Role of Ca²⁺-binding sites for secretion of SiiE.

<p>A) Schematic overview of fusion proteins analyzed in this study. GLuc fused to SiiE contains BIg50–BIg53. Conserved aspartate residues exchanged to serine are shown in blue and the position of the residues is indicated in subscript. GLuc Big50-53Δ2 is a mutant form lacking two Ca²⁺-binding sites. B) GLuc assay for secretion of fusion proteins. <i>Salmonella</i> WT and Δ<i>siiF</i> strains harboring plasmids for synthesis of GLuc-SiiE fusions or empty vector were subcultured for 6 h. Samples were processed and GLuc activity determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006418#ppat.1006418.s005" target="_blank">S1 Fig</a>. Filled and open bars show total cell-associated GLuc activity (lysate) and secreted GLuc activity (supernatant), respectively. Experiments were performed in triplicates, one representative is 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

The position of type II Ca²⁺-binding sites is critical for SiiE function.

<p>The type II Ca²⁺-binding sites of 5 consecutive BIg domains (Δ5_typeII) were removed by D/S exchanges in various positions of SiiE as indicated (A). B) SiiE-dependent invasion of polarized epithelial MDCK cells by <i>Salmonella</i> strains expressing WT and mutant alleles of SiiE. Analysis of SiiE-dependent invasion of polarized cells was performed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006418#ppat.1006418.g002" target="_blank">Fig 2</a>.</p

Analysis of calcium content of SiiE_Cterm WT and BIg48-52Δ8_{type I + II} by ICP-AES.

<p>Analysis of calcium content of SiiE_Cterm WT and BIg48-52Δ8_{type I + II} by ICP-AES.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Role of Ca²⁺-binding sites in SiiE for folding, thermal stability and aggregation properties of SiiE.

<p>A) Schematic overview of the constructs containing the C-terminal region of SiiE which were used to examine the impact of D/S exchanges on the folding and stability of the protein <i>in vitro</i>. B) Circular dichroism (CD) spectra in the far UV region of SiiE_Cterm WT and mutant forms with mutation of type I, type II, or type I and type II Ca²⁺-binding sites. C) Thermal scanning CD analysis of the stability of the SiiE_Cterm variants. D) Electrophoretic motility on native PAGE of SiiE_Cterm WT and Ca²⁺-binding site mutants. E) The aggregation behavior of SiiE_Cterm WT and variant forms was investigated by incubating samples of each protein at increasing temperatures and analysis on native PAGE.</p

Effect of mutations of type I and II sites on the geometry of the BIg51-52 domain pair.

<p>Schematic presentation of the tilt and twist angles analyzed. A, B) Tilt angle between the BIg51 (green) and BIg52 (orange) domains in the crystal structure (A), and a tilted conformation observed during the MD simulations (B). The tilt angle (depicted as black line) was defined between the Cα-atoms of residues ⁶P and ⁹S of BIg51 and ⁵⁰V of BIg52. C, D) Twist angle between the BIg51 (green) and BIg52 (orange) domains in the crystal structure (C), and a twisted conformation observed during the MD simulations (D). The twist angle (depicted as black line) was defined as torsion angle between the Cα-atoms of residues ⁹S and ⁹¹I of BIg51 and ⁷E and ⁵⁰V of BIg52. E) Effect of mutation of the type I and II site on the geometry of the BIg51-52 domain pair. Structures were defined as tilted or twisted, if the respective interdomain angle deviates by more than 15 degree from the conformation present in the crystal structure. See A)-D) for the definition of the interdomain angles. All simulations were performed for BIg50-52. BIg50 has been omitted in the presentation for reasons of clarity. Ca²⁺ ions are indicated by spheres.</p

Functional dissection of type I and type II Ca²⁺-binding sites.

<p>SiiE was mutated in 5 consecutive BIg domains in the N-terminal (BIg1-5) or C-terminal (BIg47-52) region in 10 Ca²⁺-binding sites (D10), 5 type I Ca²⁺-binding sites only (Δ5_typeI), or 5 type II Ca²⁺-binding sites only (Δ5_typeII). A) Schematic overview of D/S exchanges introduced for deletion of Ca²⁺-binding sites in BIg1-5 or BIg 47–52. B) Western blot for analyses of synthesis of mutant forms of SiiE. C) Amounts of SiiE retained on the bacterial surface at 3.5 h, 6 h, 8 h and 24 h of subculture. D) Amounts of SiiE secreted in culture supernatant after 3.5 h and 6 h of subculture. E) SiiE-dependent invasion of polarized epithelial MDCK cells. Analyses of synthesis, surface retention and secretion and SiiE-dependent invasion of polarized cells were performed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006418#ppat.1006418.g002" target="_blank">Fig 2</a>.</p

Role of Ca²⁺-binding sites in N-terminal or central portions of SiiE.

<p>A) Schematic overview of various mutant forms of SiiE with D/S exchanges in BIg2, BIg40 or BIg1-5 for deletion of 2 (Δ2) or 10 (Δ10) Ca²⁺-binding sites. B) Western blot for analyses of synthesis of mutant forms of SiiE. C) Amounts of retained SiiE after 3.5 h and 6 h of subculture. D) Secreted SiiE after 3.5 h and 6 h of subculture. E) SiiE-dependent invasion of polarized epithelial MDCK cells. Analyses of synthesis, surface retention and secretion and SiiE-dependent invasion of polarized cells were performed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006418#ppat.1006418.g002" target="_blank">Fig 2</a>.</p

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

<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

Models for the roles of Ca²⁺-binding sites in giant adhesin SiiE.

<p>A) The binding of Ca²⁺ ions to BIg domains supports secretion by the T1SS. If only a few Ca²⁺-binding sites are absent, the defect is compensated by the remaining Ca²⁺-binding sites in BIg domains preceding or following the mutated region. Removal of Ca²⁺-binding sites in several consecutive BIg domains results in block of secretion. B) Role of type I and type II Ca²⁺-binding sites. Type II sites support proper conformation, while type I sites support secretion and rigidity. If type I sites are missing, secretion ceases, SiiE does not protrude the O-antigen layer, thus is unable to mediate adhesion to polarized cells. C) Role of type II Ca²⁺-binding sites in various positions of SiiE. BIg1-5 Δ type II has a functional C-terminal part, which protrudes beyond the O-antigen layer and can bind to target structures on host cells. Mutations BIg47-52 Δ type II render the C-terminal moiety of SiiE non-functional in mediating binding and subsequent invasion. D), E) Model for the effect of type II Ca²⁺-binding sites in interaction of SiiE with cognate glycostructures. The binding of Ca²⁺ ions leads to a conformation of BIg domains that enables the interaction with N-acetyl-glucosamine and/or α2,3-linked sialic acid. Multiple BIg domains may contribute to the binding, resulting in a sum of weak interactions between SiiE and glycostructures on the host cell apical membrane. The interactions are ablated upon removal of Ca²⁺ binding in BIg domains.</p