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

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 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

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 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

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