Murinization of Internalin Extends Its Receptor Repertoire, Altering Listeria monocytogenes Cell Tropism and Host Responses

Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been “murinized” to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlAm) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlAm-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlAm-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen.


Introduction
Co-evolution of microbes with their hosts can select stringently specific host-microbe interactions at the cell, tissue and species levels [1]. Species-specific host-microbe interactions, which are the rule rather than the exception, pose a challenge for the use of laboratory animal models to study human pathogens, including Listeria monocytogenes (Lm), the etiological agent of listeriosis, a deadly foodborne infection. Lm is able to actively cross the intestinal barrier, reach the systemic circulation and cross the blood-brain and placental barriers, leading to its dissemination to the central nervous system and the fetus [2].
The mouse is a genetically amenable model that is widely used to investigate human diseases [3,4]. To obtain a mouse model in which the pathogenic properties of a given pathogen are similar to what is observed in human, species specificity can be circumvented by humanizing the mouse by transgenesis [5,6,7,8], knock-in [9], knock-out [10] or xenograft techniques [11]. One can also adapt the pathogen to the mouse by multiple passages on cell lines [12,13] or in vivo [14], or specifically ''murinize'' a pathogen ligand so that it interacts with the mouse ortholog of a species-specific human receptor [15,16].
The Lm surface protein InlA interacts with E-cadherin (Ecad) and mediates Lm entry into epithelial cells, which express this adherens junction protein [17,18]. Cadherins constitute a family of calcium-dependent cell adhesion receptors. Ecad is expressed mainly in epithelia, whereas N-cadherin (Ncad) is found primarily in neuronal cells and endothelial cells together with VE-cadherin [19,20]. Ncad can also be coexpressed with Ecad in epithelial cells [21]. Importantly, Ncad has been reported to not act as a receptor for InlA, and so far Ecad is the only known classical cadherin acting as a receptor for InlA [18]. In contrast to Ecad from human, guinea pig, rabbit and gerbil, mouse Ecad (mEcad) and rat Ecad are not recognized by InlA and do not promote bacterial entry [9,22]. The interaction of InlB, another Lm invasion protein, with its host receptor is also species-specific [23]. InlB recognizes the hepatocyte growth factor receptor Met of human, mouse, rat and gerbil but not that of guinea pig and rabbit [9,23,24].
Two mouse lines have been established to study InlA-Ecad interaction in vivo: a transgenic mouse line expressing human Ecad (hEcad) in enterocytes (hEcad Tg) [6], and a humanized mEcad knock-in mouse line (E16P KI) with an E16P amino acid substitution which enables mEcad to interact with InlA without affecting Ecad homophilic interactions and allows Lm internalization [9,22]. Using these two humanized mouse models, we have demonstrated that InlA mediates Lm crossing of the intestinal epithelium upon targeting of luminally-accessible Ecad around goblet cells [6,9,25], and that InlA and InlB act interdependently to mediate the crossing of the placental barrier [9]. Epidemiological investigations have confirmed the relevance of these experimental findings, and shown that InlA is implicated in Lm crossing of human intestinal and placental barriers [9,26].
In 2007, Wollert et al. engineered a genetically modified InlA with the purpose of increasing its binding affinity to hEcad [16]. Two amino acid substitutions in InlA, S192N and Y369S, were shown to enhance InlA binding affinity to hEcad [16]. Neither S192N nor Y369S substitution has been observed in the more than 500 Lm isolates InlA sequences we have checked (our unpublished results). Wollert et al. published that this increased affinity for hEcad translates into an increased bacterial entry into human epithelial cells (Caco-2) [16]. Importantly, Wollert et al. also showed that this modified InlA binds the extracellular cadherin domain 1 (EC1) of mEcad in solution with a comparable affinity to that of the wild-type (wt) InlA for hEcad EC1 [16]. They hypothesized that this interaction would allow Lm expressing this ''murinized'' InlA (InlA m ) to cross intestinal barrier and would render wt mice orally permissive to Lm infection, a phenotype which is mediated by InlA in permissive models [6]. In support of this hypothesis, Wollert et al. found an increased intestinal, spleen and liver bacterial loads of wt mice orally inoculated with Lm expressing InlA m , yet only after 3 to 4 days post infection, which is later than in models permissive to InlA-Ecad interaction [6,9,16]. Moreover, the ability of InlA m to mediate mEcad-dependent Lm internalization into host cells has never been tested. In addition, InlA m unexpectedly promoted pronounced inflammation and intestinal epithelial cell damages in wt mice [16], whereas wt InlA mediates the crossing of the intestinal barrier without inducing significant intestinal response and tissue damage in hEcad transgenic mice [6,27].
This prompted us to investigate the detailed properties of InlA m in cultured cells, as well as the in vivo cell and tissue tropisms of bacteria expressing InlA m , as compared to that of its isogenic parental Lm strain that expresses wt InlA. Here, we demonstrate that InlA m promotes bacterial entry not only into mEcad-positive but also into mEcad-negative mouse cells. We show that InlA mmediated entry into mEcad-negative cells is mouse Ncad (mNcad)dependent. Importantly, InlA m -mNcad interaction allows bacteria to specifically target Ncad-positive villous M cells in vivo, a cell type which is not targeted by Lm in humanized mouse models permissive to InlA-Ecad interaction. This leads to enhanced intestinal inflammatory responses and disruption of the intestinal barrier integrity, both of which are not observed in Lm-infected humanized mice and human listeriosis. Together, these results demonstrate that the murinization of InlA not only extends Lm host range, but also broadens its receptor repertoire, consequently changing Lm cell tropism and enhancing host immune responses to Lm. These results challenge the relevance of using InlA mexpressing Lm to study human listeriosis and in vivo host responses to this human pathogen.

Murinization of InlA promotes bacterial entry into mEcad-expressing cells but has no impact on bacterial entry into hEcad-expressing cells
We first investigated whether the increased affinity of InlA m to hEcad translates into an enhanced invasion of hEcadexpressing cells, as proposed by Wollert et al. [16]. To this end, we assessed InlA m -dependent entry into LoVo cell, a human epithelial cell line expressing hEcad [22]. Lm wt strain and Lm expressing InlA m (Lm-inlA m ) invaded LoVo cells at similar levels ( Figure 1A). Because Lm can be internalized by InlA-independent pathways such as InlB-Met, we transferred either inlA or inlA m onto the chromosome of Listeria innocua (Li), a naturally non-invasive and non-pathogenic Listeria species, in which heterologous expression of inlA has been shown to confer invasiveness [17,18,28]. Li expressing either InlA (Li-inlA) or InlA m (Li-inlA m ) were equally invasive in LoVo cells ( Figure 1B). These results indicate that contrary to what is reported by Wollert et al. [16], the increased affinity of InlA m to hEcad does not translate into an increased level of bacterial entry. Both Li-inlA and Li-inlA m recruited hEcad when incubated with LoVo cells, suggesting that hEcad is involved in both InlA-and InlA mmediated entries ( Figure 1E, upper panel). Because purifed InlA m interacts with the purified EC1 domain of mEcad, Wollert et al. have proposed, although not tested, that InlA m would mediate bacterial entry into mEcad-expressing cells [16]. We therefore tested the ability of InlA m to promote bacterial entry into the mouse epithelial cell line Nme, which expresses mEcad [29]. InlA m promoted bacterial entry into mEcadexpressing Nme cells, although to a lower level than InlA in hEcad-expressing LoVo cells ( Figure 1C and D). Li-inlA m also recruited mEcad during cell invasion, whereas as expected, Li-inlA does not ( Figure 1E, lower panel). Together, these results show that (i) the increased affinity of InlA m to hEcad does not enhance bacterial entry into hEcad-expressing cells, and (ii) the murinization of InlA confers to Lm an enhanced ability to be internalized into mEcad-expressing cells [16].

Author Summary
Co-evolution of microbes with their hosts can select stringently specific host-microbe interactions at the cell, tissue and species levels. Listeria monocytogenes (Lm) is a foodborne pathogen that causes a deadly systemic infection in humans. Lm crosses the intestinal epithelium upon the interaction of its surface protein InlA with Ecadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wildtype mice, InlA has been ''murinized'' to interact with mouse Ecad. Here, we demonstrate that in addition to interacting with mouse Ecad, InlA m also uses N-cadherin as a receptor, whereas InlA does not. This artifactual InlA m -Ncadherin interaction promotes bacterial translocation across villous M cells, a cell type which is not targeted by InlA-expressing bacteria. This leads to intestinal inflammation and intestinal barrier damage, both of which are not seen in humans and humanized mouse models permissive to InlA-Ecad interaction. These results challenge the relevance of using InlA m -expressing Lm as a model to study human listeriosis and host responses to this pathogen. They also illustrate that caution must be exercised before using ''murinized'' pathogens to study human infectious diseases. InlA m promotes mEcad-independent entry into mouse cells Monk et al. have reported that Lm-inlA m invades mouse CT26 cells more efficiently than Lm [13]. Strikingly, CT26 cells do not express mEcad (Figure 2A) [30], yet we confirmed that InlA m mediates bacterial entry into these cells ( Figure 2B). Because classical cadherins exhibit a high level of conservation in their EC1 domains ( Figure S1A), we tested whether Li-inlA m would recruit another classical cadherin than mEcad in CT26 cells. We labeled CT26 cells with a pan-cadherin antibody, which recognizes the cytoplasmic domain of classical cadherins [31]. CT26 cells were strongly stained with the pan-cadherin antibody ( Figure S1B), indicating that they likely express classical cadherin proteins. Furthermore, this pan-cadherin-immunoreactive protein was recruited in CT26 cells by Li-inlA m but not Li-inlA ( Figure S1B). Immunoblotting and immunostaining revealed that CT26 cells express Ncad ( Figures 2C and D), a classical cadherin known to be expressed in endothelial cells, neurons and some transformed epithelial cells [20]. Importantly, Li-inlA m , but not Li-inlA, recruited Ncad in CT26 cells ( Figure 2D). We next tested other cell lines for Ncad expression. We found that Nme cells (which also express mEcad and are permissive to InlA m -mediated entry), human HeLa cells, and guinea pig 104C1 cells all express Ncad ( Figure 2C). As in CT26 cells, InlA m promoted bacterial entry into HeLa and 104C1 cells, although these two cell lines do not express Ecad and are therefore not permissive to InlA-dependent entry ( Figure S2) [23]. These results suggest that the murinization of InlA confers to this protein the ability to interact with Ncad from different species, and to enter into host cells expressing Ncad.

mNcad is a receptor for InlA m but not InlA
To investigate if mNcad serves as a receptor for InlA m -mediated entry into CT26 cells, CT26 cells were treated with mNcadspecific siRNAs or scrambled control siRNAs. Treatment of CT26 cells with mNcad siRNAs led to a reduced expression of mNcad which correlated with a significantly decreased InlA m -dependent entry ( Figures 3A and B). To directly assess the ability of mEcad and mNcad to act as receptors for InlA m , we used the BHK21 cell line, which is of hamster origin and does not express any known classical cadherin [32], and transfected this cell line with plasmids encoding either hEcad, mEcad or mNcad. As expected, both InlA and InlA m mediated bacterial entry into hEcad-expressing cells ( Figure 3C). Moreover, InlA m mediated entry into mEcadexpressing cells, whereas as previously shown, InlA did not ( Figure 3C) [22]. Most importantly, we also demonstrated that InlA m mediated bacterial entry into Ncad-expressing cells, whereas, as previously shown, InlA did not ( Figure 3C) [18].
To investigate whether the InlA m receptor repertoire extends to other members of classical cadherins, we tested the ability of mouse P-cadherin (mPcad) and VE-cadherin (mVEcad) to serve as receptors for InlA m ( Figure S1A). Neither mPcad nor mVEcad acted as a receptor for InlA m or InlA ( Figure 3C). Taken together, these data confirm that InlA exhibits a species-specific and narrow repertoire for Ecad and mediates entry into hEcad-but not mEcad-expressing cells, and demonstrate that by widening InlA species spectrum from human to mouse Ecad, murinization of InlA extends its receptor repertoire to Ncad.

Murinization of InlA extends the cell tropism of Lm at the intestinal level
In order to investigate if these in vitro results translate into an in vivo phenotype, and study in particular the cell tropism of InlA mexpressing bacteria, we investigated Ncad luminal accessibility at the intestinal epithelium level, which is the portal of InlA-mediated entry of Lm. In contrast to luminally-accessible Ecad which is mostly observed as rings surrounding goblet cells [25], mNcad was accessible on the apical pole of villous M cells ( Figure 4, Movie S1), but not M cells of Peyer's patches (Movie S2) in wt mice. The expression of luminally-accessible Ncad was also detected on the apical pole of villous M cells in E16P KI mice ( Figure S3, Movie S3). These results suggest that InlA m may allow bacteria to target villous M cells upon mouse oral inoculation.
To specifically investigate whether InlA m -expressing bacteria target cells that express luminally-accessible Ncad, we inoculated orally wt mice with Li-inlA or Li-inlA m , and for comparison we inoculated humanized E16P KI mice orally with Li-inlA. As expected from our recent results [25], Li-inlA were found in goblet

InlA m -mNcad interaction has an impact on Lm systemic dissemination in orally inoculated mice
To investigate the impact of InlA m -mNcad interaction on the infection process, we inoculated orally wt and E16P KI mice with Lm-inlA m or Lm. In Lm-infected E16P KI mice in which InlA-Ecad interaction is functional, InlA promoted Lm invasion of the small intestinal tissue and bacterial dissemination to spleen and liver as early as 2 days post infection (dpi) ( Figure 6). In contrast, in Lm-inlA m infected wt mice, in which both InlA m -Ecad and InlA m -Ncad interactions are functional, Lm bacterial loads in the small intestinal tissue, spleen and liver were not significantly increased at 2 dpi compared to Lm-infected wt mice, but were at 4 dpi ( Figure 6). This delayed systemic dissemination was also observed when comparing Lm-inlA m to LmDinlA in E16P KI mice ( Figure  S7). These results demonstrate that, although promoting Lm crossing of the wt mouse intestinal barrier, InlA m delays bacterial systemic dissemination relative to InlA in E16P KI mice, and therefore alters the kinetics of Lm infection in vivo.  and B) and in hEcad Tg mice ( Figures S8A and B). Importantly, neutrophil infiltration correlated only with InlA m -mediated invasion, and did not reflect bacterial load in the villi, which was actually the highest in Lm-infected humanized mice, in which no neutrophil infiltration was observed (Figures 7A-C, S8A-C). Moreover, a significant increase in IFN-c and IL-1b expression was observed in the intestinal tissue of wt mice infected with Lm-inlA m , whereas no significant increase was observed in Lm-infected wt and humanized mice (Figures 7 D and E). Together, these results indicate that InlA m -Ncad-mediated intestinal invasion per se leads to exacerbated host responses compared to InlA-Ecadmediated intestinal invasion, and are not a reflect of enhanced bacterial tissue invasion.

InlA m -mNcad interaction leads to enhanced intestinal response and compromised intestinal barrier function
We next assessed intestinal barrier integrity upon infection by testing the intratissular diffusion of biotin administered intraluminally (see Material and Methods) [33]. In wt and humanized mice infected by Lm for two days, biotin localized exclusively to the luminal side of the small intestine ( Figures 7F and S8D). In contrast, although the intestinal villi of Lm-inlA m infected wt and humanized mice were not heavily infected, biotin accessed the lamina propria ( Figures 7F and S8D). These findings indicate that InlA m -Ncad-mediated intestinal invasion leads to a disruption of intestinal barrier integrity. Together, these results demonstrate that the murinization of InlA profoundly modifies the pathogenic properties of Lm by altering its intestinal portal of entry, host intestinal responses and intestinal barrier integrity.

Discussion
InlA interaction with Ecad allows Lm translocation across the intestinal epithelium and is therefore a critical event in the development of systemic listeriosis, one of the deadliest foodborne infections in human. Because InlA does not interact with mEcad, the discovery and characterization of this key step were made in species permissive to InlA-Ecad interaction (guinea pig, gerbil) and humanized mouse models (hEcad Tg and E16P KI mouse lines) [6,9]. A genetically engineered Lm strain expressing a murinized InlA (InlA m ) enabling interaction with mEcad in vitro has been proposed to constitute an attractive alternative model to study human listeriosis in wt mice [16]. A practical advantage of this latter system is that it can be readily used to infect several different mouse lines. However, a systematic study comparing the properties of Lm expressing InlA m to that of its isogenic parental strain has not been performed, neither in vitro nor in vivo.
Here we show that InlA m is able to recruit mEcad and mediate mEcad-dependent entry into cultured cells. We also show that InlA m mediates entry into goblet cells of wt mice, which express luminally-accessible mEcad. These results confirm that the S192N and Y369S substitutions confer to InlA a phenotype in wt mice which is observed in humanized mice permissive to InlA-Ecad interaction [25].
Importantly, we also uncover that InlA m is able to recruit Ncad and mediate Ncad-dependent internalization. This artifactual interaction translates in vivo into InlA m -dependent targeting of villous M cells, intestinal inflammatory responses, disruption of intestinal barrier integrity and delayed bacterial systemic dissemination in wt mice, as well as in humanized mice. Such stricking phenotypes are not observed in humanized mice orally-inoculated with wt Lm, suggesting that they depend on InlA m -Ncad interaction and invasion of villous M cells, but not on InlA m -Ecad interaction and invasion of goblet cells (Figure 8). It is important to note that these phenotypes are also present in E16P KI and hEcad Tg mice infected with Lm-inlA m , indicating that intestinal inflammation is a direct consequence of InlA m -mediated intestinal invasion, and proving that the absence of inflammation in Lm-infected humanized mice is not a side effect of mouse humanization, but is a genuine property of InlA-dependent intestinal invasion. These results are in agreement with the observation by Wollert et al. that infection with Lm-inlA m leads to severe intestinal inflammation and tissue damage in wt mice [16], and with our earlier observation that InlA has little impact on Lm intestinal responses in mice permissive to InlA-Ecad interaction [6,27]. This indicates that the murinization of InlA, in addition to broadening the host range of Lm, also extends its receptor repertoire to another member of the classical cadherin family, Ncad, therefore modifying its cell tropism, host responses and the dynamics of infection.
The engineering of InlA m was based on the rational protein design of a modified InlA that would increase InlA-hEcad binding affinity [16]. Indeed, S192N and Y369S substitutions in InlA lead to a 6,700-fold increase in the binding affinity of InlA to hEcad [16]. Here we have shown that this does not translate into increased invasion of hEcad-expressing cells. Before drawing this conclusion, we ensured that the BHK21 cell line we used does not express other cadherins than the one we intended to study. A possible reason for the observed increased level of invasion of Lm-inlA m in Caco-2 cells observed by Wollert et al. is the coexpression of Ecad and Ncad in these cells [21]. These results suggest that InlA-hEcad interaction, although it is of relatively low affinity (K D = 864 mM) [16], has been naturally selected to mediate an optimal level of infection.
We have shown that InlB, another major invasion protein of Lm, does not play a significant role for the crossing of the intestinal barrier [23]. In contrast, InlB has been reported to promote Lm expressing InlA m to invade intestinal villi [34]. Our results shed light onto these apparent contradictory results and raise the possibilty that InlA m -Ncad mediated invasion of villous M cells may involve the InlB pathway.
Shigella flexneri, the etiological agent of bacillary dysentery is associated with strong polymorphonuclear infiltration, severe local inflammation, disruption of intestinal barrier integrity, yet no systemic dissemination [35,36]. In contrast, listeriosis in human and humanized mice is characterized by the paucity of intestinal symptoms, the absence of polymorphonuclear intestinal infiltration, little local inflammation, the absence of intestinal barrier disruption, but systemic dissemination [6,27,36,37]. We have demonstrated that Lm-inlA m triggers pro-inflammatory response and disrupts epithelial integrity in intestinal tissue of wt and humanized mice, and exhibits a delayed systemic dissemination, compared to Lm-infected humanized mice. These observations strongly suggest that the targeting of villous M cells by InlA mexpressing bacteria triggers pro-inflammatory host responses which contain bacterial invasion but lead to intestinal epithelium damages. This fits with the observation that antigen delivery via villous M cells stimulates immune reponses [38]. Like InlA m , Als3 is a Candida albicans invasin that binds both Ecad and Ncad to invade host cells [39]. Candida albicans has been shown to favor gut inflammation and promotes food allergy accompanied by gut epithelial barrier hyperpermeability, the underlying mechanisms of which are so far unclear [40,41]. Our study indicates that Candida albicans may use Als3 to target Ncad-positive villous M cells, and thereby trigger intestinal inflammation. The specific functions of villous M cells remain poorly understood, yet villous M cells are a particularly abundant constituent of the intestinal epithelium. Our results show that InlA m -and Als3-expressing microorganisms would be particularly instrumental to study villous M cell functions.
Repeated infection of mice in vivo or mouse cells in vitro allows the obtention of ''murinized'' pathogens adapted to the mouse. Despite the great adaptability of microbes, evolutionary constraints limit pathogen variability [42]. A mutation beneficial under certain environmental conditions may end up as disadvantageous in another, highlighting the fine-tuning of host-microbe interactions. The structure-based rational design of InlA m was proposed as a subtle and elegant way to electively ''murinize'' a microbial ligand with least impact on the pathogen. However, we provide here evidence that the rationally designed InlA m has gained the unfortunate ability to interact with another surface protein than its cognate receptor Ecad. Even though InlA m mediates Lm crossing of the intestinal barrier, a phenotype which is strictly dependent on InlA-Ecad interaction, the way by which Lm crosses the intestinal barrier in an InlA m -dependent manner differs from what observed with wt Lm in humanized mice and humans, as does the resulting infection process. This illustrates that murinization of human-specific pathogens, although an elegant and rational approach, may unfortunately mislead rather than ease the understanding of human infectious diseases' pathophysiology. Caution must therefore be exercised before engineering and using ''murinized'' pathogens to study human infectious diseases.

Bacterial and cell culture
Bacterial strains, plasmids and primers are listed in Table S1. Note that the sequences of inlA, inlA m in Lm and in Li were confirmed by sequencing, as well as the integration sites of inlA and  inlA m in Li and the deletion site of inlA in Lm. Listeria and Escherichia coli strains were respectively cultivated in BHI and LB at 37uC with shaking at 180 rpm. To deliver plasmids into Li, E. coli S17-1 (colistin and nalidixic acid sensitive) cells were transformed with the plasmids followed by conjugation with Li (colistin and nalidixic acid resistant). Mammalian cell lines used in this study were routinely cultured at 37uC in 5% CO 2 . Except for the culture medium for BHK21 which was supplemented with 5% fetal bovine serum, all the cell culture media were supplemented with 10% fetal bovine serum. Human epithelium LoVo cells were cultured in F12K nutrient GlutaMax medium. Mouse epithelium Nme cells were cultured in DMEM GlutaMax medium supplemented with 10 mg/ml insulin. Mouse CT26 and guinea pig 104C1 cells were cultured in RPMI1640 GlutaMax medium supplemented with HEPES buffer and sodium pyruvate. Human HeLa cells were cultured in MEM GlutaMax medium. Hamster BHK21 cells were cultured in GMEM GlutaMax medium supplemented with tryptose phosphate buffer and HEPES buffer. All the culture medium and related chemicals were purchased from Gibco (Invitrogen). Transient transfection of mammalian cells was performed with jetPRIME transfection kit (Polyplus transfection). The scrambled (sc-37007) and mouse Ncad specific siRNAs (sc-35999) were purchased from Santa Cruz. For the transfection of siRNAs, mouse CT26 cells were seeded into the 24well plates for 1 day and then transfected with scrambled siRNAs (25 nM) or mNcad-specific siRNAs (25 nM) followed by 1 day incubation and replacement of transfection medium with growth medium another 1 day of incubation before infection. For the transfection of plasmid DNAs, BHK21 cells were transiently transfected with pcDNA3 expression vector harboring the cDNAs of each cadherin (1 mg DNA for each well in a 24-well plate) followed by 2 days incubation before infection.

Construction of plasmids
The strategy to express inlA or inlA m in Li is as described based on integrative plasmid pAD containing a constitutive promoter [43]. The primers EagI_UTRhly-F and UTRhly-R were used to amplify the hly 59 UTR of Lm EGDe. Full length of inlA and inlA m were amplified from the genomic DNA of Lm EGDe and Lm-inlA m , respectively, with the primers UTRhly_inlA-F and SalI_inlA-R2. The resulting PCR products were ligated to hly 59 UTR by splicing-by-overlap-extension (SOE) PCR. The final SOE PCR products, containing the entire hly 59 UTR sequence fused to the start codon of the inlA (hly 59 UTR-inlA) or inlA m , (hly 59 UTR-inlA m ), were then cloned in pCR-Blunt (Invitrogen) and verified by sequencing. Plasmids containing correct sequence and pAD-cGFP were digested by EagI and SalI. The backbone of pAD-cGFP was ligated with hly 59 UTR-inlA and hly 59 UTR-inlA m to form pAD-inlA and pAD-inlA m .

Invasion assay
Cell suspensions from confluent monolayers were seeded at a concentration of 5610 4 cells per well in 24-well tissue culture plates and grown for 40-48 hr in an antibiotics-free medium at 37uC. Lm and Li strains were grown to OD600 at 0.8 and 0.6 in BHI, respectively. Bacterial culture were then washed with PBS and diluted in cell culture medium without serum. Bacterial suspensions were added to the cells at a multiplicity of infection (MOI) of approximately 50 and incubated for 1 hr. Following wash with complete medium, 10 mg/ml of gentamicin was added to kill the extracellular bacteria for 1 hr. The cells were then washed by complete medium and PBS, and homogenized in PBS supplemented with 0.4% Triton X-100, followed by serial dilution and colony forming units (CFUs) counting. For cadherin recruitment assay, the procedure was the same as the invasion assay except that the cell attachment buffer (HEPES 20 mM, NaCl 150 mM, glucose 50 mM, MgCl 2 1 mM, CaCl 2 2 mM, MnCl 2 1 mM, 0.1% BSA) was used for infection and PBS (Ca 2+ / Mg 2+ ) (Gibco) was applied to wash the non-attached bacteria stringently followed by fixation.

Animals
Eight to 10-week old C57BL/6 female mice (JANVIER) and isogenic mEcad E16P KI female mice were food restricted overnight but allowed free access to water. Lm culture was prepared as described [6], and inoculated with a feeding needle intragastrically [44]. Mice were then immediately allowed free access to food and water. All the procedures were in agreement with the guidelines of the European Commission for the handling of laboratory animals, directive 86/609/EEC (http://ec.europa. eu/environment/chemicals/lab_animals/home_en.htm) and were approved by the Animal Care and Use Committee of the Institut Pasteur, as well as by the ethical committee of ''Paris Centre et Sud'' under the number 2010-0020.

Immunofluorescence labeling and immunoblotting
Preparation of tissue sections and whole mount tissues were as described [9,25]. The following antibodies and fluorescent probes were used for immunostaining and Western blot: anti-hEcad clone HECD-1 mouse monoclonal antibody (Invitrogen), anti-mEcad clone ECCD-2 rat monoclonal antibody (Invitrogen), anti-b-actin clone AC-15 mouse monoclonal antibody (Sigma), anti-Ncad clone 32/N-cadherin mouse monoclonal antibody (BD), anti-Ncad clone GC-4 mouse monoclonal antibody (Sigma), anti-pan cadherin clone CH-19 monoclonal antibody (Sigma), anti-M cell clone NKM 16-2-4 rat monoclonal antibody (Miltenyl Biotec), R6 anti-Li rabbit polyclonal antibody and R11 anti-Lm rabbit polyclonal antibody [45], Rat anti-mouse Ly-6G (BD), wheat germ agglutinin (WGA) conjugated with Alexa Fluor 647 (Jackson 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-c (D) and IL-1b (E) expression was observed among PBS-treated, Lm and LmDinlA-infected E16P KI mice. In contrast, Lm-inlA m oral infection induced 5 to 15 fold increase of IFN-c and IL-1b gene expression in intestinal tissue compared to Lminfected and PBS-treated wt mice. Statistical analysis was performed with the unpaired Student's t 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 Lm (green, highlighted by the arrows) and counterstained with WGA (grey) for goblet cells, respectively. Biotin is located within lamina propria of the villi from Lm-inlA m infected mice but not Lm infected wt and E16P KI mice. Scale bar, 20 mm. See also Figure S8. doi:10.1371/journal.ppat.1003381.g007

Biotin penetration experiment
Biotin was used as a molecule to address the integrity of intestinal epithelium as described previously [33]. Briefly, 2 mg/ ml of EZ-link Sulfo-NHS-Biotin (Pierce) in PBS was slowly injected into the lumen of ileum loop via the open end adjacent to cecum immediatedly after removal of the entire ileum. After 3 min, the loop was opened followed by PBS wash and 4% paraformaldehye fixation.

Intestinal tissue genes expression quantification
Four mice for each condition were sacrificed 2 days post infection. 1 cm-long of ileal loop of each animal was applied for RNA extraction. The RNA isolation, reverse transcription and quantitative real time PCR (qRT-PCR) were performed as described [46]. Primers used for qRT-PCR were pre-designed, validated RT 2 qPCR primer pairs (SABioSciences, Qiagen) as follows: IFNG (IFN-c, PPM03121A), IL1B (IL-1b, PPM03109F) and GAPDH (PPM02946E).

Statistical analysis
Values are expressed as mean + SD. Statistical comparisons were made using the unpaired Student's t test, Mann-Whitney u test or the x 2 test as indicated. p values,0.05 were considered significant. Significant differences are marked with an asterisk for p,0.05, two asterisks for p,0.01, three asterisks for p,0.001 and four asterisks for p,0.0001.  Figures S4A and B, respectively) and in E16P KI mice infected by Lm (C, related to Figure S4C) presented in Figure  S5 were shown. These images demonstrate that the bacteria highlighted in the Figure S4  The number of bacteria in each infected villus was also quantified. Bacteria load of Lm in the intestinal villi was higher than that of Lm-inlA m in both E16P KI and hEcad Tg mice upon oral infection 24 hpi. In order to compare the result of Lm-inlA m with Lm in E16P KI mice, the data of Lm-infected E16P KI mice shown here in B and C were from figure 7B and C, respectively. Statistical analysis was done with Mann-Whitney u test (n = 20 villi from 2 mice). (D) Biotin was injected into ileum loop followed by PBS wash and fixation. Tissues were stained for Lm (green, highlighted by the arrows) and counterstained with WGA (grey) for goblet cells and epithelia. Biotin is located within lamina propria of the villi from Lm-inlA m infected mice but not Lm infected mice. Scale bar, 20 mm.

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Movie S1 Luminally accessible Ncad is expressed on the apical poles of villous M cells in wt mice, related to Figure 4. Whole mount intestinal tissue of a wt mouse was stained before permeabilization for accessible mNcad (green) and NKM 16-2-4 for M cells (red), and after permeabilization for nuclei (blue) and WGA for goblet cells (grey). Intestinal villus is oriented with the villus tip facing the viewer. The luminally accessible apical surface of villous M cells is labeled with the anti-Ncad antibody. Images were acquired as a z stack by confocal microscopy and assembled as a three-dimensional reconstruction with Imaris software.

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Movie S2 Peyer's patch M cells do not express luminally accessible Ncad in wt mice, related to Figure 4. Whole mount intestinal tissue of a wt mouse was stained before permeabilization for accessible mNcad (green) and NKM 16-2-4 for M cells (red), and after permeabilization for nuclei (blue) and WGA for goblet cells (grey). The luminally accessible apical surface of Peyer's patch M cells is not labeled with the anti-Ncad antibody. Intestinal Peyer's patch is oriented with the tip facing the viewer. Images were acquired as a z stack by confocal microscopy and assembled as a three-dimensional reconstruction with Imaris software. (MOV) Movie S3 Luminally accessible Ncad is expressed on the apical poles of villous M cells in E16P KI mice, related to Figure 4. Whole mount intestinal tissue of an E16P KI mouse was stained before permeabilization for accessible mNcad (green) and NKM 16-2-4 for M cells (red), and after permeabilization for nuclei (blue) and WGA for goblet cells (grey). Intestinal villus is oriented with the villus tip facing the viewer. The luminally accessible apical surface of villous M cells is labeled with the anti-Ncad antibody. Images were acquired as a z stack by confocal microscopy and assembled as a three-dimensional reconstruction with Imaris software. (MOV) Movie S4 Li-inlA m targets both villous M cells and goblet cells in the intestinal villi upon oral inoculation of wt mice, related to Figure 5. Ileal loop of a wt mouse orally infected by Li-inlA m was taken 5 hr post infection, followed by fixation and staining for Li (green), M cells (red), goblet cells (grey) and nuclei (blue) after permeabilization. Images were acquired and assembled as described for Movie S1.

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Movie S5 Lm-inlA m targets goblet cells in the intestinal villi upon oral inoculation of wt mice, related to Figure 5. Ileal loop of a wt mouse orally infected by Lm-inlA m was taken 5 hr post infection, followed by fixation. Vibratome section was stained for Lm-inlA m (green), M cells (red), goblet cells (grey) and nuclei (blue) after permeabilization. Images were acquired and assembled as described for Movie S1. (MOV) Movie S6 Lm-inlA m targets villous M cells in the intestinal villi upon oral inoculation of wt mice, related to Figure 5. Ileal loop of a wt mouse orally infected by Lm-inlA m was taken 5 hr post infection, followed by fixation. Vibratome section was stained for Lm-inlA m (green), M cells (red), goblet cells (grey) and nuclei (blue) after permeabilization. Images were acquired and assembled as described for Movie S1. (MOV) Movie S7 Lm targets goblet cells in the intestinal villi upon oral inoculation of E16P KI mice, related to Figure 5. Ileal loop of a wt mouse orally infected by Lm was taken 5 hr post infection, followed by fixation. Vibratome section was stained for Lm (green), M cells (red), goblet cells (grey) and nuclei (blue) after permeabilization. Images were acquired and assembled as described for Movie S1. (MOV)