LETTERS

Conjugated action of two species-specific invasionproteins for fetoplacental listeriosisOlivier Disson1,2*, Solene Grayo1,2,3*, Eugenie Huillet4,5,6*, Georgios Nikitas1,2, Francina Langa-Vives7,Olivier Dussurget4,5,6, Marie Ragon3, Alban Le Monnier3, Charles Babinet8{, Pascale Cossart4,5,6 & Marc Lecuit1,2,3,9

The ability to cross host barriers is an essential virulence deter-minant of invasive microbial pathogens. Listeria monocytogenes isa model microorganism that crosses human intestinal and placen-tal barriers, and causes severe maternofetal infections by anunknown mechanism1. Several studies have helped to characterizethe bacterial invasion proteins InlA and InlB2. However, theirrespective species specificity has complicated investigations ontheir in vivo role3,4. Here we describe two novel and complemen-tary animal models for human listeriosis: the gerbil, a natural hostfor L. monocytogenes, and a knock-in mouse line ubiquitouslyexpressing humanized E-cadherin. Using these two models, weuncover the essential and interdependent roles of InlA and InlBin fetoplacental listeriosis, and thereby decipher the molecularmechanism underlying the ability of a microbe to target and crossthe placental barrier.

Several microorganisms that cross the intestinal barrier also crossthe placental barrier. Yet little is known about the mechanismsunderlying their fetoplacental tropism. As for most vertically trans-mitted infections, maternofetal listeriosis is associated with high neo-natal mortality, even when adequate antimicrobial therapy isadministered1,5. Pregnancy-associated immunosuppression isthought to favour systemic microbial dissemination6, but an electivemicrobial targeting and crossing of the placental barrier relying onspecific interactions of microbial ligands with placental receptors,although attractive, has never been demonstrated in vivo.

Listeria monocytogenes is a model microorganism for in vitro andin vivo studies2. Two bacterial proteins, InlA and InlB, mediate itsinternalization into human cultured non-phagocytic cells2,7,8. Thereceptor for InlA is E-cadherin (Ecad), whereas the receptor for InlBis Met, together with gC1qR and proteoglycans9–12. InlA and InlBinteractions with their respective receptors are species-specific: InlAinteracts with human and guinea-pig Ecad, but not mouse and ratEcad3; whereas InlB interacts with human and mouse Met, but notguinea-pig Met4. In transgenic mice expressing human Ecad inenterocytes (iFABP-hEcad mice), InlA has a critical role in L. mono-cytogenes crossing of the intestinal barrier13, whereas InlB has norole4.

Epidemiological investigations also support a role for InlA inhuman listeriosis14, as clinical strains express a functional InlA farmore frequently (96%) than strains recovered from food products(65%) (P , 0.001)14. To specifically address the contribution of InlAin pregnancy-related cases, we performed a larger epidemiologicalsurvey (Supplementary Methods). This demonstrated that expression

of full-length InlA is significantly associated with L. monocytogenesisolates from pregnancy-related cases, if compared with those of bac-teraemia origin (P 5 0.01, Supplementary Table 1), a result in favourof a role for InlA in human fetoplacental infection. Results of ex vivoinfections of human placental explants also suggest a role for InlA, aswell as InlB, in placental infection15. In line with these results, thehuman trophoblastic cell line JAR is permissive to both InlA andInlB pathways (Supplementary Fig. 1). Yet, in contradiction with theseepidemiological, ex vivo and in vitro results, InlA has no role inL. monocytogenes fetoplacental infection in the guinea-pig, despiteits permissiveness to the InlA pathway16. Similarly, InlB has no rolein L. monocytogenes fetoplacental infection in the mouse, despite itspermissiveness to the InlB pathway17.

Given our observations in human placental explants that InlAexerts a role in placental tissue invasion in the presence of InlB, wehypothesized that both InlA and InlB might be critical for fetopla-cental invasion in vivo. Accordingly, only an animal model in whichboth pathways are functional would constitute an appropriate systemto study fetoplacental listeriosis and its molecular determinants. Herewe characterize two novel and complementary animal models forhuman listeriosis: a natural host for L. monocytogenes, the gerbil,and a novel knock-in mouse line, and we reveal the criticaland interdependent roles of InlA and InlB in fetoplacental listerio-sis. Ligand–receptor interactions mediating microbial targetingand crossing of the placental barrier have not been documentedpreviously.

In 1927, J. H. Harvey Pirie discovered a novel Gram-positive bacil-lus, which he named Listerella hepatolytica (later renamed L. mono-cytogenes), as a cause of lethality in wild gerbils, indicating theirnatural susceptibility to this bacterial species18. From this seminalobservation and the fact that gerbils are closely related to rodentspermissive to either InlA or InlB pathway (Supplementary Fig. 2), wetested the hypothesis that the gerbil might be permissive to bothpathways and therefore constitute a suitable model to test therespective contributions of InlA and InlB in vivo, notably in thecontext of fetoplacental listeriosis. First, we isolated primary gerbilintestinal epithelial cells and showed that they co-express the InlAand InlB receptors Ecad and Met (Fig. 1a–c). We next conducted agentamicin survival assay and demonstrated that, in contrast to whatis observed in the rat and guinea-pig, respectively, both InlA andInlB pathways are functional in the gerbil, as in humans (Fig. 1d,Supplementary Fig. 3). We determined the amino-acid sequence ofgerbil Ecad first extracellular domain (EC1): it harbours a proline at

{Deceased.

1Institut Pasteur, Groupe Microorganismes et Barrieres de l’Hote, Unite des Interactions Bacteries-Cellules, F-75015 Paris, France. 2Inserm Avenir U604, F-75015 Paris, France.3Institut Pasteur, Centre National de Reference des Listeria, F-75015 Paris, France. 4Institut Pasteur, Unite des Interactions Bacteries-Cellules, F-75015 Paris, France. 5Inserm U604,F-75015 Paris, France. 6INRA USC2020, F-75015 Paris, France. 7Institut Pasteur, Centre d’Ingenierie Genetique Murine, F-75015 Paris, France. 8Institut Pasteur, Unite de Biologie duDeveloppement, F-75015 Paris, France. 9Centre d’Infectiologie Necker-Pasteur, Service des Maladies Infectieuses et Tropicales, Hopital Necker-Enfants malades, AssistancePublique-Hopitaux de Paris, Universite Paris Descartes, F-75015 Paris, France.*These authors contributed equally to this work.

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position 16, as for other Ecads able to interact with InlA3,19

(Supplementary Fig. 2a,b). We also determined the amino-acidsequence of the gerbil Met region orthologous to the human oneinteracting with InlB20: it clusters with species permissive to InlBpathway, whereas species non-permissive to InlB (guinea-pig andrabbit) harbour key amino-acid differences in the InlB recognitiondomain of Met (Supplementary Fig. 2c,d). Together, these in vitroresults demonstrate that the gerbil is naturally permissive to bothInlA and InlB pathways, and thus constitutes an appropriate modelto address the roles of these two invasion proteins in the successivesteps of listeriosis.

We next performed oral inoculation of gerbils and showed thatInlA plays a critical role in the crossing of the intestinal barrier(Supplementary Fig. 4), as in guinea-pig and iFABP-hEcad mice13.Interestingly, we demonstrated that InlA-dependent invasion byL. monocytogenes is not restricted to the small intestine, but alsooccurs in the caecum and colon. This is in contrast to what isobserved in iFABP-hEcad mice, in which hEcad expression isrestricted to enterocytes of the small intestine. We also showed thatInlB is not involved in the crossing of the intestinal barrier, as iniFABP-hEcad mice13,21. These results in orally inoculated gerbilsprompted us to investigate maternofetal listeriosis in pregnantgerbils.

We first tested gerbil permissiveness to listerial fetoplacental infec-tion after oral inoculation, the natural route for human infection. Byusing bioluminescent L. monocytogenes, we showed that fetoplacentallisteriosis develops in orally inoculated gerbils, and leads to 100%fetal lethality with wild-type (WT) L. monocytogenes. In contrast, theinlAB isogenic mutant led to a weaker fetoplacental infection and nofetal lethality (Supplementary Fig. 5). To assess directly the role ofInlA and InlB in fetoplacental infection, and bypass the filter effect ofthe intestinal barrier, we intravenously inoculated pregnant gerbils.Real-time imaging of fetoplacental listeriosis in these gerbils clearlyrevealed the contribution of the inlAB operon in fetoplacental infec-tion (Fig. 2a–c). Histological analysis of the placentas showed the

co-expression of Ecad and Met at the syncytiotrophoblast level,which is the epithelial tissue that constitutes the maternal blood–fetalinterface (Fig. 2d–f). At 24 h after infection, WT L. monocytogeneswere detected at the syncytiotrophoblast–fetal vessel interface,whereas none were observed with the inlAB mutant. At 42 h afterinfection, we detected small abscesses with WT L. monocytogenes,whereas no abscess was detectable with the inlAB mutant (12 vibra-tome sections examined, four abscesses with 20–200 bacteria perabscess for L. monocytogenes, none for the inlAB mutant; P 5 0.02)(Fig. 2g–i and Supplementary Movies 1–3).

To evaluate and quantify the contribution of InlA and InlB infetoplacental infection, we used the competition index method, andco-infected pregnant gerbils intravenously with a chloramphenicol-resistant L. monocytogenes strain and its parental WT strain, or eachisogenic inlA, inlB or inlAB deletion mutant (see Methods). Usingthis technique, a competition index equal to 1 indicates a similarvirulence of the two strains, a competition index significantly greaterthan 1 indicates a defect in virulence of the co-administered Listeriastrain and a competition index significantly less than 1 indicates thecontrary. Fetoplacental infection was steadily observed for inoculagreater than 2 3 105 (Supplementary Fig. 6). In contrast to what wasobserved with WT L. monocytogenes, the inlA, inlB and inlABmutants all showed a significant defect in placental invasion(Fig. 3a and Supplementary Fig. 7a) and fetal infection (Fig. 3band Supplementary Fig. 7b), with a competition index very signifi-cantly different from 1 (P , 0.001) at the placental level 72 h afterinfection and at the fetal level 96 h after infection, whereas no sig-nificant difference was observed in maternal liver (SupplementaryFig. 7c). These results sharply contrast with those observed in preg-nant mice and guinea-pigs, for which, as previously reported16,17,neither InlA nor InlB played any role in fetoplacental invasion(Supplementary Fig. 8). Together, these results establish that inspecies in which either the InlA or the InlB internalization pathwayis non-functional, neither InlB nor InlA, respectively, can mediateinvasion at the placental level. In contrast, in a species in which bothpathways are functional, both InlA and InlB are critical for placentalinvasion and fetal infection. These in vivo results obtained in anaturally permissive model, the gerbil, are in full agreement withhuman epidemiological and ex vivo data (refs 14 and 15, andSupplementary Table 1).

To assess the causal relation between permissiveness to InlA andInlB pathways and InlA- and InlB-dependent placental invasion andfetal dissemination, we followed a gain-of-function approach andgenerated a humanized E16P knock-in mouse line in which theendogenous mouse Ecad is punctually modified (‘humanized’) sothat it harbours a proline at position 16 in place of a glutamic acid(see Methods and Supplementary Fig. 9). This single amino-acidsubstitution is sufficient to convert mouse Ecad into an InlA rece-ptor3. We first infected primary enterocytes isolated from homo-zygote E16P1/1 mice: bacteria expressing InlA specifically adheredto and entered these cells, and recruited E16PmEcad, in contrast towhat was observed with primary enterocytes of WT mice (Fig. 4a).This demonstrated that mutated E16P mEcad acts as an InlA recep-tor, in contrast to WT mEcad, as previously reported3. The func-tionality of the InlA and InlB pathways in homozygote E16P1/1

mice was also demonstrated by gentamicin protection assay(Fig. 4b). We next inoculated knock-in mice orally, and showed,as in iFABP-hEcad mice, guinea-pigs13 and gerbils, that InlA med-iates the crossing of the intestinal barrier (Fig. 4c). As observed ingerbils, but in contrast to what was observed in iFABP-hEcad micein which hEcad expression is restricted to the small intestine13, InlA-dependent translocation was observed at the caecum and colonlevel, that is in tissues in which Ecad is physiologically expressed(Fig. 4c).

We next assessed competition indexes in pregnant E16P1/1 miceinoculated intravenously. In sharp contrast to WT mice (ref. 17 andSupplementary Fig. 8), knock-in mice exhibit striking InlA- and

EGD WTEGD inlA∆EGD inlB∆EGD inlAB∆

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Figure 1 | InlA and InlB internalization pathways in gerbils. Primary gerbilenterocytes were labelled with Dapi (blue), an anti-Ecad (red, a) and ananti-Met (green, b) antibody, (c: overlay). d, Entry of L. monocytogenes(EGD WT) and isogenic mutants DinlA, DinlB and DinlAB in human(Caco-2), rat (IEC-6), guinea-pig (GPC16) and gerbil primary enterocytes.Cells were incubated for 1 h with bacteria, 1 h with gentamicin and lysed.CFUs of gentamicin-resistant bacteria were enumerated. Values formutants are expressed as the percentage of recovered bacteria relative toEGD WT, for which the value is normalized to 100. Asterisks indicate asignificant difference (P , 0.05) relative to EGD WT. Scale bar, 10 mm.Error bars, s.d.

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InlB-dependent fetoplacental invasion phenotypes (P , 0.001)revealed by the acquisition of a functional InlA pathway, in thepresence of a pre-existing InlB pathway (Fig. 4d). In agreement withthese results, when comparing the barrier function of the placenta—ratio of bacterial counts between placentas and fetuses—of non-permissive models (guinea-pig, mouse) with a permissive model(E16P mEcad), a difference of more than one order of magnitudeis observed (Supplementary Fig. 10).

Our study demonstrates the concordance of epidemiological,in vitro, ex vivo and in vivo data and establishes that L. monocytogenesspecifically targets the placenta in vivo, if and only if both InlA andInlB pathways are functional. These data shed light on previousreports using the mouse or guinea-pig as models that found no rolefor InlA or InlB in placental invasion16,17: these studies had not takeninto account the double species specificity of L. monocytogenes andthe possibility that both InlA and InlB pathways had to be functionalto unveil specific invasion of the placental tissue by L. monocytogenes.To our knowledge, this is the first report demonstrating the species-and tissue-specific recognition of the placenta by a microbial patho-gen enabling it to breach the placental barrier.

The inlAB operon is located in the L. monocytogenes genome awayfrom the ‘virulence locus’, which harbours the genes critical forL. monocytogenes intracellular life22,23. The precise roles of InlA andInlB in vivo have long remained elusive. We had shown the role ofInlA at the intestinal barrier level13, but found no role for InlB at thisearly step of the infection4. Here we demonstrate a role for InlB,in combination with InlA, in fetoplacental invasion, a key step oflisteriosis in its natural hosts. This demonstrates that fetoplacental-specific tropism of L. monocytogenes correlates with host permissive-ness to both InlA and InlB. Accordingly, cattle and sheep, which arenatural hosts for L. monocytogenes and develop fetoplacental lister-iosis in natura, are both permissive to InlA and InlB (unpublisheddata).

Some questions remain about the mechanism of L. monocytogenestargeting and crossing the placental barrier. Indeed, in vitro, bothInlA and InlB are sufficient to mediate entry into cells expressingtheir respective receptors24,25. In addition, at the intestinal barrierlevel, InlA does not require InlB to exert its function4. This suggeststhat InlA could be sufficient for placental targeting and crossing.Here we show that this is not the case. To our knowledge, the placentais the only tissue—with brain microvessels, although this is contro-versial26—for which Ecad is accessible from the blood systemiccirculation. This suggests that InlA could mediate adherence ofmaternal blood-borne bacteria to the syncytiotrophoblast maternal–fetal interface. However, InlA seems insufficient to mediateinvasion in the absence of InlB, possibly because of the non-functionality, at the syncytiotrophoblast level, of the downstreameffectors needed for InlA-mediated internalization2. On the otherhand, InlB, in the absence of a functional InlA pathway, also appearsunable to fulfil its function in vivo, contrary to what is observed invitro25. This suggests that InlA-mediated bacterial adhesion is a pre-requisite for InlB-mediated placental tissue invasion. In line with thishypothesis, InlB is a weak adhesin loosely attached to the bacterialsurface27, and InlB-mediated adherence of L. monocytogenes to theplacenta could thus be insufficient in vivo because of the shear stressat the maternal blood–placenta interface. Understanding how InlAand InlB act in concert to mediate placental invasion and why thiscontrasts with the InlA-dependent, albeit InlB-independent, intest-inal invasion will be critical to our complete understanding of thebehaviour of L. monocytogenes in vivo.

Other microbial pathogens share with L. monocytogenes the abilityto breach host barriers. Identification of the ligand–receptor inter-actions mediating microbial crossing of host barriers may helpin developing inhibitors to these interactions. Conversely, theseinteractions may also be exploited to target therapeutic moleculesacross host barriers.

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Figure 2 | In vivo real-time imaging offetoplacental listeriosis in gerbils. a, Timecourse of infection of pregnant gerbils inoculatedintravenously with 2 3 106 bioluminescent EGDWT or EGD DinlAB mutant. b, Dissection at day5 after infection of the same animals, showing afar greater placental bioluminescence with EGDWT than DinlAB. c, Fetuses retrieved from twoEGD WT or one EGD DinlAB-inoculatedpregnant gerbil at day 5 after infection.d–i, Immunolabelling of gerbil placentas.d–f, Expression of Ecad (red) and Met (green) bythe syncytiotrophoblast barrier bathed inmaternal blood and surrounding fetal capillaries(isolectin B4, cyan) (g). Nuclei are labelled withDapi (blue) (g–i). h, i, Sections of placentas 24and 72 h after intravenous infection with EGDWT: h, a bacterium located at thesyncytiotrophoblast–fetoplacental capillaryinterface; i, a placental abscess 72 h after infectionwith EGD WT. Scale bar, 10mm.

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METHODS SUMMARYBacteria and cell culture conditions. The Listeria strains and cell lines used are

listed in the Supplementary Methods. Primary enterocytes were prepared as

previously described28.

Cell and tissue immunolabelling. Cell and tissue immunolabelling was per-

formed as previously described15. Mouse and gerbil E-cadherins were stained

with the ECCD2 antibody (Takara). Met was stained with the SP-260 antibody

(Santa Cruz). Endothelial cells were stained with isolectin B4, and bacteria with

R11 (L. monocytogenes) and R6 (Listeria innocua) antibodies29.

Animal experiments: infection. Animal experiments were performed according

to the Institut Pasteur guidelines for the husbandry of laboratory animals.

Pregnant gerbils were infected at day 18/26 of gestation, pregnant mice at day

14/21 and pregnant guinea-pigs at day 36/60. Oral infections were performed as

previously described13. In gerbils, intravenous infections were performed surgic-

ally in the femoral vein as described30.

Animal experiments: bacterial enumeration. At indicated times, animals were

killed and their organs harvested. The intestine, colon and caecum were rinsed,

incubated in DMEM medium containing gentamicin and rinsed again. All

organs were then homogenized in PBS buffer and colony-forming units

(CFUs) enumerated.

Animal experiments: bioluminescence real-time imaging. Animals were

infected with the luminescent EGD WT (BUG2473) and EGD DinlAB

(BUG2474) strains. Bioluminescence, measured in relative light units (photons

per second), was monitored each day in a Xenogen IVIS 100 system. At indicated

times, animals were killed and the bioluminescence of the internal organs mea-

sured.

Animal experiments: competition index assay. Both CmR reference EGD WT

(BUG2472) and chloramphenicol-sensitive (CmS) test strains were mixed in a

1:1 ratio to perform infection. At indicated times, animals were killed and their

organs homogenized in PBS. Serial dilutions of the homogenates were plated

onto brain–heart infusion (BHI) agar plates with or without chloramphenicol.

Competition indexes were calculated by dividing the number of CmR CFUs by

the number of CmS CFUs.

Received 19 March; accepted 30 July 2008.Published online 17 September 2008.

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4. Khelef, N., Lecuit, M., Bierne, H. & Cossart, P. Species specificity of the Listeriamonocytogenes InlB protein. Cell. Microbiol. 8, 457–470 (2006).

NS NSNS

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Figure 4 | Infection of primary epithelial cells and mice expressing ahumanized mEcad able to interact with InlA. a, InlA-mediated adhesion oncells expressing E16P mEcad, but not on those expressing WT mEcad.L. innocua (InlA) recruits E16P mEcad (boxed area and inset). Scale bar,10 mm. b, Entry of L. monocytogenes (EGD WT) and DinlA, DinlB andDinlAB mutants in enterocytes of E16P1/1 mice. c, InlA-dependentintestinal invasion in E16P1/1 mice infected orally with 109 CFUs anddissected 48 h after infection. In a, b, asterisks indicate significant differencesrelative to EGD WT (*P , 0.05; **P , 0.01). Error bars, s.d. (b, c).d, Competition indexes of DinlA, DinlB and DinlAB deletion mutantsagainst EGD WTr at 72 h after infection. E16P1/1 pregnant mice wereinoculated intravenously, inoculum 2 3 105 CFUs. Each dot represents aninfected organ, a cross an uninfected one. Refer to Fig. 3 for details.

Placenta

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EGD WTr / EGD inlABs∆EGD WTr / EGD inlAs∆EGD WTr / EGD inlBs∆

Figure 3 | Interdependent roles of InlA and InlB in placental invasion andfetal infection in gerbils. Competition index of EGD WT and DinlA, DinlBand DinlAB mutants tested against chloramphenicol-resistant EGD WTr.Pregnant gerbils were inoculated intravenously with a 1:1 mixture of the twostrains (total dose 2 3 106 CFUs). Bacteria were enumerated at 72 and 96 hafter infection and competition indexes calculated for placenta (a) andfetuses (b). Each dot represents one placenta or fetus, a cross an uninfectedorgan. Each vertical dot alignment originates from one mother. Small,coloured bars represent mean competition indexes for placentas or fetuses ofone animal. Large, black bars represent mean competition indexes for threeanimals. Asterisks indicate that the competition index is significantly greaterthan one (*P , 0.05; **P , 0.01; ***P , 0.001), NS a non-significantdifference.

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements This paper is dedicated to the memory of Charles Babinet, ourfriend and colleague, who died a few days before the submission of this manuscript.We thank his collaborators S. Vandormael-Pournin, C. Kress andM. Cohen-Tannoudji, as well as L. Larue and S. Tajbakhsh for help in generating theknock-in mice. We thank C. Hill for the gift of the pPL2lux-PhlyA plasmid, M.-A.Nahori for help with animal experiments, P. Roux for help with confocal imaging,P.-M. Lledo and M. Gabellec for help with vibratome sectioning, V. Masse for helpwith statistical analysis and O. Lortholary for his support. We also thankS. Mostowy for reading the paper. This work received financial support from theInstitut Pasteur, Inserm and INRA. O. Disson received financial support from theFondation pour la Recherche Medicale (FRM) and Inserm, P.C. is an HowardHughes Medical Institute international research scholar and M.L. is a recipient ofan Inserm interface contract.

Author Contributions M.L. planned the project and analysed the experiments,together with P.C., as well as O. Disson, S.G., E.H. and G.N. O. Disson, S.G., E.H. andG.N. performed the experiments. Engineering of knock-in mice was done withF.L.-V. and C.B. O. Dussurget was involved in bioluminescence imaging; M.R. andA.L.M. were involved in the epidemiological study. M.L. wrote the manuscript andall co-authors commented on it.

Author Information The Gerbil Ecad and Gerbil Met nucleotide sequences aredeposited in GenBank under accession numbers EU878370 and EU878371,respectively. Reprints and permissions information is available atwww.nature.com/reprints. Correspondence and requests for materials should beaddressed to M.L. (mlecuit@pasteur.fr).

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