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

III型分泌系统（T3SS）是革兰氏阴性菌常见的毒力决定因素，遗传信息常聚集在致病性岛或毒力质粒上。我们分析了沙门氏菌致病性岛1 （SPI-1）编码的T3SS，该T3SS是鼠伤寒沙门氏菌侵袭上皮细胞所必需的。SipC是鼠伤寒沙门氏菌中由spi -1编码的T3SS分泌的一种409个氨基酸的蛋白。SipC是鼠伤寒沙门氏菌跨宿主质膜转运一组分泌效应蛋白的必要条件，这些效应蛋白的功能是抵消免疫信号反应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 ~ 140和残基173 ~ 192），它们被认为在易位过程中插入宿主质膜。为了阐明膜插入对SipC功能的重要性，SipC蛋白在跨膜中含有缺失和点突变。域被构造。用表达SipC突变蛋白的鼠伤寒沙门氏菌感染上皮细胞。研究了效应物易位、孔形成和宿主细胞信号反应。在SipC的跨膜编辑中引入缺失和点突变（S165P和S165A），导致III型机制不分泌无功能蛋白。此外，这些SipC突变体衍生物在与宿主细胞膜结合方面存在缺陷。此外，在小鼠感染模型中，表达SipC衍生物宿主细胞膜插入缺陷的沙门氏菌突变株未能引起严重结肠炎。这些结果强烈提示SipC的所有功能都与插入宿主膜有关。

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.