Functional analysis of Salmonella intracellular survival and proliferation in macrophages via the type III secretion system encoded by Salmonella pathogenicity island 2

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Type III secretion system (T3SS) is a common virulence determinant in Gram-negative bacteria and the genetic information is often clustered in pathogenicity islands or on virulence plasmids. We have analyzed the T3SS encoded by Salmonella pathogenicity island 2 (SPI-2) that is indispensable for systemic disease of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mice. Since the low abundance of this secretion system restricted direct analysis by proteomic approaches, we have adopted the two-dimensional electrophoresis (2-DE) using agarose IEF for first dimension (agarose 2-DE). (1) Proteins prepared from supernatant grown the S. Typhimurium wild-type strain under SPI-2 inducing conditions were separated by agarose 2-DE and 17 protein spots were detected on agarose 2-DE gel stained with CBB. However, 16 out of 17 spots were originated from FliC, and remaining was PagC, an outer membrane protein of S. Typhimurium. (2)Next, to identify proteins whose expression is regulated by SsrA/SsrB two-component regulatory system encoded by SPI-2, we constructed S. Typhimurium strain carrying ssrB mutation. Whole-cell lysates prepared from S. Typhimurium wild-type and ssrB mutant strains, grown under SPI-2 inducing conditions were fractionated by using partial bacterial proteome extraction kit (Calbio Chem). and proteomic analysis was performed by agarose 2-DE. A total 107 proteins that were strongly reduced by an ssrB mutation have been identified. However, out of 107 proteins, only one protein was included as known SsrB-regulated protein. Thus, further analysis is required to reveal the ssrB regulon in S. Typhimurium. (3) We further characterize the protein-protein interaction among SPI-2 effectors, including SifA, SifB, SseF, SseG, and SseJ. Using bacterial two-hybrid system and pull-down assays, we have found the interaction between SifB and SseJ. In addition, the results indicate that SifB and SseJ form homodimer (or homoconcatemer).