Salmonella enterica relies on carbon metabolism to adapt to agricultural environments

Salmonella enterica, a foodborne and human pathogen, is a constant threat to human health. Agricultural environments, for example, soil and plants, can be ecological niches and vectors for Salmonella transmission. Salmonella persistence in such environments increases the risk for consumers. Therefore, it is necessary to investigate the mechanisms used by Salmonella to adapt to agricultural environments. We assessed the adaptation strategy of S. enterica serovar Typhimurium strain 14028s to agricultural-relevant situations by analyzing the abundance of intermediates in glycolysis and the tricarboxylic acid pathway in tested environments (diluvial sand soil suspension and leaf-based media from tomato and lettuce), as well as in bacterial cells grown in such conditions. By reanalyzing the transcriptome data of Salmonella grown in those environments and using an independent RT-qPCR approach for verification, several genes were identified as important for persistence in root or leaf tissues, including the pyruvate dehydrogenase subunit E1 encoding gene aceE. In vivo persistence assay in tomato leaves confirmed the crucial role of aceE. A mutant in another tomato leaf persistence-related gene, aceB, encoding malate synthase A, displayed opposite persistence features. By comparing the metabolites and gene expression of the wild-type strain and its aceB mutant, fumarate accumulation was discovered as a potential way to replenish the effects of the aceB mutation. Our research interprets the mechanism of S. enterica adaptation to agriculture by adapting its carbon metabolism to the carbon sources available in the environment. These insights may assist in the development of strategies aimed at diminishing Salmonella persistence in food production systems

Interresidual Distance Determination by Four-Pulse Double Electron-Electron Resonance in an Integral Membrane Protein: The Na(+)/Proline Transporter PutP of Escherichia coli

Proximity relationships within three doubly spin-labeled variants of the Na(+)/proline transporter PutP of Escherichia coli were studied by means of four-pulse double electron-electron resonance spectroscopy. The large value of 4.8 nm for the interspin distance determined between positions 107 in loop 4 and 223 in loop 7 strongly supports the idea of these positions being located on opposite sides of the membrane. Significant smaller values of between 1.8 and 2.5 nm were found for the average interspin distances between spin labels attached to the cytoplasmic loops 2 and 4 (position 37 and 107) and loops 2 and 6 (position 37 and 187). The large distance distribution widths visible in the pair correlation functions reveal a high flexibility of the studied loop regions. An increase of the distance between positions 37 and 187 upon Na(+) binding suggests ligand-induced structural alterations of PutP. The results demonstrate that four-pulse double electron-electron resonance spectroscopy is a powerful means to investigate the structure and conformational changes of integral membrane proteins reconstituted in proteoliposomes

Minimal SPI1-T3SS effector requirement for Salmonella enterocyte invasion and intracellular proliferation in vivo

Effector molecules translocated by the Salmonella pathogenicity island (SPI)1-encoded type 3 secretion system (T3SS) critically contribute to the pathogenesis of human Salmonella infection. They facilitate internalization by non-phagocytic enterocytes rendering the intestinal epithelium an entry site for infection. Their function in vivo has remained ill-defined due to the lack of a suitable animal model that allows visualization of intraepithelial Salmonella. Here, we took advantage of our novel neonatal mouse model and analyzed various bacterial mutants and reporter strains as well as gene deficient mice. Our results demonstrate the critical but redundant role of SopE2 and SipA for enterocyte invasion, prerequisite for transcriptional stimulation and mucosal translocation in vivo. In contrast, the generation of a replicative intraepithelial endosomal compartment required the cooperative action of SipA and SopE2 or SipA and SopB but was independent of SopA or host MyD88 signaling. Intraepithelial growth had no critical influence on systemic spread. Our results define the role of SPI1-T3SS effector molecules during enterocyte invasion and intraepithelial proliferation in vivo providing novel insight in the early course of Salmonella infection

The role of SopB in the interaction between <i>Salmonella</i> and the epithelium.

<p>1-day-old C57BL/6 mice were orally infected with 100 CFU wild type (WT) (filled circles), isogenic <i>sopB</i> mutant (filled triangles), or p<i>sopB</i>-complemented Δ<i>sopB</i> (open triangles) <i>S</i>. Typhimurium. Viable counts in <b>(A)</b> isolated gentamicin-treated enterocytes, <b>(B)</b> total MLN and <b>(C)</b> total liver tissue homogenate at 2 days p.i.. <b>(D)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 2 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 3–5 animals per group). <b>(E)</b> Quantitative analysis of the number of cleaved caspase 3- and <b>(F)</b> cleaved caspase 8 positive cells per 200 times magnification image field. Positive cells from 20 image fields from one section were analyzed per infected neonate (n = 3–6) at day 3 p.i.. Results represent the mean ± SD. <b>(G)</b> Immunostaining for <i>S</i>. Typhimurium (red) in small intestinal tissue sections at 3 days p.i. with 100 CFU WT and Δ<i>sopB S</i>. Typhimurium. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. <b>(H)</b> Co-immunostaining for Δ<i>sopB S</i>. Typhimurium (green) and LAMP1 (red) in small intestinal tissue sections at day 3 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(I)</b> Quantitative evaluation of the percentage of intraepithelial <i>S</i>. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3) at day 3 p.i.. Results represent the mean ± SD. <b>(J)</b> Co-immumostaining for Δ<i>sopB S</i>. Typhimurium (red) and the GFP expressing SPI2 reporter (pM973; green) in small intestinal tissue sections at day 3 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(K)</b> Quantitative analysis of the percentage of intraepithelial <i>S</i>. Typhimurium expressing the SPI2 reporter. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3) at day 3 p.i.. Results represent the mean ± SD.</p

Analysis of <i>sopBE</i>_<i>2</i> and <i>sopAE</i>_<i>2</i> double mutant <i>S</i>. Typhimurium.

<p><b>(A-C)</b> 1-day-old C57BL/6 mice were orally infected with 100 CFU wild type (WT) (filled circles), isogenic Δ<i>sopBE</i>_<i>2</i> (inverted open triangles), or Δ<i>sopAE</i>_<i>2</i> (open triangles) <i>S</i>. Typhimurium. Viable counts in <b>(A)</b> isolated gentamicin-treated enterocytes and <b>(B)</b> total liver tissue homogenate at 4 days p.i.. <b>(C)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 3–6 animals per group). The data for uninfected control animals and <i>Salmonella</i> WT infected mice are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1A–1C</a>. <b>(D)</b> Immunostaining for <i>Salmonella</i> (red) in small intestinal tissue sections at 4 days p.i. with 100 CFU Δ<i>sopE</i>_<i>2</i>, Δ<i>sopBE</i>_<i>2</i>, or Δ<i>sopAE</i>_<i>2</i> <i>S</i>. Typhimurium. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. <b>(E)</b> Percentage of epithelial cells positive for single bacteria or microcolonies (>1 intraepithelial bacterium) at 4 days p.i. with Δ<i>sopBE</i>_<i>2</i> or Δ<i>sopAE</i>_<i>2</i> <i>S</i>. Typhimurium. 30 <i>Salmonella-</i>positive epithelial cells per infected neonate (n = 8–13) were analyzed. Results represent the mean ± SD. <b>(F)</b> Co-immunostaining for <i>Salmonella</i> Δ<i>sopBE</i>_<i>2</i> and Δ<i>sopAE</i>_<i>2</i> (green) and LAMP1 (red) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(G)</b> Quantitative evaluation of the percentage of intraepithelial Δ<i>sopBE</i>_<i>2</i> and Δ<i>sopAE</i>_<i>2</i> <i>S</i>. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3–4) at day 4 p.i.. Results represent the mean ± SD. <b>(H)</b> Co-immunostaining for Δ<i>sopBE</i>_<i>2</i> and Δ<i>sopAE</i>_<i>2</i> <i>Salmonella</i> (red) and the GFP-expressing SPI2 reporter (pM973; green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(I)</b> Quantitative analysis of the percentage of intraepithelial <i>S</i>. Typhimurium expressing the SPI2 reporter. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3–4) at day 4 p.i.. Results represent the mean ± SD.</p

The redundant role of SipA and SopE₂ for enterocyte invasion.

<p><b>(A-C)</b> 1-day-old C57BL/6 mice were orally infected with 100 CFU wild type (WT) (filled circles) or isogenic <i>sopE</i>_<i>2</i><i>sipA</i> mutant <i>S</i>. Typhimurium (filled triangles). Viable counts in <b>(A)</b> isolated gentamicin-treated enterocytes and <b>(B)</b> total liver tissue homogenate at 4 days p.i.. <b>(C)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 4–6 animals per group). The data for uninfected control animals and WT <i>Salmonella</i> infected mice are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1A–1C</a>. <b>(D-F)</b> 1-day-old C57BL/6 mice were orally infected with 100 CFU WT (filled circles), isogenic <i>sipA</i> mutant (open squares), complemented Δ<i>sipA</i> p<i>sipA</i> (filled squares), isogenic <i>sopE</i>_<i>2</i> mutant (open diamonds), or complemented Δ<i>sopE</i>_<i>2</i> p<i>sopE</i>_<i>2</i> (filled diamonds) <i>Salmonella</i>. Viable counts in <b>(D)</b> isolated gentamicin-treated enterocytes and <b>(E)</b> total liver tissue homogenate at 4 days p.i.. <b>(F)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 3–5 animals per group). The data for uninfected control animals and <i>Salmonella</i> WT infected mice are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1A–1C</a>. <b>(G)</b> Immunostaining for <i>S</i>. Typhimurium (red) in small intestinal tissue sections at 4 days p.i. with 100 CFU Δ<i>sopE</i>_<i>2</i><i>sipA</i>, Δ<i>sipA</i>, or Δs<i>opE</i>_<i>2</i> <i>S</i>. Typhimurium <i>Salmonella</i>. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. <b>(H)</b> Co-immunostaining for <i>Salmonella</i> Δ<i>sipA</i>, Δ<i>sopE</i>_<i>2</i> (green) and LAMP1 (red) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(I)</b> Quantitative evaluation of the percentage of intraepithelial <i>S</i>. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 5) at day 4 p.i.. Results represent the mean ± SD. <b>(J)</b> Co-immunostaining for <i>Salmonella</i> Δ<i>sipA</i> or Δ<i>sopE</i>_<i>2</i> (red) and the GFP-expressing SPI2 reporter (pM973; green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5μm. <b>(K)</b> Quantitative analysis of the percentage of intraepithelial <i>S</i>. Typhimurium expressing the SPI2 reporter. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3) at day 4 p.i.. Results represent the mean ± SD.</p

The influence of MyD88-dependent innate immune signaling on intraepithelial microcolony formation.

<p>1-day-old MyD88^+/+ and MyD88^-/- mice were orally infected with 100 CFU <i>S</i>. Typhimurium WT. <b>(A)</b> 4 days after infection, small intestinal tissues were collected and analyzed by immunostaining. Three representative images showing <i>S</i>. Typhimurium (red) forming intraepithelial microcolonies in MyD88^-/- mice. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 10 μm. For wild type controls see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1D</a>. <b>(B)</b> Quantitative evaluation of the number of intraepithelial microcolonies per villus in MyD88^+/+ and MyD88^-/- mice at 4 days p.i.. <i>S</i>. Typhimurium microcolonies were quantified in 20–30 villi per animal (n = 6–8). Results represent the mean ± SD. <b>(C)</b> Transmission electron microscopy (TEM) images of intraepithelial <i>Salmonella</i> in MyD88^+/+ (left panel) and MyD88^-/- mice (right panel). Asterisks highlight bacteria. Bar, 2 μm. <b>(D)</b> Co-immunostaining for <i>Salmonella</i> (green) and LAMP1 (red) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(E)</b> Quantitative evaluation of the percentage of intraepithelial <i>S</i>. Typhimurium associated with LAMP1 staining. Four neonates were analyzed at day 4 p.i.. Results represent the mean ± SD. <b>(F)</b> Co-immumostaining for <i>Salmonella</i> (red) and the GFP expressing SPI2 reporter (pM973, green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(G)</b> Quantitative analysis of the percentage of intraepithelial <i>S</i>. Typhimurium expressing the SPI2 reporter. Microcolonies from tissue sections from four neonates were analyzed at day 4 p.i.. Results represent the mean ± SD.</p

The role of SipA for intraepithelial microcolony formation.

<p><b>(A)</b> Mucosal barrier integrity tested by serum quantification 4 hours after oral administration of FITC labeled-4kDa dextran. 1-day-old C57BL/6 mice were left untreated (crosses) or infected with WT (filled circles) or Δ<i>sopABE</i>_<i>2</i> (open squares) <i>S</i>. Typhimurium. FITC labeled-4 kDa dextran was quantified in serum at day 3 p.i. as indicated. <b>(B and C)</b> Flow cytometric analysis of lamina propria immune cells. 4-day-old mice were left untreated or orally infected with 100 CFU WT or Δ<i>sopABE</i>_<i>2</i> <i>S</i>. Typhimurium and total small intestinal leukocytes were analyzed by flow cytometry at day 3 p.i.. <b>(B)</b> Monocytes (Ly6C^hiLy6G^-CD11b⁺ MHCII^lo/-CD45⁺DAPI^-) and <b>(C)</b> neutrophils (Ly6G⁺Ly6C^intCD11b⁺ MHCII^lo/-CD45⁺DAPI^-) are depicted as percentage of CD45⁺ cells in non-infected (crosses), WT (filled circles) and Δ<i>sopABE</i>_<i>2</i> <i>Salmonella</i> (open squares). The results represent the mean values from at least two independent experiments (n = 4–6 per group). <b>(D)</b> Immunostaining for <i>Salmonella</i> in small intestinal tissue sections at 4 days after co-infection with 100 CFU GFP-expressing WT (yellow) and Δ<i>sopABsipA</i> (red) <i>S</i>. Typhimurium. WT <i>Salmonella</i> appear in yellow due to simultaneous staining for O4/O5 antigen (red) and GFP (green). Δ<i>sopABsipA Salmonella</i> appear in red due to simultaneous staining for O4/O5 antigen (red). Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 10 μm. <b>(E-J)</b> 1-day-old C57BL/6 mice were orally infected with 100 CFU WT (filled circles), Δ<i>sipA</i> (open diamonds), Δ<i>sipA</i> complemented with p<i>sipA</i>^{K635A E637W} (half-filled diamonds), Δ<i>sipA</i> complemented with p<i>sipA</i>^D434A (filled squares), or Δ<i>sipA</i> complemented with p<i>sipA</i>^{D434A K635A E637W} (filled triangles) <i>S</i>. Typhimurium. Viable counts in <b>(E)</b> isolated gentamicin-treated enterocytes and <b>(F)</b> total liver tissue homogenate at 4 days p.i.. <b>(G)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 4–7 animals per group). The data for WT <i>Salmonella</i> infected mice and uninfected control animals are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1A–1C</a>. <b>(H)</b> Immunostaining for <i>Salmonella</i> (red) in small intestinal tissue sections at 4 days p.i. with 100 CFU Δ<i>sipA</i>, Δ<i>sipA</i> complemented with p<i>sipA</i>^{K635A E637W}, Δ<i>sipA</i> complemented with p<i>sipA</i>^D434A, or Δ<i>sipA</i> complemented with p<i>sipA</i>^{D434A K635A E637W} <i>S</i>. Typhimurium. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. <b>(I)</b> Co-immunostaining for LAMP1 (red) and Δ<i>sipA</i> complemented with p<i>sipA</i>^{K635A E637W}, Δ<i>sipA</i> complemented with p<i>sipA</i>^D434A, or Δ<i>sipA</i> complemented with p<i>sipA</i>^{D434A K635A E637W} <i>S</i>. Typhimurium (green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(J)</b> Quantitative evaluation of the percentage of intraepithelial <i>S</i>. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3) at day 4 p.i.. Results represent the mean ± SD.</p

Graphical illustration of the role of the SPI1-T3SS effectors SopA, SopB, SopE₂ and SipA during enterocyte invasion and intraepithelial proliferation <i>in vivo</i>.

<p><b>(1)</b> SipA (red dots) promotes the early recruitment of PMNs and causes barrier disruption and disease progression. This has been reported to occur via stimulation of the epithelial surface molecule p53-effector related to PMP-22 (PERP) and activation of the chemotactic eicosanoid hepoxillin A₃ (HXA₃₎ [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.ref068" target="_blank">68</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.ref089" target="_blank">89</a>]. This effect appears to be invasion-independent and strongly enhanced in the absence of SopA, SopB and SopE₂ suggesting that these effectors exert regulatory functions. <b>(2)</b> Among the studied effector molecules, expression of SipA (red dots), SopE₂ (green dots), or SopE (not shown here) alone is sufficient to facilitate enterocyte invasion. Intraepithelial <i>Salmonella</i> then reside within a LAMP1 negative endosomal compartment and fail to proliferate or express SPI2 encoded genes. <b>(3a)</b> SipA together with SopE₂ or SipA together with SopB (blue dots) facilitate the recruitment of LAMP1 (yellow membrane) from the Golgi apparatus (GA) and the generation of a replicative compartment with intraepithelial bacterial proliferation and expression of SPI2 effector molecules. <b>(3b)</b> Enterocyte invasion or, alternatively, penetration of the epithelial barrier <i>via</i> innate stimulation and signaling through MyD88 induce expression of the chemokines <i>Cxcl2</i> and <i>Cxcl5</i> in the epithelium. <b>(3c)</b> SopB appears to directly or indirectly inhibit caspase 3 and caspase 8 mediated epithelial cell apoptosis. GA, golgi apparatus; HXA₃, hepoxilin A₃; LAMP1, lysosomal-associated membrane protein 1; PERP, p53-effector related to PMP-22; SPI2, <i>Salmonella</i> pathogenicity island 2.</p