Analysis of Cells Targeted by Salmonella Type III Secretion In Vivo

Abstract

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.

MonitoringShigella flexnerivacuolar escape by flow cytometry

“…Previous studies on translocation of effector proteins have shown that the FRET-based β-lactamase reporter assay is compatible with analysis by flow cytometry. [10][11][12] In the present report we show that our vacuolar rupture assay can also be analyzed by flow cytometry, which constitutes an important alternative and complementary read-out to microscopy-based data acquisition. Figure 1.…”

“…7 Recently, this assay has been adapted to measure effector translocation or internalization of invasive bacteria in vitro and in vivo by creating β-lactamase fusion proteins with bacterial effectors. [8][9][10][11][12][13] Using Shigella invasion of host cells as a model system, we have extended these approaches by establishing a novel fluorescence microscopy assay that allows the study of vacuole rupture with high spatiotemporal precision (Fig. 1).…”

The small GTPase Rab29 is a common regulator of immune synapse assembly and ciliogenesis

“…36 Moreover, both CD4 and CD8 T cells have been shown to internalize S. thyphimurium in infected mice. 37 The implication of Rab29 in IS assembly and T-cell activation suggests a potential novel target for immune evasion by Salmonella involving the protease GtgE. By preventing the delivery to the IS of endosomal TCRs, which requires Rab29, in infected T cells, GtgE would be expected to effectively suppress the long-lasting signaling required for T-cell activation.…”

Novel Highly Sensitive IL-10–β-Lactamase Reporter Mouse Reveals Cells of the Innate Immune System as a Substantial Source of IL-10 In Vivo

“…Y. enterocolitica is known to disseminate, among other locations, to the spleen, where they replicate predominately extracellularly and form microcolonies consisting primarily of extracellular residing Yersinia-bacteria and neutrophils (66). In contrast, S. Typhimuruim is an intracellular living pathogen that has been recently shown to specifically target splenic neutrophils and to survive and replicate inside them (67). However, despite the different life styles of Yersinia and Salmonella, Ly6G + neutrophils represented also a major IL-10-producing population in the spleen of Salmonella-infected ITIB mice.…”