eukaryotic cell. To test this hypothesis, we are undertaking a three-prong approach in attempting to determine the basis of YpkA's cellular activity. (1) YpkA variants with specific deficiencies will be identified using various genetic, structural, and biochemical techniques. (2) Y. pseudotuberculosis mutant strains will be generated expressing the YpkA variants that come through our genetic and biochemical screens or identified in our structure-based analysis. And (3), proteomic analysis of host cells infected with the Y. pseudotuberculosis ypkA mutant strains will be performed in order to uncover YpkA-mediated changes in host cell protein phosphorylation activity. We are seeking to understand how YpkA enhances Yersinia's ability to withstand the microbial killing activity of the host cell, which in turn will undoubtedly provide key insights into YpkA's role in pathogenesis. ? ? ?
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