ZirT is arranged into a β-barrel structure in the outer membrane.

<p>A. ZirT is localized into the cell envelope. <i>S.</i> Typhimurium SL1344 expressing ZirT-HA was grown in LB to late-logarithmic phase followed by cellular fractionation as explained in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000036#s4" target="_blank">Materials and Methods</a> section. Proteins from the whole bacterial cell pellets (P), cytoplasmic fraction (C), and membranes (M) were analyzed by SDS–10% PAGE and immunoblotted with anti-HA. As a control for cytoplasmic proteins, the blot was probed with anti-DnaK. Antisera raised against the β-subunit of ATPase were used as a control for membrane proteins. B. ZirT is an outer membrane protein. Total membranes fraction (TM) was isolated from SL1344 expressing ZirT-HA by ultracentrifugation, applied to the top of a sucrose density gradient and subjected to ultracentrifugation at 100,000 <i>g</i> for 16 h. Fractions were collected from the bottom of the gradient and aliquots were separated on SDS-10% PAGE followed by Western blotting. As controls for OM and IM proteins we used polyclonal antisera raised against OmpA (two OmpA isoforms are shown) and the β-subunit of ATPase, respectively. C. ZirT is expected to adopt a compact β-barrel secondary structure. Total protein extract from whole cell lysate of <i>S.</i> Typhimurium expressing ZirT-HA was subjected to heat-modifiable electrophoretic mobility analysis. Equal protein portions were heated for 10 min at 25, 37, 42, 65, and 100°C (left panel) or boiled for 0, 1, 2 or 4 min (right panel), immediately placed on ice and separated in SDS-8% PAGE followed by Western blotting using an anti-HA antibody. The ZirT-HA bands representing the folded and the denatured forms are indicated.</p

<i>zirS</i> and <i>zirT</i> are organized in a conserved <i>Salmonella</i> genomic island.

<p>The <i>zirTS</i> corresponding region was compared between different <i>Salmonella</i> species and serovars, whose genome sequence is currently available, including: <i>S. enterica</i> serovar Typhimurium LT2; <i>S. enterica</i> serovar Choleraesuis str. SC-B67; <i>S. enterica</i> serovar Paratyphi A str. ATCC 9150; <i>S. enterica</i> serovar Typhi str. CT18; <i>S. enterica</i> serovar Enteritidis PT4 NCTC 13349; <i>S. enterica</i> serovar Gallinarum 287/91 NCTC 13346; and <i>S. bongori</i> 12419 ATCC 43975. Spotted arrows indicate possible frameshift mutations. ORF annotations (when available) and chromosomal position of the coresponding regions are indicated.</p

<i>zirT</i> is induced and displays a unique expression pattern in vivo.

<p>Two groups of female C57BL/6 mice were infected orally with ∼10⁷ cfu of wild-type <i>S.</i> Typhimurium expressing <i>rpoD::lux</i> (A) or <i>zirT::lux</i> (B) reporter strains. At day 3 p.i. mice were sacrificed and their intact GI tracts as well as systemic organs were removed and imaged immediately. The detected bioluminescence signal is shown as pseudocolor images, with variations in color representing light intensity at a given location. The color bar indicates relative signal intensity (as photons s⁻¹ cm⁻² sr⁻¹). Different organs are indicated as follow: liver (L); spleen (S); mesenteric lymph nodes (LN); colon (C); ileum (I); and jejunum (J). The experiment was repeated twice (with 9 mice in total for each reporter strain), and representative images are shown. Bacterial load is indicated by a cfu per organ in a table below each image.</p

The ZirTS pathway is negatively regulated by zinc and OxyR.

<p>A. OxyR negatively regulates the expression of <i>zirTS</i>. Wild-type <i>S.</i> Typhimurium SL1344 (wild-type) and an isogenic strain harboring a mutation in <i>oxyR</i> (<i>oxyR</i>) expressing <i>zirTS::lacZ</i> were grown in M9 minimal medium to late stationary phase. β-galactosidase assay was performed to evaluate the expression of <i>zirTS::lacZ</i> in each strain. The presented values represent the average of at least 7 independent cultures with a standard deviation shown by the error bars. B. Expression of <i>zirT</i> and <i>zirS</i> in the <i>oxyR</i> background vs. the wild-type strain as determined by q-RT-PCR. RNA was harvested from SL1344 and the <i>oxyR</i> mutant strains grown in M9; reverse-transcribed and the expression of <i>zirT</i> and <i>zirS</i> was examined by quantitative real-time PCR. The fold change in the expression of <i>zirT</i> and <i>zirS</i> in the <i>oxyR</i> background, relative to their expression in the wild-type strain is presented. Expression was normalized using the housekeeping <i>rpoD</i> gene as a control. The results represent the average of 4 independent experiments (each included 3–5 replicates) with a standard deviation shown by the error bars. C. Expression of <i>zirTS</i> in the presence of metals. <i>S.</i> Typhimurium SL1344 expressing <i>zirTS::lacZ</i> was grown in M9 medium (M9) or M9 supplemented with the following metal salts: 0.1 mM MgSO₄ (Mg), 0.1 mM FeCl₃ (Fe³⁺), 0.1 mM FeSO₄ (Fe²⁺), and ZnSO₄ (Zn) in the indicated concentrations (0.01, 0.1, or 0.5 mM). Cultures were grown to a late stationary phase and β-galactosidase assay was performed to evaluate the expression levels of <i>zirTS::lacZ</i> under each condition. The presented values represent the average of at least 8 independent cultures with a standard deviation shown by the error bars. D. Expression in the presence of metal chelators. <i>S.</i> Typhimurium SL1344 expressing <i>zirTS::lacZ</i> was grown in LB broth (LB) or LB supplemented with the following compounds: 1 mM ZnSO₄ (LB+Zn), 0.1 mM metal chelators (DTPA, EDDA, or TPEN), metals chelators with 1 mM ZnSO₄ (Zn), or metal chelators with 1 mM FeSO₄ (Fe). Cultures were grown to a late stationary phase following by β-galactosidase assay. The presented values represent the average of at least 8 independent cultures with a standard deviation shown by the error bars. E. Expression of <i>zirT</i> and <i>zirS</i> in the presence and absence of zinc and metal chelators as determined by q-RT-PCR. RNA was harvested from wild-type <i>Salmonella</i> cultures that were grown either in M9 medium in the presence or absence of 0.5 mM ZnSO₄; or in LB supplemented with or lacking DTPA (0.1 mM) or TPEN (0.05 mM). RNA was reverse-transcribed and the expression of <i>zirT</i> and <i>zirS</i> was examined by real-time PCR. The fold change in the expression of <i>zirT</i> and <i>zirS</i> in cultures that were grown in the presence of zinc (M9+Zn) or metal chelators (LB+DTPA/TPEN) relative to their expression in the appropriate unsupplemented media is presented. Expression was normalized using the housekeeping <i>rpoD</i> gene as a control. The results represent the average of 3 independent experiments (each included 3–5 replicates) with a standard deviation shown by the error bars.</p

ZirS is an exoprotein secreted in a Sec-dependent manner.

<p>A. ZirS is secreted into the extracellular environment. <i>S.</i> Typhimurium strains harboring ZirS-HA in the presence of <i>zirT</i> (pOG-zirTS-HA; ZirS) or the empty vector (pWSK29; vector) were grown in LB to late-logarithmic phase. Whole bacterial cell pellets (P), culture media supernatant (S), and the periplasmic fraction (PP) were analyzed by Western-blot using an anti-HA antibody (bottom panel). To show that the ZirS-HA detected in the supernatant is not a result of cells lysis, the same blot was reprobed for the cytoplasmic protein, DnaK (top panel). B. ZirS is secreted in a Sec translocase-dependent manner. <i>S.</i> Typhimurium harboring pOG-zirTS-HA (ZirS) or the empty vector (vector) were grown in LB to late exponential phase, washed and incubated for additional 90 or 120 min in fresh LB supplemented with (+) or lacking (−) 2 mM sodium azide. The cellular and the secreted levels of ZirS were evaluated in the whole bacterial cell pellets (P) and the secreted fractions (S) by Western-blot using an anti-HA antibody (bottom panel). As a fractionation control, the blot was probed against the β-ATPase membrane protein (top panel). Protein samples were normalized according to the optical density (O.D.₆₀₀) of the cultures and separated on SDS-13.5% PAGE.</p

Bacterial strains and plasmids used in the study.

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

ZirS modulates <i>Salmonella</i> virulence during systemic disease in mice.

<p>The geometrical means of competitive index values are shown for the spleen and liver of mice that were infected orally (panels A–C) or i.p. (panel D). Six to seven weeks old 129X1/SvJ (panels A and D), <i>Nramp1</i>^+/+ (panel B), or <i>Nramp1</i>^−/− (panel C) female mice were infected with a 1:1 mixed inoculum of marked wild-type SL1344 strain and a Δ<i>zirS</i> mutant. For oral infection, mice were inoculated with 1×10⁶ cfu in 0.1 ml of infection buffer (0.1 M HEPES pH 8.0, 0.9% NaCl) and sacrificed after 6 days. For i.p. infection, 2×10⁴ cfu were injected in 0.2 ml PBS and mice were sacrificed after 3 days. Panels A and D represent pooled results from 2 independent experiments.</p

List of primers used in the study.

<p>List of primers used in the study.</p

ZirS and ZirT compose an autonomous secretion system in <i>Salmonella.</i>

<p>A. The secretion of ZirS is ZirT-dependent. Wild-type <i>S.</i> Typhimurium (wild-type) or its isogenic strains harboring a deletion mutation in <i>zirS</i> (Δ<i>zirS</i>) or <i>zirT</i> (Δ<i>zirT</i>) expressing ZirS-HA only (pOG-zirS-HA; ZirS) or both ZirT and ZirS-HA (pOG-zirTS-HA; ZirS+ZirT) were grown in LB to late-logarithmic phase. To evaluate the expression and the secretion of ZirS-HA, whole bacterial cell pellets (P) and culture media supernatant (S) were analyzed by immunoblot using antibodies against hemagglutinin (bottom panel). To show that the ZirS-HA detected in the supernatant is not a result of bacterial cell lysis and that equal amounts of protein were loaded, the same blot was reprobed for the bacterial cytoplasmic protein, DnaK (top panel). B. Complementation of ZirS secretion in the presence of ZirT in <i>cis</i> and <i>trans</i>. The secretion of ZirS-HA was analyzed in wild-type <i>S.</i> Typhimurium (wild-type) or in a <i>zirT</i> mutant strain (Δ<i>zirT</i>) expressing ZirT and ZirS-HA either in <i>cis</i> from a single low copy number construct (pOG-zirTS-HA; ZirS+ZirT) or in <i>trans</i>, from two different, but similar-copy numbered vectors (pOG-zirS-HA and pOG-zirT-4; V₁ZirS+V₂ZirT). Whole bacterial cell pellets (P) and the secreted fractions (S) were analyzed by Western-blot using antibodies against HA (bottom panel) or DnaK (top panel). C. Expression of ZirS and ZirT in a heterologous host leads to the secretion of ZirS to the extracellular milieu. <i>E. coli</i> DH5α harboring an empty vector (vector), ZirS-HA only (pOG-zirS-HA; ZirS), or ZirS-HA and ZirT (pOG-zirTS-HA; ZirS+ZirT) were grown in LB. Whole bacterial cell pellets (P) and the secreted fractions (S) were analyzed by Western-blot as outlined above.</p

The expression of <i>zirT</i> is induced in shed <i>Salmonella</i> within fecal pellets during persistent and acute infection.

<p><i>S.</i> Typhimurium expressing <i>zirT::lux</i> was used to infect 129X1/SvJ and C57BL/6 mice as explained above. (A) Fresh fecal pellets were collected from 129X1/SvJ mice with persistent <i>Salmonella</i> infection and imaged immediately. Bioluminescence of fecal pellets is shown for 1, 28, 42, and 56 days p.i.. An asterisk at day 42 p.i. indicates low levels of <i>Salmonella</i> shedding in the collected stool as was determined by cfu count. (B) In vivo imaging of 129X1/SvJ mouse, 1 day p.i. The induced expression of <i>zirT::lux</i> in the stool is shown. (C) Bioluminescence of fecal pellets from C57BL/6 mice developing an acute <i>Salmonella</i> infection is shown for day 4 p.i. Fresh pellets were harvested directly from the animal prior to imaging and dried stools were collected from the cage.</p