Yersinia pestis, the causative agent of plague, is one of the most pathogenic bacteria known to mankind. Due to its high pathogenicity, Yersinia has been recognized as a potential weapon for bioterrorism. An aerosolized form of the bacteria in particular could cause a pneumonic infection, which could infect a large number of people very rapidly. While early antibiotic treatment is usually effective in the bubonic form of plaque, the pneumonic plaque is less responsive and often results in death. Additional modes of treatment would therefore be required to combat plague following exposure of a population to weaponized Yersinia. Yersinia's high pathogenicity is due to its capability to evade the immune system. Thus, while most other bacteria are effectively ingested by integrin-mediated engulfment and destroyed by phagocytes, Yersinia blocks phagocytosis. The mechanism of """"""""anti-phagocytosis"""""""" involves the virulence factor YopH, a highly), effective tyrosine phosphatase. One crucial target for YopH is thought to be the docking protein pl30cas(Cas), but the precise mechanism of its action remains unknown.
In Aim 1, we will utilize our familiarity with integrin signaling and the Cas protein to examine the molecular mechanism antiphagocytosis. Our studies indicate that Cas is required for activation of the Rac GTPase in response to integrin ligation, and that this activation is essential for phagocytosis. Our data support the notion that the N-terminal domain of YopH binds to Cas, which correlates with YopH-mediated anti-phagocytosis. We will test the hypothesis that YopH functions by inhibiting a complex formation between Cas and its binding partner Crk and subsequent Rac activation. We will also test an alternative, although not mutually exclusive hypothesis that the YopH-Cas interaction results in targeting of YopH to the sites of phagocytosis, and in the subsequent inactivation of yet-to-be-identified target molecules. Substrate-trapping technology and proteomics approaches combined with functional assays will be used to identify and characterize these target proteins.
Aim 2 will take advantage of the expertise of Dr. Maurizio Pellecchia, our collaborator, in NMR-based drug discovery and structural biology with YopH. Our goal is to develop YopH-specific small molecule inhibitors that block the function of the N-terminal domain of YopH. A combination of chemical library screening and NMR-based design will be used. Lead compounds will be evaluated for efficacy in the phagocytosis and signaling assays established in Aim 1 above. This application will combine complementary expertise of a cell biological and a structural biological laboratory to address a fundamentally important question in the Yersinia pathogenesis in a unique manner. We anticipate that these studies, which are supported by a significant amount of preliminary data, will allow us to better understand the mechanisms by which Yersinia block the antimicrobial function of phagocytes. We further expect that the inhibitors to be identified will be a valuable starting point for the further development of novel drugs that can be used to combat plague mortality.
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| Pellecchia, Maurizio; Becattini, Barbara; Crowell, Kevin J et al. (2004) NMR-based techniques in the hit identification and optimisation processes. Expert Opin Ther Targets 8:597-611 |
