Role of Ca²⁺-binding sites in the C-terminal moiety for secretion and function of chromosomally encoded SiiE.

Abstract

<p>A) Schematic overview of the domain organization of SiiE and chromosomally encoded forms of SiiE with mutated Ca²⁺-binding sites. The positions of deleted Ca²⁺-binding sites is shown in blue and Δ1, Δ2, Δ4 and Δ10 indicate 1, 2, 4, or 10 mutated Ca²⁺-binding sites, respectively. Mutant alleles were generated using the λ Red-mediated replacement of I-<i>Sce</i>I <i>aph</i> cassette by DNA fragments harboring sequence alterations. B) Western blot detection of SiiE in whole bacterial lysates of a 3.5 h subculture for analysis of protein synthesis. C) The amounts of SiiE retained on the bacterial surface of <i>Salmonella</i> WT and various mutant strains at 3.5 h and 6 h subculture was determined by dot blot analysis. Representative dot blots are shown. The loading of dot blots was normalized by parallel analysis of the signal for LPS. D) The amounts of secreted SiiE for <i>Salmonella</i> WT and various mutant strains at 3.5 h and 6 h subculture were determined by dot blot analysis. After TCA precipitation, the pellet was resuspended according to OD₆₀₀ of the culture and equal amounts of samples were loaded onto a nitrocellulose membrane. E) Comparison of activities of secreted GLuc-SiiE reporters obtained for plasmid-encoded GLuc conjugated SiiE by GLuc assay (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006418#ppat.1006418.s005" target="_blank">S1 Fig</a>) or chromosomally encoded AA exchanges by dot blot analysis depicted in D). F) SiiE-dependent invasion of polarized epithelial MDCK cells by <i>Salmonella</i> WT, Δ<i>siiF</i>, and strains expressing various mutant alleles of <i>siiE</i>. Cells were infected with the indicated strains at a MOI of 5. Non-internalized bacteria were removed by washing and remaining bacteria were killed by addition of gentamicin for 1 h. Subsequently, cells were lysed and serial dilutions were plated onto agar plates for determination of colony-forming units (CFU). Invasion is depicted as percentage of the inoculum that was internalized by host cells. Experiments were performed in duplicates (C, D) or triplicates (E, F), and means and standard deviations are shown. Statistical analysis was performed by one-way ANOVA with Bonferroni t-test and is indicated as follows: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.001.</p