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 beta-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.

Figure 1. Visualization of Secretion in HeLa Cells (A) and J774s (B) by Microscopy (60× Magnification)

S. typhimurium 14028 strains expressing Bla fusions to SipA, SptP, SlrP, SteA, SteC, and SseJ; WT 14028, or 14028 harboring pWKS30 were used to infect HeLa cells for 2 hours using SPI-1-inducing conditions and J774s for 10 hours using SPI-2- inducing conditions. Following the infections, cells were loaded with CCF2-AM and visualized for green and blue fluorescence by microscopy. Green fluorescence indicates CCF2-AM was loaded and the presence of blue cells is evidence of secretion. SPI-1 TTSS- and SPI-2 TTSS-dependent secretion detected by FACS analysis (C). HeLa cells and J774s were infected as described above with the six effector-Bla fusions that were expressed in WT 14028, invA::cat and ssaK::cat backgrounds, mutations in structural components of SPI-1 and SPI-2 TTSS, respectively. FACS analysis was performed on CCF2-AM loaded cells to determine the percentage of blue cells (positive for secretion). Background percentage of blue cells was set using WT 14028–infected cells. At least 10,000 cells were analyzed for each sample. Each bar represents the mean percentage of blue cells from triplicate samples and the error bars are ± one standard error of the mean. FACS analysis was also performed on HeLa cells infected with each Bla fusion strain for 1, 8, or 20 hours using SPI-1 inducing conditions (D).

Figure 2. Microscopic Analysis of a Blue Spleen Cell Containing Intracellular Salmonella

Mice were infected i.p. for 48 hours with 14028 expressing a chromosomal SteA-Bla construct and harboring pWKS30-Tomato, a red fluorescent protein expression vector (shown in red). Spleen cell suspensions were prepared then loaded with CCF2-AM (blue) to detect secretion and a DNA stain, DRAQ5 (green), to visualize nuclei. The image was taken at 60× magnification.

Figure 3. Detection of Secretion in C57BL/6 Spleen Cells

FACS data with levels of green and blue CCF2-AM fluorescence in spleen cells from mice infected with each fusion strain (A). Green fluorescence indicates CCF2-AM is present within the cells. The percentage of blue cells, positive for secretion, is shown in the lower right corner of each dot plot. The graph below shows the percentage of total spleen cells emitting blue fluorescence as detected by FACS analysis (B). Each X represents the value for one mouse infected with the indicated strain and horizontal bars represent the average value of seven mice. The * denotes samples for which the Student's t-test returned a value where p < 0.05 when compared to 14028-infected mice.

Figure 4. GR-1+/CD11b+ Cells Consist of Neutrophils and Monocytes

FACS analysis was performed on spleen cells from uninfected C57BL/6 mice or mice infected with 14028 for 2 days (A). The level of GR-1 and CD11b in total viable spleen cells is shown in the density plots on the left. GR-1+/CD11b+ cells (R1 gate) were analyzed for CD11c and GR-1 expression levels in the density plots to the right. The level of F4/80 expression for ungated cells (all analyzed cells), R2 and R3 gated cells are shown in the histograms (B). GR-1 Hi/CD11c Lo (R2) cells and GR-1 Int/CD11c Hi (R3) cells from infected mice were FACS sorted, then cytospun onto slides, and stained with Wright-Giemsa stain and visualized by microscopy (C). 300 cells from Wright-Giemsa stained slides were analyzed and the percentage of neutrophils and monocytes was determined. The percentage of total intracellular Salmonella in GR-1+/CD11b+ cells was estimated (D). GR-1+/CD11b+ cells (R1), GR-1 Hi / CD11c Lo (R2), GR-1 Int / CD11c Hi (R3) populations were FACS sorted, lysed, then plated on LB to determine the number of intracellular CFU. The percentage of total recovered CFU was then calculated for each FACS sorted population (see Materials and Methods). The graph shows the average percentage total recovered CFU present in each FACS sorted population from three mice and the error bars represent one standard error of the mean.

Figure 5. Microscopic Analysis Reveals That FACS Sorted Blue Spleen Cells Contain Intracellular Red-Fluorescent Salmonella

Mice were infected as described in Figure 2. Spleen cell suspensions were prepared then loaded with CCF2-AM to detect secretion (shown in blue). FACS sorted secretion positive cells (blue cells) were then stained with a DNA stain, DRAQ5 (shown in green), to visualize nuclei. Images were taken at 60× magnification and a single cell is shown in each horizontal row. Red reference bars represent 2 μm.

Figure 6. CD3+ and CD19+ Cells Contain Salmonella

Blue cells that are CD3+ or CD19+ were FACS sorted and visualized for the presence of intracellular red-fluorescent Salmonella using fluorescence microscopy. Experiments were performed as described in Figure 5 and red reference bars represent 2 μm.