Figure 3. T3SS-mediated secretion of OrgC is required for its function.

(A and B) Removal of its first 21 amino acids (A) or N-terminal addition of MBP (B) prevents the secretion of OrgC. Whole cell lysates (w. c. l.) and culture supernatants (c. s.) of wild-type S. Typhimurium expressing, C-terminally FLAG-tagged full length OrgC or an equivalently tagged deletion mutant lacking its first 21 amino acids (N21) were analyzed by western immunoblot with a monoclonal antibody directed to the FLAG tag (A). Alternatively, whole cell lysates (w. c. l.) or culture supernatants of wild-type S. Typhimurium, or the indicated isogenic mutants carrying an empty pWSK129 plasmid or its derivatives expressing either maltose-binding protein (MBP) or MBP-OrgC fusion were analyzed by western-blot using specific antibodies directed to the MBP tag or the protein translocase SipB (as a secretion control) (B). (C) Non-secretable forms of OrgC are non-functional. The S. Typhimurium ∆invJ ∆orgC mutant strains carrying the indicated plasmids were analyzed by the clumping assay as indicated in Figure 2. Values represent OD₆₀₀ before (grey bars) and after (black bars) vortexing and are the mean ± SEM of three independent measurements. Asterisks indicate statistically significant differences from the values of the vortexed sample (*p<0.001, Student t test).