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predicted domains in SseB or SseD are required for the function as translocon subunit, while secretion by the SPI2-T3SS can still take place after deletion of various protein domains. Results Deletional analyses of translocon proteins SseB and SseD Based on the previous observation that SseB, SseC and SseD are required for the translocation of effector proteins by LY3023414 ic50 intracellular Salmonella [7], we started deletional analyses for the identification of functionally essential domains of the proteins. Here we focused on SseB and SseD. Since SseB and SseD are most likely membrane-associated or integral proteins with hydrophobic character, the analysis of the hydrophobicity was a main consideration for the positions of deletions. In addition, coiled-coil domains are

commonly found BMN 673 purchase in substrate proteins of T3SS and LCZ696 cell line have been shown as required for protein-protein interactions. The location of predicted coiled-coil domains in the sequence of SseB and SseD was also considered for the design of mutations. The hydropathy plots, predictions of coiled-coil domains and the positions of deletions are displayed in Fig. 1A. Briefly, SseBΔN1 lacked the N-terminal aa residues 2-14 and SseBΔ1 the N-terminal residues 15-30. SseBΔ2 was deleted for a hydrophobic region predicted as transmembrane region (aa 38-57), SseBΔ3 lacked the region containing coiled-coil domains (aa 58-90) and SseBΔ4 lacked both regions (aa 38-90). Constructs SseBΔ5 and SseBΔ6 were deleted for aa 91-115 or aa 116-136, respectively,

both regions were without specific functional or structural predictions. SseΔ7 was deleted for the putative chaperone binding site, i.e. aa 137-182. Finally, SseBΔC1 was deleted for the C-terminal region of aa 183-196. Figure 1 Bioinformatic analyses of SPI2 translocon protein SseB and characteristics of deletion variants of SseB. A) Using the program TMpred, putative transmembrane (TM) domains of the translocon protein SseB was predicted. o-i indicate the strongly preferred model, with N-terminus outside (aa 38-57), i-o indicates the alternative model. B) Using the program COILS, coiled-coil regions in SseB were predicted. As output option the default Sunitinib clinical trial parameters were selected that gave residue number, residue type and the frame and coiled-coil forming probability obtained in scanning windows of 14, 21 and 28 residues (as described on the Swiss EMBnet homepage). The region spanning aa 58-90 was considered as coiled-coil domain. C) Schematic representation of the amino acid sequence of wild-type SseB and positions of deletions analyzed in this study. The predicted TM domain, coiled-coil region, as well as the chaperone-binding site [10] are indicated. The deleted regions within sseB variants are indicated by arrows and C- or N-terminal truncations are indicated by vertical red lines.