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. 2012 Nov 23;14(1):95–102. doi: 10.1038/embor.2012.186

Figure 4.

Figure 4

Identification of functional specificity determinants in TtsA. (A) Functional complementation analysis of TtsA homologues. Henle-407 cells were infected with WT S. Typhi or a ΔttsA mutant derivative expressing chromosomally encoded FLAG-tagged cdtB and carrying plasmids encoding TtsA, Sty0016 or Sen1395. Twenty-two hours post infection, infected cells were fixed and stained with a mouse monoclonal antibody directed to the FLAG epitope (to visualize CdtB), a rabbit antibody directed to S. Typhi LPS and DAPI for DNA detection. The bar represents 10 μm. (B) Western blot analysis of the expression levels of the indicated proteins in the strains used in panel (A). (C) Ability of TtsA-Sen1395 chimeras to complement a S. Typhi ΔttsA mutant for typhoid toxin secretion. Henle-407 cells were infected with wild-type (WT) S. Typhi or a ΔttsA mutant derivative expressing chromosomally encoded FLAG-tagged cdtB and carrying a plasmid encoding the indicated chimeric proteins. Twenty-two hours post infection, infected cells were fixed and stained with a mouse monoclonal antibody directed to the FLAG epitope (to visualize CdtB), a rabbit antibody directed to S. Typhi LPS and DAPI for DNA detection. The bar represents 10 μm. (D) Western blot analysis of the expression levels of the indicated proteins in the strains used in (C). (E) Schematic representation and summary of the typhoid toxin secretion phenotype of the chimeras analysed in panels (C) and (D). DAPI, 4,6-diamidino-2-phenylindole; LPS, lipopolysaccharide; WT, wild-type.