The PhoP from a <i>S. bongori</i> SARC 12 strain is impaired in regulating the <i>phoPQ</i> regulon.

<p><i>S.</i> Typhimurium (ST), <i>S.</i> Typhimurium <i>phoP</i> (ST phoP), <i>S. bongori phoP</i> (SB phoP) and <i>S. bongori phoP</i> complemented with a <i>S. bongori phoPQ</i> (pPhoP_SB), <i>S.</i> Typhimurium <i>phoPQ</i> (pPhoP_ST), or the <i>S. bongori phoPQ</i> harboring V169 (pPhoP_SB169V) expressing <i>ybjX::lacZ</i> (A), <i>mig-5::lacZ</i> (B) or <i>pagO::lacZ</i> (C) were examined for their β-galactosidase activity following growth under inducing conditions. The means with a standard error shown by the error bars are presented.</p

Bacterial strains and plasmids used in the study.

<p>*Sm, streptomycin; Cm, chloramphenicol; Kan, kanamycin.</p

The PhoP regulator from <i>S. enterica</i> and a <i>S. bongori</i> SARC 12 strain are functionally different.

<p>(A) <i>S.</i> Typhimurium (ST), <i>E. coli</i> MC1061 (EC), <i>S. bongori</i> SARC 12 (SB) harboring <i>sseL::lacZ</i> in the presence or absence of SsrAB (pSsrAB) were tested for their β-galactosidase activities following growth in LPM medium. (B) <i>S. bongori</i> SARC 12 (SB), <i>S. bongori phoP</i> (SB phoP), and <i>S. bongori phoP</i> strain complemented with a <i>S. bongori phoPQ</i> (pPhoP_SB), <i>S.</i> Typhimurium <i>phoPQ</i> (pPhoP_ST), or the <i>S. bongori phoPQ</i> harboring V169 (pPhoP_SB169V) all carrying <i>sseL::lacZ</i> were tested for their β-galactosidase activity following growth under inducing conditions. (C) <i>sseL::lacZ</i> expression is shown in a <i>S.</i> Typhimurium <i>phoP ssrB</i> background without complementation (<i>phoP ssrB</i>), in the presence of the <i>S. bongori</i> PhoP (<i>phoP ssrB</i>+pPhoP_SB) or in the presence of the <i>S.</i> Typhimurium PhoP (<i>phoP ssrB</i>+pPhoP_ST). (D) Amino acid alignment of PhoP sequences from <i>S. bongori</i> SARC 12 (SB), <i>S.</i> Typhimurium SL1344 (ST), and <i>E. coli</i> (EC; P23836) is shown. Identical, similar and disparate amino acids are shown in black, grey, and white, respectively. Secondary structural elements (α-helices in white and β-strands in grey) within the C-terminus domain were predicted using the SOPMA program and are correlated with the <i>E. coli</i> OmpRc structure. Amino acid variation between <i>S. bongori</i> and <i>S.</i> Typhimurium at position 169 is indicated by the black arrow.</p

<i>sseL::lacZ</i> is induced in response to low phosphate, low magnesium and acidic pH environmental cues.

<p><i>S.</i> Typhimurium strains carrying <i>sseL:lacZ</i> were grown for 16 h at 37°C in LB, LPM (pH 7.4) LPM (pH 5.8) supplemented with 10 mM MgCl₂, and LPM (pH 5.8), and were assayed for β-galactosidase activity presented in Miller units (M.U.). Basal <i>lacZ</i> expression of <i>S.</i> Typhimurium harboring pMC1403 (vector) that was grown in LPM (pH 5.8) is also shown.</p

His-PhoP binds to the promoter regions of <i>sseL</i>.

<p>(A) 5 µg of the purified His-tagged PhoP protein was analyzed on an SDS-PAGE by Coomassie Blue staining to assess its purity. Molecular weight markers (kDa) are shown on the left. (B) Electrophoretic mobility shift assay analysis of <i>sseL</i> promoter. A 128-bp DNA fragment containing the putative PhoP binding sites of the <i>sseL</i> promoter was Dig-labeled. 15 fmol of the labeled probe was incubated at 37°C in a binding buffer containing a 100-fold excess of dI-dC as a nonspecific DNA competitor, in the presence of increasing amounts of phosphorylated His-PhoP protein (0, 12.5, 25, 50, 100, 200, 400 pmol). The PhoP-DNA mixtures were subjected to a 6% native polyacrylamide gel electrophoresis and imaged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020024#s4" target="_blank">Materials and Methods</a>.</p

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Heterologous expression of Ipf and Klf in surrogate <i>E</i>. <i>coli</i> cells.

<p>Non-fimbriated <i>E</i>. <i>coli</i> strain ORN172 carrying the entire <i>ipf</i> operon (pBAD18::<i>ipf</i>, lanes 1 and 2), the <i>klf</i> operon (pBAD18::<i>klf</i>, lanes 4 and 5), or the empty vector (pBAD18, lane 3) that was used as a negative control was grown in N-minimal medium supplemented with 50 mM L-arabinose (inducing conditions) or 1 M glucose (suppressing conditions). Cultures supernatants that were enriched with surface structures were collected after a shearing treatment, subjected to TCA precipitation and separated on a 12% SDS-PAGE. Arrows show the bands that were isolated from the gel and identified by LC-MS/MS. The proteins that were detected by LC-MS/MS are summarized in the table below. The score value presents the cumulative protein score based on summing the ion scores of the unique peptides identified for that protein. Coverage displays the percentage of the protein sequence covered by the identified peptides. PSMs show the total number of identified peptide sequences (peptide spectrum matches) for the protein, including those redundantly identified. The area displays the average area of the three unique peptides with the largest peak area.</p

<i>klf</i> and <i>ipf</i> are significantly expressed in the cecum of infected chicks.

<p>Two groups of one day old White Leghorns chicks were infected orally with ∼1×10⁷ CFU of wild-type <i>S</i>. Infantis harboring the <i>ipf</i>::<i>lux</i> (<b>A, C</b>) and <i>klf</i>::<i>lux</i> (<b>B, D</b>) reporter strains. Twenty four hours p.i. chicks were sacrificed and their intact GI tracts as well as their liver were removed and imaged immediately using a photon-counting <i>in-vivo</i> imaging system. (<b>A</b> and <b>B)</b> Bacterial loads in the duodenum, jejunum and ileum, cecum, colon and liver is indicated by a CFU count per organ. The geometrical mean in each organ is shown by a solid horizontal line. <b>(C</b> and <b>D</b>) The color bar indicates relative signal intensity and the minimal and maximal values measured are shown in the box below the color bar. Different organs are designated as follow: duodenum (D) jejunum (J); ileum (I); cecum (CE); colon (C) and liver (L).</p

The plasmid-encoded Ipf and Klf fimbriae display different expression and varying roles in the virulence of <i>Salmonella enterica</i> serovar Infantis in mouse vs. avian hosts

<div><p><i>Salmonella enterica</i> serovar Infantis is one of the prevalent <i>Salmonella</i> serovars worldwide. Different emergent clones of <i>S</i>. Infantis were shown to acquire the pESI virulence-resistance megaplasmid affecting its ecology and pathogenicity. Here, we studied two previously uncharacterized pESI-encoded chaperone-usher fimbriae, named Ipf and Klf. While Ipf homologs are rare and were found only in <i>S</i>. <i>enterica</i> subspecies diarizonae and subspecies VII, Klf is related to the known K88-Fae fimbria and <i>klf</i> clusters were identified in seven <i>S</i>. <i>enterica</i> subspecies I serovars, harboring interchanging alleles of the fimbria major subunit, KlfG. Regulation studies showed that the <i>klf</i> genes expression is negatively and positively controlled by the pESI-encoded regulators KlfL and KlfB, respectively, and are activated by the ancestral leucine-responsive regulator (Lrp). <i>ipf</i> genes are negatively regulated by Fur and activated by OmpR. Furthermore, induced expression of both <i>klf</i> and <i>ipf</i> clusters occurs under microaerobic conditions and at 41°C compared to 37°C, <i>in-vitro</i>. Consistent with these results, we demonstrate higher expression of <i>ipf</i> and <i>klf</i> in chicks compared to mice, characterized by physiological temperature of 41.2°C and 37°C, respectively. Interestingly, while Klf was dispensable for <i>S</i>. Infantis colonization in the mouse, Ipf was required for maximal colonization in the murine ileum. In contrast to these phenotypes in mice, both Klf and Ipf contributed to a restrained infection in chicks, where the absence of these fimbriae has led to moderately higher bacterial burden in the avian host. Taken together, these data suggest that physiological differences between host species, such as the body temperature, can confer differences in fimbriome expression, affecting <i>Salmonella</i> colonization and other host-pathogen interplays.</p></div