Analysis of Cells Targeted by <em>Salmonella</em> Type III Secretion In Vivo

The type III secretion systems (TTSS) encoded in Salmonella pathogenicity island-1 and -2 (SPI-1 and -2) are virulence factors required for specific phases of Salmonella infection in animal hosts. However, the host cell types targeted by the TTSS have not been determined. To investigate this, we have constructed translational fusions between the ß-lactamase reporter and a broad array of TTSS effectors secreted via SPI-1, SPI-2, or both. Secretion of the fusion protein to a host cell was determined by cleavage of a specific fluorescent substrate. In cultured cells, secretion of all six effectors could be observed. However, two to four days following i.p. infection of mice, only effectors secreted by SPI-2 were detected in spleen cells. The cells targeted were identified via staining with nine different cell surface markers followed by FACS analysis as well as by conventional cytological methods. The targeted cells include B and T lymphocytes, neutrophils, monocytes, and dendritic cells, but not mature macrophages. To further investigate replication in these various cell types, Salmonella derivatives were constructed that express a red fluorescent protein. Bacteria could be seen in each of the cell types above; however, most viable bacteria were present in neutrophils. We find that Salmonella is capable of targeting most phagocytic and non-phagocytic cells in the spleen but has a surprisingly high preference for neutrophils. These findings suggest that Salmonella specifically target splenic neutrophils presumably to attenuate their microbicidal functions, thereby promoting intracellular survival and replication in the mouse.沙门氏菌致病岛1和2（SPI-1和SPI-2）所编码的III型分泌系统（Type III Secretion System, TTSS）是沙门氏菌在动物宿主中完成感染特定阶段所必需的毒力因子。然而，目前尚未明确该III型分泌系统所靶向的宿主细胞类型。为探究这一科学问题，本研究构建了β-内酰胺酶报告基因（β-lactamase reporter）与一系列经SPI-1、SPI-2或二者共同分泌的III型分泌系统效应蛋白之间的翻译融合体。通过特定荧光底物的切割反应，可检测融合蛋白是否被分泌至宿主细胞。在体外培养细胞中，可观测到全部六种效应蛋白的分泌。然而，在小鼠腹腔（intraperitoneal, i.p.）感染后的2至4天，仅在脾细胞中检测到由SPI-2分泌的效应蛋白。靶向的细胞通过九种不同细胞表面标志物染色、随后的荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分析以及常规细胞学方法得以鉴定，其包括B淋巴细胞、T淋巴细胞、中性粒细胞、单核细胞以及树突状细胞，但不包括成熟巨噬细胞。为进一步研究沙门氏菌在这些不同细胞类型中的增殖情况，本研究构建了表达红色荧光蛋白的沙门氏菌衍生菌株。实验结果显示，虽可在上述所有细胞类型中观察到细菌，但绝大多数活菌存在于中性粒细胞中。本研究发现，沙门氏菌可靶向脾脏内的大多数吞噬细胞与非吞噬细胞，但对中性粒细胞表现出惊人的高度偏好性。这些结果表明，沙门氏菌会特异性靶向脾脏内的中性粒细胞，推测其目的是削弱中性粒细胞的杀菌功能，从而促进自身在小鼠体内的胞内存活与增殖。