Salmonella Modulation of Host Cell Gene Expression Promotes Its Intracellular Growth
Figure 5
Host cell gene expression reprogramming is required for Salmonella intracellular fitness and replication.
(A) and (B) STAT3 activity is required for the formation of S. Typhimurium-induced filaments (SIFs). Henle-407 cells treated with the STAT3 inhibitor S31-201 (100 µM) or DMSO were infected (MOI = 5) with wild-type S. Typhimurium for 1 h, chased for additional 8 h in gentamicin-containing medium, in the presence of the inhibitor or DMSO. Cells were then fixed, immuno stained for LAMP1 (red) and LPS (green) and the number of infected cells showing the presence of SIFs enumerated by epifluorescence microscopy (arrows denote the presence of SIFs). Values are the mean percentages (± SD) of infected cells showing SIF formation and represent three independent experiments in which at least 100 cells per condition were examined. *: indicates statistically significant differences (p≤0.005). (C–E) STAT3 activity is required for intracellular bacterial growth. Henle-407 cells were treated with the STAT3 inhibitor S31-201 (100 µM) or DMSO and then infected (MOI = 5) with wild-type S. Typhimurium for 1 h, chased for the indicated times in the presence of gentamicin and the inhibitor, and intracellular c.f.u. was enumerated by plating dilutions in the appropriate media (c. f. u. at time 0.5 h were as follows: DMSO treated: 7.6×105±3.2×105; S31-201 treated: 9.6×105±4.9×105 (C). Alternatively, S. Typhimurium growth was examined in HeLa cells transfected with an shRNA construct targeting STAT3 following the same procedure (c. f. u. at time 0.5 h were as follows: vector treated: 6.6×105±2.4×105; STAT3 shRNA treated: 7.3×105±1.8×105 (D). Numbers represent fold replication and are the mean (± SD) of three independent determinations (C and D). *: indicates statistically significant differences (p≤0.007). Levels of STAT3 in shRNA-targeted cells were determined by immunoblotting with the indicated antibodies (E). (F) S. Typhimurium shows increased fitness and greater replication in SerpinB3-positive (i. e. transcriptionally reprogrammed) cells. Henle-407 cells were infected (MOI = 10) for 1 h with S. Typhimurium encoding dsRed under the control of an arabinose inducible promoter, chased for 17 h in gentamicin supplemented medium, and further incubated for 3 h in the presence of 0.1% arabinose. Cells were then fixed, immuno stained for LPS, endogenous SerpinB3, and DNA, and bacteria expressing dsRed in SerpinB3-positive or negative cells were enumerated by epifluorescence microscopy. Fitness was measured by evaluating the ability of intracellular bacteria to produce dsRed protein. Values are the means (± SD) of the percentages of the bacteria showing dsRed expression after addition of arabinose in SerpinB3-positive or negative cells, and represent data from three independent experiments in which at least 100 infected cells were examined (F) (*: p≤0.007). (G) Increased Salmonella-induced filament (SIFs) formation in SerpinB3-positive (i. e. transcriptionally reprogrammed) cells. Cultured epithelial cells were infected (MOI = 10) for 1 h with S. Typhimurium, chased for 20 h in gentamicin-supplemented medium, fixed, immuno stained for LAMP1 (to stain for SIFs), LPS and SerpinB3, and analyzed by epifluorescence microscopy. Depicted are the mean percentages (± SD) of infected, SerpinB3-positive cells that show the presence (SIF+) or absence (SIF−) of Salmonella-induced filaments (SIFs) from three independent experiments in which at least 100 cells per condition were analyzed (*: p≤0.001). (H) Increased bacterial replication in SerpinB3-positive cells. Cells were infected with wild type S. Typhimurium, (MOI = 10) chased for 20 h in gentamicin supplemented medium, fixed, immuno stained for LPS (to stain for Salmonella), endogenous SerpinB3 and DNA, and the total number of bacteria in SerpinB3-positive or negative cells were enumerated by epifluorescence microscopy. Values are the means (± SD) of the percentages of SerpinB3-positive or negative cells that had a bacterial load of up to 10 bacteria or more than 10 bacteria, and represent three independent experiments in which at least 100 cells per condition were examined (*: p≤0.00002).