InlA^m mediates mouse Ncad-dependent internalization.

<p>(A) Mouse CT26 cells were transfected with scrambled siRNAs or mNcad-specific siRNAs. Bacteria internalization was evaluated by counting intracellular gentamicin resistant bacteria. Values are expressed as a mean + SD (n = 3). Statistical analysis was performed with the unpaired Student's <i>t</i> test. (B) The expression of mNcad was decreased in the cells transfected with mNcad–specific siRNAs compared to that transfected with scrambled siRNAs. Decrease of mNcad expression in CT26 cells reduced the entry of InlA^m-expressing bacteria but not that of InlA-expressing bacteria into CT26 cells. (C) To evaluate the function of different cadherins as receptors, BHK21 cells were transiently transfected with pcDNA3 expression vector harboring the cDNAs of each cadherin. Bacterial invasion were evaluated by counting intracellular gentamicin resistant bacteria. Expression of hEcad provides gain-of-function for both InlA- and InlA^m-expressing <i>Li</i> to invade. InlA^m but not InlA expression promotes bacterial entry to mEcad- and mNcad-expressing cells. Neither mouse P-cadherin (mPcad) nor mouse VE-cadherin (mVEcad) expression promotes InlA- and InlA^m-dependent entry. Values are expressed as a mean + SD (n = 3). Statistical analysis was performed with the unpaired Student's <i>t</i> test. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s001" target="_blank">Figure S1</a>.</p

<i>Lm</i> expressing InlA^m stimulate inflammatory response in the small intestine and compromise intestinal epithelial barrier integrity.

<p>The intestinal ileum was taken from E16P KI and wt mice orally inoculated by 10¹⁰<i>Lm</i> and <i>Lm-inlA^m</i> at 24 (A–C) or 48 (D–F) hr post infection, respectively. (A) Anti-Ly6G antibody staining indicates neutrophils (red, highlighted by the open arrowheads). Tissues were stained for <i>Lm</i> (green, highlighted by the arrows) and counterstained with WGA (grey) for goblet cells, respectively. Scale bar, 20 µm. (B) No obvious difference on neutrophil numbers was observed between wt and E16P KI mice infected by <i>Lm</i>, whereas orally <i>Lm-inlA^m</i> infection in wt mice induced neutrophil recruitment at 24 hr post infection. Statistical analysis was performed with the Mann-Whitney <i>u</i> test (n = 20 from 2 mice). (C) No obvious difference on bacterial numbers was observed between <i>Lm</i> and <i>Lm-inlA^m</i> in wt mice intestinal villi, whereas a significantly increased invasion of <i>Lm</i> in the villi was obsevred in E16P KI mice at 24 hr post infection. Statistical analysis was performed with the Mann-Whitney <i>u</i> test (n = 20 from 2 mice). (D and E) RNA was extracted from the ileum loops of infected or PBS-treated mice 48 hr post infection (n = 4). Following reverse transcription reaction, gene expression was quantified by qPCR with normalization to the GAPDH transcript. Values are expressed as a mean + SD of the fold change relative to that in PBS-treated mice. No significant difference on IFN-γ (D) and IL-1β (E) expression was observed among PBS-treated, <i>Lm</i> and <i>Lm</i>Δ<i>inlA</i>-infected E16P KI mice. In contrast, <i>Lm</i>-<i>inlA^m</i> oral infection induced 5 to 15 fold increase of IFN-γ and IL-1β gene expression in intestinal tissue compared to <i>Lm</i>-infected and PBS-treated wt mice. Statistical analysis was performed with the unpaired Student's <i>t</i> test. (F) Biotin (red) penetration into intestinal lamina propria was done to address intestinal barrier integrity during infection. Mice were sacrificed 2 days post infection. Biotin was injected into ileum loop followed by PBS wash and fixation. Tissues were stained for <i>Lm</i> (green, highlighted by the arrows) and counterstained with WGA (grey) for goblet cells, respectively. Biotin is located within lamina propria of the villi from <i>Lm</i>-<i>inlA^m</i> infected mice but not <i>Lm</i> infected wt and E16P KI mice. Scale bar, 20 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s008" target="_blank">Figure S8</a>.</p

Respective ability of InlA and InlA^m to promote bacterial entry into hEcad- and mEcad-expressing cells.

<p>Bacterial entry into hEcad-expressing human epithelial cells (LoVo) (A for <i>Lm</i> and B for <i>Li</i>) and mEcad-expressing mouse epithelial cells (Nme) (C for <i>Lm</i> and D for <i>Li</i>) was performed by counting intracellular gentamicin resistant bacteria following 1 hr of infection and 1 hr of gentamicin incubation. No significant difference was observed between InlA- and InlA^m-expressing bacteria when invading LoVo cells, whereas InlA^m expression promoted bacterial entry into Nme cells. Values are expressed as a mean + SD (n = 3). Statistical analysis was performed with the unpaired Student's <i>t</i> test. (E) Recruitment of Ecad was performed by incubating <i>Li-inlA</i> or <i>Li-inlA^m</i> with the cells cultured on coverslips for 1 hr, followed by PBS wash and fixation before staining. The cells were stained with the anti-<i>Li</i> antibody and anti-hEcad or anti-mEcad antibody. Right panels show separated channels and merge of boxed regions, showing the recruitment of cadherin by bacteria. Both <i>Li-inlA</i> and <i>Li-inlA^m</i> recruit hEcad when incubated with LoVo cells. <i>Li-inlA^m</i> but not <i>Li-inlA</i> is able to recruit endogenous mEcad in Nme cells. Scale bar, 20 µm.</p

Revisited model of InlA^m-expressing bacteria at the intestinal epithelium.

<p>In humanized mEcad-expressing E16P mouse, as well as in human, <i>Lm</i> targets accessible Ecad around intestinal goblet cells to cross intestinal epithelium, without inducing significant intestinal response. The wt InlA of <i>Lm</i> does not interact with mEcad, thus limits the ability of <i>Lm</i> to cross wt mouse intestinal epithelium. Murinization of InlA enables <i>Lm</i>-<i>inlA^m</i> to interact with mEcad and also Ncad of mouse. <i>Lm-inlA^m</i> therefore invades not only the goblet cells but also the villous M cells expressing accessible Ncad in the intestinal epithelia of mice. Targeting of villous M cells, which is not observed in <i>Lm</i>-infected E16P KI mice and is not predicted to occur in humans, results in severe intestinal inflammation which induces polymorphonuclear neutrophil (PMN) infiltration, intestinal barrier damage and a delayed systemic dissemination in wt mice, all of which are not observed in human listeriosis and <i>Lm</i>-infected humanized mice.</p

Respective invasive potential of <i>Lm</i> and <i>Lm</i>-<i>inlA</i>^m in orally inoculated E16P KI and wt mice.

<p>Mice were orally inoculated with 10¹⁰ bacteria for 2 (n = 8, upper panel) or 4 (n = 6, lower panel) days. Bacterial loads in the ileum loops of small intestine, in spleens and in livers were shown. Statistical analysis was performed with the Mann-Whitney <i>u</i> test. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s007" target="_blank">Figure S7</a>.</p

InlA^m mediates targeting of villous M cells.

<p>The intestinal ileum was taken from E16P KI mice and wt mice orally inoculated by 10¹⁰<i>Li-inlA</i> and <i>Li-inlA^m</i> at 5 hr post infection, respectively. The intestinal tissues were fixed and stained with WGA for goblet cells, NKM 16-2-4 monoclonal antibody for M cells, and for bacteria and nuclei. (A and B) The confocal Z-plane of an ileal villus from <i>Li-inlA^m</i> infected wt mice demonstrates that <i>Li-inlA^m</i> was able to target goblet cells (A) and villous M cells (B). Right panels show separated channels and merge of boxed regions, showing the location of bacteria in villous epithelia. (C) The confocal Z-plane of an ileal villus from <i>Li-inlA</i> infected E16P KI mice shows that <i>Li-inlA</i> targeted goblet cells. (D) Relative location of bacteria in mice intestinal epithelia of villi is shown. The total number of <i>Li-inlA^m</i> in wt mice intestinal villi epithelia was set to 100. 20 villi from two mice ileal loops were counted in each set. Scale bar, 20 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s004" target="_blank"> Figures S4</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s005" target="_blank">S5</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s006" target="_blank">S6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s011" target="_blank">Movies S4</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s012" target="_blank">S5</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s013" target="_blank">S6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s014" target="_blank">S7</a>.</p

InlA^m promotes bacterial entry into mEcad-negative mouse cells and recruits endogenous Ncad.

<p>(A) Nme cells express mEcad, whereas no mEcad expression can be detected in CT26 cells. Actin expression was used as a loading control. (B) Bacterial entry was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat-1003381-g001" target="_blank">Figure 1</a>. InlA^m- but not InlA-expression promotes bacterial entry into mouse CT26 cells. Values are expressed as a mean + SD (n = 3). Statistical analysis was performed with the unpaired Student's <i>t</i> test. (C) Nme cells, CT26 cells, HeLa cells and 104C1 cells express Ncad. The lysate of BHK21 cells which do not express any detectable classical cadherins was used as a negative control. (D) Recruitment of Ncad was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat-1003381-g001" target="_blank">Figure 1</a>. The coverslips were stained with the anti-<i>Li</i> antibody and anti-Ncad antibody. Right panels show separated channels and merge of boxed regions, showing the recruitment of Ncad by <i>Li-inlA^m</i>. Scale bar, 20 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s001" target="_blank">Figures S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s002" target="_blank">S2</a>.</p

Ncad expression is detected on the apical pole of villous M cells.

<p>Immunolabeling of the small intestine tissue section (A) and whole mount tissue of a part of ileum (B and C) of wt mice was performed. (A) The small intestine tissue section was stained by anti-mouse IgG followed by anti-Ncad mouse IgG and anti-mouse IgG conjugated with another fluorophore to distinguish mouse IgG-positive cells and Ncad-positive cells. Scale bar, 50 µm. (B and C) Intestinal tissue of wt mice was fixed and stained for luminally accessible (acc) Ncad with antibody against extracellular domain of Ncad (clone GC4) before tissue permeabilization, M cells with NKM 16-2-4 antibody, wheat germ agglutinin (WGA) and nuclei after tissue permeabilization. Projection of a 25 µm thick reconstructed intestinal villus (B) and one of the xy plane (C) are shown. Right panels show separated channels and merge of boxed regions in (C), showing Ncad on the apical side of NKM 16-2-4-positive cells. NKM 16-2-4 antibody is a monoclonal antibody raised against α(1,2) fucose moiety in absence of neighboring sialic acids, a specific marker on M cells surface. WGA was used to stain the mucus of goblet cells and cell membrane. Scale bar, 20 µm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s003" target="_blank">Figure S3</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s009" target="_blank">Movies S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s010" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003381#ppat.1003381.s011" target="_blank">S3</a>.</p

Clinical signs.

<p><b><i>Group A1: RT PCR CHIKV positive patients. Group A2: RT PCR CHIKV negative, IgM anti CHIKV positive, IgG anti CHIKV negative patients. Group B: RT PCR CHIKV negative, IgM anti CHIKV negative, IgG anti CHIKV negative patients.</i></b></p><p><b><i>Group A1 vs Group A2: ^¤¤¤ p<0.001, ^¤¤ p<0.01, ^¤ p<0,05.</i></b></p><p><b><i>Group A1 vs. Group B:</i></b> *** <b><i>p<0.001, ** p<0.01, * p<0,05.</i></b></p><p><b><i>Average, standard deviation, percentage in parenthesis are indicated.</i></b></p

Biological parameters.

<p><b><i>Group A1: RT PCR CHIKV positive patients. Group A2: RT PCR CHIKV negative, IgM anti CHIKV positive, IgG anti CHIKV negative patients. Group B: RT PCR CHIKV negative, IgM anti CHIKV negative, IgG anti CHIKV negative patients.</i></b></p><p><b><i>Group A1 vs. Group A2: ^¤¤¤ p<0.001, ^¤¤ p<0.01, ^¤ p<0,05.</i></b></p><p><b><i>Group A1 vs. Group B:</i></b> *** <b><i>p<0.001, ** p<0.01, * p<0,05.</i></b></p><p><b><i>Average, standard deviation, percentage in parenthesis are indicated.</i></b></p><p>^#<b><i>Physiological ranges.</i></b></p