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

III型分泌系统（T3 SS）是革兰氏阴性菌中常见的毒力决定因子，其遗传信息通常聚集在毒力岛或毒力质粒上。我们分析了沙门氏菌致病岛2（SPI-2）编码的T3 SS，它是肠道沙门氏菌血清型鼠伤寒（S.鼠伤寒沙门氏菌）。由于该分泌系统的低丰度限制了通过蛋白质组学方法的直接分析，我们采用了使用琼脂糖IEF的二维电泳（2-DE）用于第一维（琼脂糖2-DE）。(1)从S.对SPI-2诱导条件下的鼠伤寒沙门氏菌野生型菌株进行琼脂糖2-DE分离，经CBB染色，在琼脂糖2-DE凝胶上检测到17个蛋白质点。然而，17个点中有16个来自FliC，其余为S.鼠伤寒（2）为了鉴定受SPI-2编码的SsrA/SsrB双组分调控系统调控的蛋白质，我们构建了S.携带ssrB突变的鼠伤寒杆菌菌株。从S.通过使用部分细菌蛋白质组提取试剂盒（Calbio Chem）分级分离在SPI-2诱导条件下生长的鼠伤寒野生型和ssrB突变菌株。琼脂糖2-DE进行蛋白质组学分析。已经鉴定了总共107种被ssrB突变强烈减少的蛋白质。然而，在107种蛋白质中，仅包括一种蛋白质作为已知的SsrB调节蛋白。因此，需要进一步的分析来揭示S.鼠伤寒(3)我们进一步表征了SPI-2效应子（包括SifA、SifB、SseF、SseG和SseJ）之间的蛋白质-蛋白质相互作用。利用细菌双杂交系统和pull-down实验，我们发现SifB和SseJ之间存在相互作用。此外，结果表明，SifB和SseJ形成同源二聚体（或同源串联体）。

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).