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.
Table 1.
FACS Analysis of Spleen Cells from Uninfected and Infected Mice
Table 2.
Percentage of Blue Spleen Cells Represented by Specific Cell Types from Mice Infected with Effector-Bla Fusion Expressing Salmonella Strains
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.