|Keywords:||yersinia spp.; yersinia protein kinase A; YpkA inhibitors; anti-plague drugs|
|Full text PDF:||http://hdl.handle.net/10209/94|
Yersinia spp. cause gastroenteritis and the plague, representing historically devastating pathogens that are currently an important biodefense and antibiotic resistance concern. Although several antibiotic therapies exist, the emergence of strains that have garnered multiple drug resistances in combination with the weaponization of Yersinia, make understanding the biology of this pathogen a high priority. Yersinia, along with other pathogenic bacteria such as Salmonella, utilize a macromolecular complex, called a type III secretion apparatus, to deliver virulence proteins directly into cells. These factors commandeer several signaling pathways, often targeting the Rho family of small GTPases which regulate actin cytoskeletal dynamics. A critical virulence determinant in Yersinia species is the Yersinia protein kinase A, or YpkA, a multi-domain protein that disrupts the eukaryotic actin cytoskeleton. YpkA contains a Ser/Thr kinase domain whose activity modulates pathogenicity and a domain that binds to both Rac1 and RhoA of the Rho family of small GTPases. The crystal structure of a YpkA-Rac1 complex reveals that YpkA possesses a novel Rac1-binding domain that mimics the interactions of host guanine nucleotide dissociation inhibitors (GDIs) of the Rho GTPases. YpkA inhibits the exchange of nucleotide in Rac1 and RhoA, and mutations that disrupt the YpkA-GTPase interface abolish this activity in vitro and significantly impair in vivo YpkA-induced cytoskeletal disruption. A Yersinia pseudotuberculosis mutant lacking the GDI activity of YpkA was significantly attenuated for virulence in a mouse infection assay as compared to wild type bacteria. We conclude that virulence in Yersinia depends strongly upon a novel mimicry of host GDI proteins by YpkA. Finally, the YpkA kinase domain has homology to known eukaryotic Ser/Thr kinases and thus could be targeted for small molecule inhibitor design. An efficient approach integrating a machine learning method, homology modeling, and multiple conformational high throughput docking was used for the discovery of YpkA inhibitors. The resultant small molecule compounds, which are the first reported inhibitors for YpkA, not only provide a useful means in probing the function and mechanism of YpkA in bacterial pathogenesis, but also are potential candidates for further development of novel anti-plague drugs. A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.