Various serotypes of the enteric Gram-negative bacterial pathogen Salmonella are responsible for a number of diseases of public health significance, including acute gastroenteritis, as well as typhoid fever. During the course of infection, these organisms invade intestinal epithelial cells, dendritic cells and macrophages of the host. In doing so, the bacteria introduce specific effector proteins into the host cells through a specialized secretory apparatus. The cytoskeletal changes and activation of cellular signaling pathways induced by these proteins facilitate bacterial invasion and also elicit the production of host pro-inflammatory molecules. Elucidating exactly how the effector proteins carry out these functions would help to clarify the pathogenesis of, and might suggest new approaches to treating, Salmonella -associated disease. In preliminary experiments, I have found that the effector SopE2, a guanine nucleotide exchange factor for mammalian Rho GTPases, is necessary for the Salmonella -dependent upregulation of inducible nitric oxide synthase (iNOS), the enzyme that is responsible for controlling the production of nitric oxide (NO) in macrophages. The pro-inflammatory and immunomodulatory effects of NO contribute to anti-microbial defense, as well as to the tissue damage that is associated with infection. In further studies, I have found that SopE2 activates the transcription factor NF-kappa-B, both on its own, and in a synergistic interaction with TRAF6, an adaptor molecule involved in signaling via members of the Toll-like receptor, and TNF receptor families. The experiments proposed in this application will extend these preliminary observations to elucidate the function of SopE2, and its homolog SopE, by (a) examining the mechanism by which SopE2 and SopE initiate signals leading to iNOS induction, particularly the role of the Rho GTPases in this process (b) elucidating how SopE2 and SopE activate NF-kappa-B, and characterizing their influence on TRAF-dependent signals, (c) identifying cis-acting transcriptional regulatory elements in the iNOS promoter that respond to Salmonella infection, and to Sop-induced signals, and (d) examining the role of SopE2 and SopE in iNOS induction by Salmonella in primary macrophages and dendritic cells. The results of these studies will shed light on a novel function of SopE and SopE2 and will also improve understanding of the mechanisms that regulate iNOS expression.
