Salmonella enterica serovar Typhimurium is one of the most common causes of enterocolitis in humans. Pathogenesis of this facultative intracellular pathogen is dependent on the ability to invade non-phagocytic cells, such as those found in the intestinal epithelium. Invasion is dependent on a type III secretion system (T3SS1), which is used to translocate a set of bacterial effector proteins into the host cell. Following internalization, intracellular Salmonella survive and replicate within a modified phagosome, the Salmonella-containing vacuole (SCV). A second type III system (T3SS2) is induced intracellularly and is associated with intracellular survival/replication and biogenesis of the SCV. To understand Salmonella pathogenesis we must dissect the roles of the individual T3SS1 and T3SS2 effector proteins as well as the mechanisms that control their expression and activity inside host cells. Since the expression and function of these virulence factors is exquisitely dependent on the intracellular environment, we are focusing on developing appropriate in vitro systems to study their activities at the molecular level. This year we have established the primary human macrophage model system. We can detect growth of Salmonella Typhimurium in these cells and have identified novel mutants that are replication defective. We are currently characterizing these mutants. Another cell system that we have been developing for studying Salmonella-host cells interactions is polarized epithelial cells. We have previously used this model to show that Salmonella can escape from the SCV and replicate in the cytosol of epithelial cells. We are now using this system to investigate the role of T3SS in this cytosolic population. A separate part of this project is to develop novel tools for studying effector function. One approach we have taken is to raise monoclonal antibodies by injecting mice with pooled effector proteins. We are now completing the screening of the hybridomas. Approximately half of the monoclonals that we have characterized are specific for the actin-binding effector SipA. A peptide scanning method has been used to identify the epitopes recognized by these antibodies. The epitopes are located within all three characterized domains of SipA, including the actin binding domain. We have also screened the anti-SipA antibodies for by immunofluorescence microscopy, ELISA and immunoprecipitation. And these screens indicate that within this group of 19 antibodies there are ones that will be useful for specific techniques. For example, three of the antibodies specifically detect translocated SipA in the cytosol of Salmonella-infected HeLa cells. Most of the other antibodies recognize known effector proteins but some recognize proteins that have not been identified as T3SS1 effectors.