8 to induce SPI2 expression as well as protein secretion by the SPI2-T3SS. For analyses of protein synthesis, equal amounts of bacterial cells as adjusted by OD600 were harvested and resuspended in SDS-PAGE sample buffer (T, total cell fraction). Secreted protein bound to the bacterial surface was released by mechanical shearing and precipitated from bacteria-free supernatant (D, detached fraction) and secreted proteins in the supernatant were precipitated by addition of LOXO-101 nmr 10% TCA (final concentration).

For Western blot analysis, samples corresponding to equal amount of bacteria or supernatant were separated by SDS-PAGE and transferred to nitrocellulose membranes and protein was detected with antiserum raised against SseB. As loading control and control for cell lysis, the bacterial heat shock protein DnaK was detected. The position of SseB and various mutant forms of SseB was indicated by asterisks. The quantification of the signal intensities is shown in Additional Combretastatin A4 research buy file 1. We then investigated the effect of the various deletions in SseB on the secretion of SseC and SseD and the partitioning of secreted

SseC and SseD between the soluble and cell-bound fractions. For unknown reasons, larger amounts of DnaK were observed in the detached fraction of the sseB strain, but the mutation per se did not affect cell integrity since the complemented strains did not show increased release of cytosolic protein. In accordance with our previous report [7] we observed that the majority of SseD secreted by WT Salmonella is present in the detached fraction (Fig. 3). Strains Torin 1 supplier expressing sseBΔ5, sseBΔ6 and, to a certain extend sseBΔ3 resulted in reduced amounts of

the secreted SseD in the supernatant fraction. The expression of the other deletion alleles of sseB resulted in the presence of secreted SseD in the culture supernatant as well as in the detached fraction (Fig. 3). Deletions in sseBΔ5, sseBΔ6 affect the binding site for SseA that acts as chaperone for SseB and SseD [9]. The altered partitioning observed for strains expressing these alleles may be due to the altered binding of chaperone SseA to its targets and altered Ergoloid stability of these proteins. The partitioning of SseD in the complemented sseB strain was different from that observed for the WT strain and most of SseD was present in the total cell fraction rather than in the detached fraction. This may be due to the over expression of sseB in the sseB [psseB] strain leading to more secretion of SseB in the supernatant and reduces the binding of SseB to the surface (compare Fig. 2). Therefore, the binding of SseD to the surface would be reduced and the release of SseD in the supernatant is increased. Most of the mutant alleles of sseB also resulted in higher amounts secreted SseD in the supernatant. Figure 3 Effect of various deletions in sseB on secretion and partitioning of SseD in vitro. S.