Role of Adapter Protein in Infectious Diseases

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DESCRIPTION (provided by applicant): Listeria monocytogenes is a food-borne pathogen that can cause meningitis, meningoencephalitis, septicemias, abortions and, in some cases, gastroenteritis. The overall mortality rate is >20% and fetal or neonatal infection with Listeria has an even higher mortality. Listeria invades a broad range of cell types. Intracellular Listeria replicates in the cytoplasm of host cells and induces the polymerization of host actin filaments ("actin tails") at the bacteria surface using bacterial protein ActA. Actin-based motility allows Listeria to spread from cell to cell without leaving the protective intracellular niche, and is essential for pathogenesis. However, the mechanism underlying Listeria motility and spreading remains elusive. The adapter protein SH2- Bbeta regulates cell motility. I have implicated SH2- Bbeta in the motility of Listeria. Preliminary data revealed that Listeria in cells overexpressing wild type SH2- Bbeta demonstrates increased velocity (225% of control) while expression of SH2 domain-deficient mutants of SH2- Bbeta in host cells inhibits Listeria movement (by approximately 60%). In a cell-free system using Xenopus ooeyte extracts and purified GST-SH2-Bbeta, SH2-Ba increased the velocity of Listeria by 140% of control. I have shown that SH2- Bbeta binds to VASP/profilin two proteins that have been shown to participate in actin-dependent Listeria motility. This application tests the hypothesis that SH2- Bbeta promotes Listeria infection by stimulating actin-based motility. The first aim will determine whether VASP/ profilin directly bind(s) to SH2- Bbeta. The second aim will determine whether SH2- Bbeta interaction with VASP/profilin is required for Listeria motility. The third aim will test whether SH2- Ba is required for spreading of Listeria infection. The fourth aim will examine whether SH2- Bbeta is required for the virulence of Listeria. In addition to providing insight into the molecular mechanism by which SH2- Bbeta contributes to Listeria motility, the results of the application studies will increase our understanding of the fundamental mechanism by which Listeria spreads. These studies designed to identify new proteins and signaling pathways involved in Listeria motility may identify new therapeutic targets for preventing the rapid distribution of Listeria infection and thereby protect people from listeriosis.