This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.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 compromised in their ability to proliferate in a cell culture infection assay compared to the wild-type strain. Proliferation is dependent on a catalytically-active YpkA as well as an intact type III section system. These data mimic what is observed in animal infection experiments. These observations have generated the hypothesis that YpkA's proliferation-promoting activity is based on its ability to alter host cell physiology and is dependent on YpkA physically contacting specific eukaryotic proteins following its injection into the eukaryotic cell. To test this hypothesis, we will take a three-prong approach in attempting to account for YpkA's proliferation-enhancing activity vis-?-vis the eukaryotic cell. (1) We will utilize high-resolution electrophoretic techniques to uncover YpkA-mediated changes in host cell protein phosphorylation activity. (2) We will determine whether YpkA targeting of RhoA regulatory GTPases, actin, and the calcium- and integrin-binding protein (CIB), three YpkA-interacting host proteins that have been identified in protein-protein interaction screens, play a role in YpkA's proliferation-enhancing activity. And (3), we will perform a structure base analysis of the known domains of YpkA in order to determine YpkA's relationship to eukaryotic proteins that share sequence similarity. Overall 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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