Brucella abortus is a facultative intracellular pathogen that is highly infectious by the aerosol route and causes chronic, debilitating disease. A key step in B. abortus infection is the establishment of persistent infection within macrophages. The bacterial genes encoding virulence mechanisms required for specific interactions between Brucella and the macrophage remain largely undiscovered. We have identified a genetic locus of B. abortus, virB, that is required for establishing infection both in macrophages and In the mouse model. The B. abortus virB locus is predicted by sequence homology to encode a type IV secretion system. Our long- range goal is to elucidate the mechanism by which the virB locus mediates intracellular survival and persistent infection. The objective of this application is to study the expression of the virB genes and compare the interaction of wild type B. abortus and virB mutants with regard to vacuolar trafficking in the macrophage. The central hypothesis of this application is that the virB locus mediates a critical interaction with the macrophage that allows B. abortus to establish infection. The rationale for the proposed research is that characterization of B. abortus virulence factors mediating specific interactions with macrophages will form the basis for new approaches to treat or prevent brucellosis. We are uniquely prepared to undertake the proposed research, because we have generated tools for studying virB expression at both the transcriptional and translational level. Furthermore, the work will be performed in an excellent research environment that is conducive to its completion. Our Department contains several funded investigators working on intracellular bacterial pathogens and excellent BL-3 facilities, as well as other shared resources available for the study of host/pathogen interactions. The central hypothesis will be tested, and the objectives of this application accomplished by pursuing the following two specific aims: (1) Identify conditions for In vitro and in vivo expression of the B. abortus virB locus and localize protein products in the bacterium, and (2) Determine the mechanism by which the virB locus enables B. abortus to survive and grow intracellularly within macrophages. We expect that the results of this work will provide the first direct evidence for expression of the B. abortus virB proteins as well as define the environmental signals that induce expression of this locus. Furthermore, our results will provide information essential to defining the cellular interaction mediated by the virB locus. These results will be significant, because they are expected to provide new targets for preventive or therapeutic interventions to be employed in the case of illegitimate use of this bacterial pathogen. In addition, it is expected that these results will advance our knowledge of type IV secretion systems, which are used by a number of different bacterial pathogens to subvert the host's defense mechanisms.
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