(B) Western blot was performed as above and lysates probed for SseB in wild type (wt) S. Typhimurium SL1344, ΔrpoE and in ΔrpoE complemented with pWSK29 carrying full length rpoE with endogenous promoters. Caspase Inhibitor VI clinical trial (C) Wild type S. Typhimurium SL1344 and ΔrpoE cells were immunoblotted

as above and lysates probed for SseL-2HA, SrfN-2HA and SifA-2HA which were expressed from their endogenous promoters in pWSK29. Blots were probed for DnaK as a control. The experiment was performed three times with similar results. An unmarked in-frame learn more deletion of rpoE was then generated in S. Typhimurium strain SL1344 and we verified that this in-frame deletion had the same effect on SseB as the rpoE::cat mutant used previously (Figure 1B). A low-copy plasmid selleck chemical containing full-length rpoE and the three endogenous promoters that can drive its expression [20] was able to restore wild type levels of SseB to ΔrpoE cells (Figure 1B) demonstrating that the results were specific to the rpoE deletion. In these complementation experiments, attempts were made to examine the levels of SseB secreted into the culture supernatant [21], however consistent with previous observations [22, 23] perturbations to the rpoE pathway increased cell lysis resulting in contamination of secreted fractions with cytosolic proteins which

precluded accurate interpretation (data not shown). In order to examine the effect of σE (rpoE) on the expression of a broad range of SsrB-regulated virulence genes, we tested whether or not the effect of rpoE deletion was specific to sseB or if it extended to other SsrB-regulated genes. To do this we examined the levels of SseL-2HA, SifA-2HA and SrfN-2HA expressed from their endogenous promoters under SPI-2 inducing conditions (Figure 1C). Consistent with the results for SseB, there was a decrease in SifA-2HA levels in ΔrpoE compared to wild type, although deletion PAK5 of rpoE did not have an effect on SseL-2HA. Relative to its expression in wild type cells,

the level of SrfN-2HA was reproducibly increased in the ΔrpoE cells, suggesting a role for σE in the repression of SrfN, although it is unlikely that this is through a direct mechanism. RpoE is involved in transcriptional activity of a subset of virulence genes In order to confirm the effect of σE on the expression of a broad range of SsrB-regulated virulence genes, we used wild type and ΔrpoE cells and integrated into the chromosome individually six single-copy transcriptional fusions representing promoters for four classes of SsrB-dependent genes or operons ((i) type III secretion effector operon (sseA); (ii) structural operon I (ssaB); (iii) structural operon II (ssaG); (iv and v) effectors encoded outside of SPI-2 (sseL and sifA); and (vi) integrated virulence genes unlinked to SPI-2 (srfN) [9].