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，其遗传信息往往聚集在致病岛或毒力质粒上。我们分析了沙门氏菌致病岛1(SPI-1)编码的T3SS，它是鼠伤寒沙门氏菌(S.typhimurium)上皮细胞入侵所必需的。SIPC是由鼠伤寒沙门氏菌SPI-1编码的T3SS分泌的一种409个氨基酸的蛋白质。SIPC是鼠伤寒沙门氏菌通过宿主质膜转运一组分泌的效应蛋白所必需的，这些蛋白具有对抗免疫信号反应和诱导宿主肌动蛋白重排的功能。SIPC包含两个预测的跨膜螺旋(残基122到140和173到192)，它们被认为在转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，对含有SIPC跨膜缺失和点突变的SIPC蛋白进行了研究。构建了结构域。用表达突变SIPC蛋白的鼠伤寒沙门氏菌感染上皮细胞。研究了效应器移位、气孔形成和宿主细胞信号反应。将缺失和点突变(S165P和S165A)引入SIPC的跨膜反应中，导致了一个不能由III型机械分泌的无功能蛋白。此外，这些SIPC突变衍生物与宿主细胞膜的结合存在缺陷。此外，一种表达SIPC衍生物的突变沙门氏菌株在宿主细胞膜插入方面存在缺陷，但在小鼠感染模型中未能导致严重结肠炎。这些结果有力地表明，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.