Figure 1.
SPI-2 T3SS mutants can grow to high intracellular densities in CD18+ cells in vivo.
(A) Model of the SPI-2 T3SS. SseB forms part of the translocon; SsaV is located in the inner membrane. (B–E) Representative fluorescence micrographs of Salmonella within phagocytes in infected livers of C57BL/6 mice at 72 h p.i. (B) S12023; (C) S12023 ssaV; (D) S12023 sseB; (E) S12023 sseB(psseB) [CD18+ cells (red), Salmonella (green), nucleic acid is stained with DAPI (blue), scale bars, 5 µm]. (F and G) C57BL/6 mice were infected i.v. with ∼Log10 4.3 CFU ( = 2.15×104 CFU) of S12023, ∼Log10 6.4 CFU ( = 2.45×106 CFU) of S12023 sseB, or ∼Log10 4.1 CFU ( = 1.27×104 CFU) of S12023 sseB(psseB). (F) Net bacterial numbers in livers (unbroken lines) and spleens (dotted lines) were determined between 6 to 72 h p.i. from 4 mice per strain per time point (results are expressed as mean Log10 viable count ± standard deviation). (G) The proportion of infected phagocytes relative to the number of bacteria contained within each phagocyte is shown for livers (solid bars) and spleens (diagonal shading) at 72 h p.i. based on the counts obtained from 400 infected phagocytes per strain, from tissue obtained from 4 mice per strain [S12023 - red; S12023 sseB - blue; S12023 sseB(psseB) – green].
Figure 2.
SPI-2 T3SS mutants are present in far fewer infected foci per organ than the wild-type.
(A to C) Representative fluorescence micrographs of Salmonella within phagocytes in infected livers of C57BL/6 mice at 72 h p.i.. (A) S12023; (B) S12023 sseB; (C) S12023 sseB(psseB) [CD18+ cells (red), Salmonella (green), nucleic acid is indicated by DAPI (blue), scale bars, 75 µm]. (D and E) Box and whisker plots showing the median, interquartile range and maximum and minimum number of infected cells per field-of-view in spleens for (D) S12023, S12023 sseB and S12023 sseB p(sseB) at 72 h p.i., obtained from 100 random fields from 4 mice for S12023 and S12023 sseB(psseB) infected tissue and 200 random fields from 7 mice for S12023 sseB infected tissue, and (E) S12023 sseB at 0.5 and 72 h p.i., obtained from 100 random fields from 3 mice for S12023 sseB infected tissue at 0.5 h p.i., and 200 random fields from 7 mice for S12023 sseB infected tissue at 72 h p.i.
Figure 3.
Accumulation of SPI-2 T3SS mutants inside cells is due to clonal expansion of a bacterium.
(A) C57BL/6 mice were infected i.v. with ∼Log10 6.4 CFU ( = 2.44×106 CFU) of Salmonella S12023, ∼Log10 6.5 CFU ( = 2.86×106 CFU) of S12023 sseB, or ∼Log10 6.3 CFU ( = 2.17×106 CFU) of S12023 sseB(psseB). The proportion of infected phagocytes relative to the number of bacteria contained within each phagocyte is shown for livers (solid bars) and spleens (diagonal shading) at 0.5 h p.i. based on the counts obtained from 150 infected phagocytes per strain, from tissue obtained from 3 mice per strain [S12023 - red; S12023 sseB - blue; S12023 sseB(psseB) – green]. (B) C57BL/6 mice were infected i.v. with ∼Log10 6.4 CFU ( = 2.64×106 CFU) of S12023 sseB. The proportion of infected phagocytes relative to the numbers of bacteria contained within each phagocyte is shown for livers (solid bars) and spleens (diagonal shading) at different time points between 0.5 and 72 h p.i. inclusive, based on the counts obtained from 100 infected phagocytes per organ, per time point, from tissue obtained from 4 mice per time point. (C) C57BL/6 mice were infected i.v. with ∼Log10 6.3 CFU ( = 1.99×106 CFU) of SL5559 sseB and SL5560 sseB bacteria into the same animal via a single injection. Representative fluorescence micrograph of Salmonella SL5559 sseB and SL5560 sseB within phagocytes in an infected spleen at 72 h p.i. [SL5559 sseB (green), SL5560 sseB (red), nucleic acid is stained with DAPI (blue). Scale bar, 75 µm].
Figure 4.
Similar net growth kinetics can be the result of very different intracellular infection dynamics.
C57BL/6 mice were infected i.v. with ∼Log10 6.2 CFU ( = 1.56×106 CFU) of S12023, ∼Log10 6.4 CFU ( = 2.64×106 CFU) of S12023 sseB, ∼Log10 6 CFU ( = 1.10×106 CFU) of S12023 aroA, ∼Log10 7.2 CFU ( = 1.69×107 CFU) of S12023 purA, ∼Log10 6.1 CFU ( = 1.37×106 CFU) of S12023 sseB aroA, or ∼Log10 6.9 CFU ( = 7.93×106 CFU) of S12023 sseB purA. (A) Net bacterial numbers in livers (unbroken lines) and spleens (dotted lines) were determined between 0.5 and 72 h p.i. inclusive. The net bacterial growth of S12023 sseB was obtained from 4 mice per time point, S12023 (high input dose) was obtained from 3 mice per time point at 0.5, 6 and 24 h p.i. and 6 mice at the 48 h p.i. time point, S12023 aroA, S12023 purA, S12023 sseB aroA and S12023 sseB purA was obtained from 3 mice per time point (results are expressed as mean Log10 viable count ± standard deviation) [S12023 - red; S12023 sseB - blue; S12023 aroA - purple; S12023 purA – orange; S12023 sseB aroA – light blue; S12023 sseB purA - grey]. (B–F) The proportion of infected phagocytes relative to the number of bacteria contained within each phagocyte is shown for livers (solid bars) and spleens (diagonal shading) at (B) 0.5 h p.i.; (C) 6 h p.i.; (D) 24 h p.i.; (E) 48 h p.i. and (F) 72 h p.i.. The intracellular bacterial distributions of each strain were based on the counts obtained from 100 infected phagocytes per organ, per time point, from tissue obtained from 3 mice per time point. (G and H) Box and whisker plots showing the median, interquartile range and maximum and minimum number of infected cells per field-of-view in spleens at 0.5 h p.i. and 72 h p.i. for (G) S12023 aroA and S12023 sseB aroA, and (H) S12023 purA and S12023 sseB purA. The number of infected cells per field of view was obtained from 100 random fields from 3 mice for each strain at each time point.
Figure 5.
Phagocyte NADPH oxidase inhibits Salmonella spread in the tissues in the absence of SPI-2 T3SS.
gp91phox−/− mice were infected i.v. with ∼Log10 4.2 CFU ( = 1.45×104 CFU) of S12023 or ∼Log10 4.1 CFU ( = 1.21×104 CFU) of S12023 sseB, C57BL/6 mice were infected i.v. with ∼Log10 4.3 CFU ( = 2.15×104 CFU) of S12023 or ∼Log10 6.4 CFU ( = 2.45×106 CFU) of S12023 sseB. (A) Net bacterial numbers in livers (unbroken lines) and spleens (dotted lines) were determined between 6 and 72 h p.i. inclusive (results are expressed as mean Log10 viable count ± standard deviation), the net bacterial growth of S12023 and S12023 sseB in gp91phox−/− mice was obtained from 3 mice per time point (the data for S12023 and S12023 sseB in C57BL/6 mice is reproduced from Figure 1F. S12023 in C57BL/6 mice – red; S12023 in gp91phox−/− mice – pink; S12023 sseB in C57BL/6 mice – dark blue; S12023 sseB in gp91phox−/− mice – light blue). (B) The proportion of infected phagocytes relative to the numbers of bacteria contained within each phagocyte for S12023 sseB in gp91phox−/− mice and S12023 sseB in C57BL/6 mice at 48 h p.i., based on the counts obtained from 100 infected phagocytes per organ, from tissue obtained from 3 mice per time point. (Livers – fully shaded; Spleens – diagonal shading). (C) Box and whisker plot showing the median, interquartile range and maximum and minimum number of infected cells per field-of-view for S12023 sseB in C57BL/6 mice and S12023 sseB in gp91phox−/− mice at 48 h p.i., from 100 random fields from 3 mice for each strain.