Systematic analysis of the SsrAB virulon of Salmonella enterica

Intracellular Salmonella enterica serovar Typhimurium deploys the Salmonella pathogenicity island 2 (SPI2)-encoded type III secretion system (T3SS) to modify host cell functions and accomplish intracellular replication. This virulence function is controlled by the two-component system SsrAB that regulates the expression of several operons in SPI2 and, in addition, a large number of genes for non-SPI2-encoded effector proteins. Here, we analyzed the relative expression levels of members of the SsrAB virulon. We used a novel reporter fusion strategy for single-copy chromosomal fusions, all done in an identical manner in order to enable direct quantitative comparison. We observed very high expression levels for sseJ and sifA; high expression levels for ssaG, steC, sseL, and sopD2; moderate expression levels for ssaB, sseA, sseG, sifB, pipB2, and sspH1; and low expression levels for sspH2, sseI, slrP, sseK1, sseK2, pipB, and gogB. The expression of the SsrAB virulon was highly dependent on the function of SsrB but also required EnvR/OmpZ. Deletion of PhoP, part of the global regulatory system PhoPQ, resulted in highly delayed expression of the SsrAB virulon under in vitro conditions; however, maximal expression was similar to that in a wild-type background. The expression levels of SsrAB-dependent genes in intracellular bacteria were in good agreement with in vitro analyses. We provide here a comprehensive and fully comparable analysis of the expression of genes in the SsrAB virulon. This information will be of interest for the selection of in vivo-activated promoters, for example, for rational design of recombinant vaccines.