Salmonella Modulation of Host Cell Gene Expression Promotes Its Intracellular Growth
Figure 2
Salmonella stimulation of transcriptional responses in infected cells requires STAT3.
(A) Interaction map of genes whose expression is stimulated by S. Typhimurium infection of Henle-407 cells. Shown is the interaction of genes whose expression increased at least 3 fold at 10 or 20 h after infection. The analysis was carried out with STRING 9.0 (http://string-db.org/) using the highest confidence (0.9) parameters. (B–E) S. Typhimurium induces STAT3 activation. Henle-407 cells were infected (MOI = 10) with wild-type S. Typhimurium for 1 h. Following chase in gentamicin containing medium, cells were lysed at the indicated times, separated by SDS-PAGE and probed by immuno blotting with antibodies to the phosphorylated (activated) form of STAT3 (P-Y705), unphosphorylated STAT3 (to detect total amount), and actin (loading control) (B). Fold activation of STAT3 (relative to uninfected cells) was quantified by scanning the blots with a LI-COR Odyssey imaging system standardizing the signals with the actin loading control. Values are the means (± SD) of three independent experiments (C). Alternatively, Henle-407 cells were infected (MOI = 5) for 1 h with wild-type S. Typhimurium, chased for 8 h in gentamicin supplemented medium, fixed, immuno stained for LPS (red), DNA (blue) and phosphorylated STAT3 (green), as indicated. Samples were then analyzed by epifluorescence microscopy (bar represents 10 µm) (D). The number of infected cells showing staining of phosphorylated STAT3 was determined and the values represent the mean (± SD) of three independent experiments in which at least 100 cells were quantified. *: indicates values that are statistically significantly different from uninfected controls (p≤0.01) (E). (F) STAT3 is required for the transcriptional responses to S. Typhimurium infection. Henle-407 cells treated with the STAT3 inhibitor S31-201 were infected (MOI = 10) with S. Typhimurium for 1 h, and chased for additional 3 h in gentamicin containing medium in the presence of DMSO or 100 µM S3I-201. mRNA levels of selected genes whose expression is induced by S. Typhimurium infection was analyzed by qRT-PCR. Values represent the mean (± SEM) of GAPDH normalized transcript levels of the indicated genes, relative to DMSO treated uninfected cells. *: indicates statistically significant differences (p≤0.0004). (n. i.: non infected).