Functional analysis of Salmonella type III secretion system translocator protein SipC and application of vaccine development

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1（SPI-1）编码的T3 SS，该致病岛1是肠沙门氏菌鼠伤寒血清型（S.鼠伤寒沙门氏菌）。SipC是一种由SPI-1编码的T3 SS分泌的409个氨基酸的蛋白质。鼠伤寒S需要SipC。鼠伤寒沙门氏菌转运穿过宿主质膜的一组分泌的效应蛋白，其功能是抵消免疫信号应答并诱导宿主肌动蛋白重排。SipC含有两个预测的跨膜螺旋（残基122至140和173至192），其被认为在易位期间插入宿主质膜。为了阐明膜插入对SipC功能的重要性，在跨膜中含有缺失和点突变的SipC蛋白。域被构建。S.表达突变SipC蛋白的鼠伤寒杆菌菌株用于感染上皮细胞。研究了效应子易位、孔形成和宿主细胞信号传导反应。将缺失和点突变（S165 P和S165 A）引入SipC的跨膜区导致不被III型机器分泌的无功能蛋白。此外，这些SipC突变体衍生物与宿主细胞膜的关联是有缺陷的。此外，表达宿主细胞膜插入缺陷的SipC衍生物的突变沙门氏菌菌株在小鼠感染模型中未能引起严重的结肠炎。这些结果有力地表明，所有的功能SipC涉及插入到宿主膜。

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 1 (SPI-1) that is required for epithelial cell invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium). SipC is a 409-amino-acid protein that is secreted by a SPI-1-encoded T3SS in S. Typhimurium. SipC is required for S. Typhimurium to translocate across the host plasma membrane a set of secreted effector proteins that function to counteract immune signaling responses and to induce host actin rearrangement. SipC contains two predicted transmembrane helices (residues 122 to 140 and 173 to 192) that are thought to insert into the host plasma membrane during translocation. To elucidate the importance of membrane insertion for SipC function, SipC proteins containing deletions and point-mutations in the transmembrane. domain were constructed. S. Typhimurium strains expressing the mutant SipC proteins were used to infect epithelial cells. Effector translocation, pore formation, and host-cell-signaling responses were studied. Introduction of deletions and point-mutations (S165P and S165A) into the transmembrane resion of SipC resulted in a nonfunctional protein that was not secreted by the type III machinery. In addition, these SipC mutant derivatives were defective for association with host cell membranes. Furthermore, a mutant Salmonella strain that expresses the SipC derivative defective in host cell membrane insertion failed to cause severe colitis in a mouse infection model. These results strongly suggest that all functions of SipC involve insertion into host membrane.