INFECTION AND IMMUNITY, Aug. 2004, p. 4401–4409 Vol. 72, No. 80019-9567/04/$08.00�0 DOI: 10.1128/IAI.72.8.4401–4409.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Sequence and Binding Activity of the Autolysin-Adhesin Ami fromEpidemic Listeria monocytogenes 4b

Eliane Milohanic,1,2,3 Renaud Jonquieres,2 Philippe Glaser,3 Pierre Dehoux,2 Christine Jacquet,4Patrick Berche,1 Pascale Cossart,2 and Jean-Louis Gaillard1,5*

Laboratoire de Microbiologie, Institut National de la Sante et de la Recherche Medicale U 411, Faculte de Medecine Necker-EnfantsMalades, 75730 Paris Cedex 15,1 Unite des Interactions Bacteries-Cellules,2 Laboratoire de Genomique des Microorganismes

Pathogenes,3 and Laboratoire des Listeria,4 Institut Pasteur, 75724 Paris Cedex 15, and Laboratoire de Microbiologie,Hopital Raymond Poincare (Assistance Publique-Hopitaux de Paris) and EA 3647, Faculte de Medecine

Paris–Ile-de-France–Ouest, 92380 Garches,5 France

Received 17 November 2003/Returned for modification 26 February 2004/Accepted 9 April 2004

Ami is an autolytic amidase from Listeria monocytogenes that is targeted to the bacterial surface via itsC-terminal cell wall anchoring (CWA) domain. We recently showed that the CWA domain from Ami of L.monocytogenes EGD (serovar 1/2a) (Ami 1/2a) mediated bacterial binding to mammalian cells. Here we studiedthe sequence and binding properties of Ami from CHUT 82337 (serovar 4b) (Ami 4b). The Ami 4b polypeptideis predicted to be 770 amino acids long (compared with the 917 amino acids of Ami 1/2a from EGD). Ami 1/2aand Ami 4b are almost identical in the N-terminal enzymatic domain (�98% amino acid identity), but thesequence is poorly conserved in the C-terminal CWA domain, with only �54% amino acid identity and eightGW modules in Ami 1/2a compared with six GW modules in Ami 4b. The purified Ami 4b CWA domainefficiently bound serovar 4b bacterial cells and only poorly bound serovar 1/2a bacterial cells. The Ami 4b CWAdomain was also significantly less able to bind Hep-G2 human hepatocytic cells than the Ami 1/2a CWAdomain. We sequenced the ami regions encoding CWA domains of reference strains belonging to the 12 L.monocytogenes serovars. The phylogenic tree constructed from the sequences yielded a binary division intogroup I (serovars 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, and 7) and group II (serovars 4a, 4b, 4c, 4d, and 4e). This is thefirst direct evidence of divergence between serovars 1/2a and 4b in a gene involved in the adhesion of L.monocytogenes to mammalian cells, as well as the first demonstration of allelic polymorphism correlated withthe somatic antigen in this species.

Listeria monocytogenes is a gram-positive bacillus that iswidespread in the environment and causes life-threatening in-fections in humans and animals, including meningoencephali-tis, bacteremia, and perinatal infections (7, 15). In humans, itis transmitted by contaminated food and may be responsiblefor large outbreaks in industrialized countries (7). L. monocy-togenes is a facultative intracellular pathogen that is able toinfect both professional (25) and nonprofessional phagocytes,such as epithelial cells (10, 11) and hepatocytes (5, 13, 43). Themolecular basis of its intracellular life cycle has largely beenelucidated (40). Early in the cycle, L. monocytogenes lyses thephagosomal vacuole and is released into the cytoplasm. It thendivides and spreads into adjacent cells by mediating actin as-sembly.

Listeria strains can be classified by their antigenic propertiesaccording to the serological scheme introduced by Paterson(31) and modified by Seeliger and Hohne (36). This schemedistinguishes various serovars on the basis of their somatic (O)and flagellar (H) antigens. It is based on hemagglutinationtests with different sera designated A to E for flagellar antigensand I to XV for somatic antigens. At least 13 serovars arerecognized within L. monocytogenes, and they are designatedserovars 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and

7 (38). While several serovars can be recovered from the en-vironment and from foods, serovars 1/2a, 1/2b, and 4b accountfor most human infections (7, 26, 34). Serovar 4b strains areespecially overrepresented among clinical isolates, accountingfor 40 to 60% of sporadic cases of human listeriosis. Serovar 4bis also the serovar that is most frequently involved in majoroutbreaks (7).

Ami is an autolytic amidase that was originally identified inL. monocytogenes serovar 1/2a (2, 27). Ami 1/2a, the predictedAmi of strain EGD (serovar 1/2a), the strain whose genomehas been sequenced (14), is a 917-amino-acid protein withthree characteristic domains: (i) a 30-amino-acid putative sig-nal sequence; (ii) a 179-amino-acid N-terminal domain similarto the alanine amidase domain of the Atl autolysin of Staphy-lococcus aureus; and (iii) a C-terminal cell wall anchoring(CWA) domain (amino acids 262 to 917) containing four re-peats, each composed of two approximately 80-amino-acidmodules called GW modules because of the presence of thedipeptide GW (2, 4). Ami 1/2a is exposed at the Listeria surface(2). Like other GW autolysins, the molecule is likely to besecreted and targeted to the bacterial surface via its CWAdomain (4).

Ami 1/2a is believed to contribute to the attachment of L.monocytogenes to eukaryotic cells. We recently showed thatami null mutants constructed with serovar 1/2a strains lackingInlA or InlB or both are five to 10 times less adherent than theparental strains in various cell types (28, 29). We also showedthat the adhesive properties of Ami 1/2a are carried by the

* Corresponding author. Mailing address: Laboratoire de Microbi-ologie, Hopital Raymond Poincare, 104 boulevard Raymond Poincare,92380 Garches, France. Phone: (1) 7 10 79 0. Fax: (1) 47 10 79 49.E-mail: jean-louis.gaillard@rpc.ap-hop-paris.fr.

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C-terminal CWA domain of the molecule (29). Expression ofthis domain by complementation fully restores the adhesioncapacity of ami null mutants in inlA and/or inlB backgrounds.Moreover, the purified CWA domain of Ami 1/2a binds eu-karyotic cells in a cell adhesion assay.

Paradoxically, to date there is no information about theproperties of Ami 4b, the Ami from L. monocytogenes serovar4b, the most prevalent serovar in human listeriosis. Here westudied the sequence and binding activity of Ami 4b. We foundthat the CWA domains of Ami 4b and Ami 1/2a are not similarand have distinct patterns of binding to eukaryotic cells. Wealso sequenced the Ami CWA domains of strains belonging toeach L. monocytogenes serovar. We showed that the Ami CWAsequences can be divided into two groups correlated with thesomatic antigen and the structure of teichoic acids (TA).

MATERIALS AND METHODS

Strains, plasmids, and growth conditions. The bacterial strains and plasmidsused in this study are listed in Table 1. All bacteria were grown at 37°C. Listeriaand Escherichia coli strains were grown in brain heart infusion (Difco Labora-tories, Detroit, Mich.) and Luria-Bertani (Difco Laboratories) broth and agar,respectively. For E. coli strains harboring pET28a� derivatives kanamycin wasadded to the medium at a final concentration of 30 mg/liter.

General genetic manipulations, nucleotide sequencing, and sequence analy-ses. Total DNA was isolated from Listeria cells as previously described (32).Plasmid DNA was prepared from E. coli by the rapid alkaline lysis method (1).Standard techniques were used for DNA fragment isolation, DNA cloning, andrestriction analysis (35). Restriction enzymes and ligase were purchased fromNew England Biolabs Inc. (Beverly, Mass.) and were used as recommended by

the manufacturer. DNA was amplified with DyNazyme EXT DNA polymerase(Finnzymes) or Vent DNA polymerase (New England Biolabs). The PCR con-ditions were as follows: 35 cycles of 30 s at 95°C, 45 s at 55°C, and 90 s at 72°Cin a PTC-200 thermal cycler (MJ Research). The ami 4b gene was amplified byusing primers AMI-F1 (5�-CAAGTGCTGCTTCCATTG-3�) and AMI-R1 (5�-CTGATTGATCACCCTTGG-3�). The CWA domain portion of the ami genewas amplified by using primers CWAsv1-F (5�-GATGGCAAAGGAACAGTC-3�) and CWAsv1-R (5�-CTGATTGATCACCCTTGG-3�) for serovars 1/2a, 1/2b,1/2c, 3a, 3b, 3c, and 7 and primers CWAsv4-F (5�-GTCTGGTCCCACGATGC-3�) and CWAsv4-R (5�-CTCGTCCAAATCGTCCGC-3�) for serovars 4a, 4b, 4c,4d, and 4e. Nucleotide sequencing was carried out with a Big Dye terminatorsequencing kit (Applied Biosystems, Perkin-Elmer) with fluorescently labeleddideoxynucleotides and primers. Labeled extension products were analyzed witha 3700 DNA sequencer (Perkin-Elmer). Multiple alignments were constructedwith the Clustal W program. Phylogenetic trees were constructed by using theNJplot program.

Preparation of bacterial extracts, SDS-PAGE, and Western blot analysis.Total bacterial extracts were prepared as follows. Listeria was grown in brainheart infusion broth to the exponential phase (A600, 0.8), and then 1 ml wascentrifuged. The resulting pellet was washed twice in cold water and sonicatedthree times for 5 min. The lysate was collected by centrifugation and suspendedin 1� sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)sample buffer (130 mM Tris-HCl [pH 6.8], 1% SDS, 7% 2-�-mercaptoethanol,7% sucrose, 0.01% bromophenol blue). To prepare a 1% SDS extract, 10 ml ofa culture was centrifuged, and the bacterial pellet was washed twice in phos-phate-buffered saline (PBS) and suspended in 0.2 ml of PBS containing 1% SDS.The bacterial cells were incubated for 15 min at 37°C. The supernatant collectedafter centrifugation was filtered and solubilized in 1� SDS-PAGE sample buffer.SDS-PAGE was carried out as previously described (21) in 10% polyacrylamideminigels (Mini Protean II; Bio-Rad, Ivry sur Seine, France). Proteins werestained with Coomassie brilliant blue. Western blotting was carried out as pre-viously described (29). Western blots were probed with rabbit affinity-purifiedanti-Ami antibodies or rabbit anti-InlB antibodies (2) diluted 1/1,000 and anti-

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Serovar Genotype or description Reference or source

L. monocytogenes strainsEGD (BUG600) 1/2a Virulent, clinical isolate 25LO28 1/2c Virulent, clinical isolate 41ATCC 19111 1/2a Virulent, clinical isolate 39CNL 880203 1/2b Virulent, clinical isolate 39CHUT 861141 1/2c Virulent, food environment isolate 39CHUT 82337 4b Virulent, clinical isolate 39CHUT 850212 4b Virulent, clinical isolate 39INRA 76 4b Virulent, clinical isolate 39EGD ami 1/2a ami null mutant 29CLIP 74902 1/2a Reference strain 36CLIP 74903 1/2b Reference strain 36CLIP 74904 1/2c Reference strain 36CLIP 74905 3a Reference strain 36CLIP 74906 3b Reference strain 36CLIP 74907 3c Reference strain 36CLIP 74908 4a Reference strain 36CLIP 74910 4b Reference strain 36CLIP 74911 4c Reference strain 36CLIP 74912 4d Reference strain 36CLIP 74913 4e Reference strain 36CLIP 74917 7 Reference strain 36

E. coli strainsBUG1756 E. coli BL21(DE3)/pET28.a-5 29BUG1963 E. coli BL21(DE3)/pET28.a-6 This study

PlasmidspUC18 44pET28a� Expression vector NovagenpET28.a-5 pET28 �amicwa 1/2a 29pET28.a-6 pET28 �amicwa 4b This study

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rabbit horseradish peroxidase-conjugated secondary antibodies. Antibody bind-ing was revealed by adding 0.05% diaminobenzidine tetrahydrochloride (Sigma)and 0.03% hydrogen peroxide.

Expression and purification of recombinant Amicwa-His6 polypeptides. TheCWA domain of Ami (Amicwa) of CHUT 82337 was produced as a recombinantHis-tagged polypeptide as previously described (29). A 1,582-bp PCR fragmentwas produced by using genomic DNA of CHUT 82337 as the template andprimers 5�-GGAATTCCATATGTTGATTAATGAAAAGTACAAAGCG-3�and 5�-CGCGGATCCATAATTGGCTGGGAG-3�, which introduced NdeI andBamHI sites (underlined), respectively. The resulting fragment was digested withNdeI and BamHI and inserted in frame upstream from the His tag sequence inthe expression vector pET28a� (Novagen). The resulting plasmid, pET28.a-6,was verified by sequencing the insert from both junctions. It was used to trans-form E. coli BL21(DE3) (Novagen), giving rise to BUG 1963. RecombinantAmicwa-His6 polypeptides originating from either EGD (29) or CHUT 82337(this study) were purified by using a two-step chromatographic procedure. Thefirst step, involving metal affinity chromatography (Novagen), was carried out aspreviously described (3). The fractions containing Amicwa-His6 were pooled andsubjected to cation-exchange chromatography with a POROS HS column (Phar-macia). The loading buffer contained 50 mM HEPES (pH 7.6) and 200 mMNaCl. Elution was performed with a 0.2 to 0.4 M NaCl gradient. The Amicwa-His6 polypeptide that eluted in the presence of 280 mM NaCl was dialyzed for18 h against loading buffer and concentrated by using Centriprep 50 devices(Amicon). The His tag was removed by thrombin digestion. The purified Amicwa

polypeptide was stored in 10% glycerol at �80°C. Protein concentrations weredetermined with the bicinchoninic acid system (Pierce).

Binding of Amicwa to bacterial cells. Binding assays were performed as previ-ously described (2), with minor modifications. One milliliter of an exponential-phase culture (A600, 0.8) of L. monocytogenes was washed twice in PBS, pelleted,and resuspended in 200 �l of 220 mM NaCl–PBS. Various concentrations of thepurified Amicwa-His6 polypeptide were added, and the mixtures were incubatedfor 45 min at room temperature with gentle agitation. Bacterial cells were thenwashed twice in 220 mM NaCl–PBS to remove unbound material and resus-pended in 1� SDS-PAGE sample buffer. Bound proteins were visualized byCoomassie staining after SDS-PAGE.

Culture of cell lines. The human colon carcinoma cell line Caco-2 (ATCCHTB 37), used between passages 25 and 35, was propagated as described pre-viously (12). The human hepatocellular carcinoma cell line Hep-G2 (ATCC HB8065) was propagated as described by Dramsi et al. (5). All incubations werecarried out in a 10% CO2 atmosphere at 37°C.

Cell binding assay with Amicwa-coated surface. A cell binding assay was per-formed as described previously (29). Maxisorp microtiter plates (Nunc) werecoated for 18 h at 4°C with 50 �l of purified Amicwa 1/2a or Ami 4b, with variousconcentrations of bovine serum albumin (BSA) (0.07, 0.14, 0.35, and 0.7 �M), orwith 10 �g of poly-L-lysine in 50 mM carbonate buffer (pH 9.6). Wells weretreated for 2 h at 37°C with 0.5% BSA in PBS for blocking and washed threetimes with PBS. The adhesion assay was performed as follows. Wells were filledwith 50 �l of a cell suspension (approximately 106 cells per ml) in Dulbeccomodified Eagle medium containing 0.4% BSA and incubated for 1 h at 37°C ina 10% CO2 atmosphere. After washing, bound cells were quantified by thehexosaminidase assay (22). The results are expressed below relative to the cellbinding obtained with poly-L-lysine-coated wells, which was arbitrarily defined as100.

Statistical analysis. The Student t test was used to compare values, and Pvalues of 0.05 were considered to be statistically significant.

RESULTS

CWA domains of Ami 4b and Ami 1/2a are not similar. Wesequenced the ami gene from L. monocytogenes CHUT 82337(serovar 4b) and compared it with that of L. monocytogenesEGD (serovar 1/2a). The two ami gene sequences were similarin the region encoding the enzymatic domain (97.2% nucleo-tide identity) but not in the region encoding the CWA domain(43.2% nucleotide identity). Moreover, the gene encoding theCWA domain of CHUT 82337 contained only three DNArepeats, compared to the four DNA repeats in the EGD gene.We verified that this difference was not due to a cloning arti-fact. The ami region encoding the CWA domain of CHUT82337 was amplified with the external primers AMIsv4-F and

PYRGsv4-R. The resulting PCR product was about 0.5 kbsmaller than the EGD PCR product. To ensure that the resultsobtained with CHUT 82337 were not strain specific, the amiregions encoding the CWA domains of three other serovar 4bstrains (ATCC 19115, CHUT 850212, and INRA 76) wereamplified by using the same primers. Each PCR product wasthe same size as the CHUT 82337 product (data not shown).The DNA repeat nearest the C-terminal extremity (588 bp) ofeach of the three strains was sequenced on both strands. Thesequences were 99.9 and 43.2% identical to those of CHUT82337 and EGD, respectively (data not shown), thus confirm-ing the previous results obtained with CHUT 82337.

The deduced Ami polypeptide of L. monocytogenes CHUT82337 is 770 amino acids long, whereas that of EGD is 917amino acids long (Fig. 1). CHUT 82337 Ami is organized likeAmi of EGD. It contains an N-terminal domain with putativealanine amidase activity and a C-terminal CWA domain com-posed of repeats, each made up of two GW modules (theN-terminal and C-terminal GW modules of each repeat areconventionally referred to as a and b, respectively; the a and bmodules of repeat 1 are designated R1a and R1b, respectively[4]). However, although the two proteins have almost identicalenzymatic domains (98.1% amino acid identity), their CWAdomains are different (54.2% amino acid identity). Moreover,the CWA domain of CHUT 82337 contains only three repeats(six GW modules), compared to four repeats (eight GW mod-ules) in the EGD CWA domain. In addition, the b modules ofCHUT 82337 are each four or five amino acids longer than theb modules of EGD. Another striking difference is the presenceof the short sequence RTSXTFI upstream from the GWdipeptide of the b modules in CHUT 82337. In EGD, thismotif is found at a similar location in InlB (6, 11) but not inAmi (Fig. 1).

L. monocytogenes serovar 4b produces an 85-kDa Amipolypeptide found in total cell extracts and SDS extracts. Weused Western blotting to analyze the Ami proteins from sero-var 1/2a and 4b strains. Western blots were probed with rabbitaffinity-purified anti-Ami antibodies or anti-InlB antibodies,which recognize Ami and give lower background values (2). Inaccordance with previous observations made with Ami 1/2afrom EGD (2, 29), Ami 4b was detected in total cell extractsand SDS extracts but not in culture supernatants. However, asexpected from sequence data, the Ami molecules were clearlyshorter in strains of serovar 4b than in strains of serovar 1/2a(�85 kDa versus �100 kDa) (Fig. 2). Similar data were re-ported in a recent study (18).

CWA domain of Ami 4b binds to the surface of serovar 4bcells more efficiently than to the surface of serovar 1/2a cells.We compared the abilities of the CWA domains of Ami 1/2aand Ami 4b to bind to the surfaces of serovar 1/2a and 4bbacterial cells using purified polypeptides. Binding experi-ments were performed by incubating EGD and CHUT 82337bacterial cells (serovar 1/2a and 4b cells) with the purified AmiCWA domain originating from either strain. After the prepa-rations were washed to remove nonspecifically bound material,bacterial protein extracts were separated by SDS-PAGE andstained with Coomassie blue (Fig. 3).

The purified Amicwa 1/2a bound efficiently to the serovar1/2a bacterial cells in a dose-dependent manner. Much lessAmicwa 1/2a bound to the serovar 4b cells, and binding was not

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dose dependent. This experiment was repeated with purifiedAmicwa 4b. Amicwa 4b bound to the serovar 4b cells very effi-ciently and in a dose-dependent manner. Less Amicwa 4bbound to the serovar 1/2a cells, but the binding remainedclearly dose dependent; overall, Amicwa 4b bound to the sero-var 1/2a cells slightly better than Amicwa 1/2a bound to theserovar 4b cells. Thus, the CWA domains of Ami 1/2a and Ami4b appear to bind autologous bacterial surfaces most effi-ciently. However, the CWA domain of Ami 4b may have lessselective binding capacity.

CWA domains of Ami 4b and Ami 1/2a show different pat-terns of binding to eukaryotic cells. It was recently shown that

the efficiencies with which purified Amicwa 1/2a binds to eu-karyotic cells are different for different cell lines (29). Forexample, in identical experimental conditions, Amicwa 1/2abinds to Hep-G2 hepatocytic cells with significantly greaterefficiency than it binds to Caco-2 enterocytic cells. Thus, westudied whether the CWA domains of Ami 1/2a and Ami 4bdisplay distinct patterns of binding to eukaryotic cells. Micro-titer plates were coated with various amounts of purifiedAmicwa 1/2a or Amicwa 4b and incubated with either Hep-G2or Caco-2 cells. After washing, cell binding was evaluated by ahexosaminidase colorimetric assay.

As previously reported (29), Amicwa 1/2a bound to Hep-G2

FIG. 1. Ami 4b molecule. (A) Amino acid sequence of the ami gene product from CHUT 82337. The signal peptide is underlined, as is the shortRTSXTFI sequence present in InlB (see panel C). The amidase domain (positions 1 to 261) and the CWA domain (positions 262 to 770) areindicated. The a and b modules of the three GW repeats (positions 277 to 770), designated R1a to R3a and R1b to R3b, are aligned. The repeatconsensus sequence (con) shows the amino acids that are identical in at least two of three repeats. (B) Amino acid sequence of the CWA domainof Ami 1/2a (strain EGD). The a and b modules of the four GW repeats (positions 274 to 917), designated R1a to R4a and R1b to R4b, are aligned.Dashes represent gaps introduced to maximize matching (see panel C). The repeat consensus sequence (con) shows the amino acids that areidentical in at least three of four repeats. Asterisks indicate identity with the repeat consensus sequence of Ami 4b. (C) Amino acid sequence ofthe CWA domain of InlB 1/2a (strain EGD). The a an b modules of the two GW repeats (positions 399 to 630) are designated R�1a, R�1b, R�2a,and R�2b. R�1a is missing, and R�1b is incomplete (68 amino acids). Asterisks indicate amino acids that are identical in R�2 and in the repeatconsensus sequence of Ami 4b. The short RTSXTFI sequence, common to InlB and Ami 4b and absent from Ami 1/2a (see panel B), is underlined.

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cells much more efficiently than it bound to Caco-2 cells (2.5-to 5.0-fold-greater efficiencies at concentrations of 0.35 and0.70 mM, respectively; P 0.001) (Fig. 4). This may have beendue to the fact that Hep-G2 cells express particularly highlevels of surface glycosaminoglycans (9, 30), which are the cellreceptors of InlB and possibly of Ami molecules (20). How-ever, interestingly, Amicwa 4b bound to Hep-G2 cells 1.5 to 2times less efficiently than Amicwa 1/2a bound to these cells (P 0.001), whereas the two polypeptides bound to Caco-2 cellssimilarly. Thus, Amicwa 4b and Amicwa 1/2a differ in the abilityto bind to Hep-G2 cells but not in the ability to bind to Caco-2cells.

Sequences of the Ami CWA domain in various serovars of L.monocytogenes correlate with the somatic antigen. The dissim-ilarity of the Ami 1/2a and Ami 4b CWA domains suggestedthat the CWA domains of the Ami molecules may vary amongthe serovars of L. monocytogenes. To approach this question,we amplified and sequenced the part of the ami gene encodingthe CWA domain (amicwa) in strains belonging to the 12 L.monocytogenes serovars. To prevent errors in serovar assign-ment, we sequenced the panel of strains that are used by theNational Reference Center for Listeria (Institut Pasteur, Paris,France) for the preparation of specific antisera.

The sizes of PCR products allowed us to distinguish twogroups, one that yielded products about 2.2 kb long (serovars

FIG. 2. Immunoblot analysis of Ami molecules produced by serovar 1/2 and 4b strains. Blots prepared from SDS bacterial extracts were probedwith anti-InlB (lanes 1 to 8) or anti-Ami (lanes 9 and 10) polyclonal antibodies. Lane 1, EGD as a control serovar 1/2a strain; lane 2, LO28 (serovar1/2c); lane 3, ATCC 19111 (serovar 1/2a); lane 4, CNL 880203 (serovar 1/2b); lane 5, CHUT 861141 (serovar 1/2c); lane 6, CHUT 82337 (serovar4b); lane 7, CHUT 850212 (serovar 4b); lane 8, INRA 76 (serovar 4b); lane 9, EGD (serovar 1/2a); lane 10, CHUT 82337 (serovar 4b). Note thatthe main band, corresponding to the complete form of Ami (arrowheads), is at �85 kDa in serovar 4b strains and at �100 kDa in serovar 1/2strains. The �65-kDa band present in all extracts probed with anti-InlB antibodies is InlB.

FIG. 3. Binding efficiencies of purified Amicwa 1/2a and Amicwa 4badded exogenously to L. monocytogenes bacterial cells. Equivalentnumbers of either EGD (serovar 1/2a) or CHUT 82337 (serovar 4b)cells were incubated with 0, 1, 5, or 10 �g of purified Ami CWAdomains. After washing, bound protein was detected by Coomassieblue staining. (A) Binding of Amicwa 1/2a. (B) Binding of Amicwa 4b.

FIG. 4. Binding of Amicwa 1/2a and Amicwa 4b to eukaryotic cells.Wells coated with various concentrations of purified Amicwa 1/2a orAmicwa 4b were incubated with Caco-2 or Hep-G2 cells for 2 h at 37°C.After washing, bound cells were quantified by a colorimetric hex-osaminidase assay. Values are expressed relative to the binding valueobtained with poly-L-lysine-coated wells, defined arbitrarily as 100. Thevalues are means and standard errors for two independent experi-ments.

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1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4c, and 7) and one that yieldedproducts about 1.7 kb long (serovars 4b, 4d, and 4e). DNAsequence analysis showed that the difference in size betweenthe two groups was mainly due to the presence of four DNArepeats in the first group and only three DNA repeats in thesecond group. A phylogenetic analysis based on the nucleotidesequence also allowed us to distinguish two groups; group Iconsisted of serovars 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, and 7 (sero-groups 1/2 and 3 and serovar 7), and group II consisted ofserovars 4a, 4b, 4c, 4d, and 4e (serogroup 4) (Fig. 5A). Allmembers of group I had an amicwa region consisting of about

2.2 kb containing four DNA repeats. Group II could be furtherdivided into two subgroups; members of subgroup IIa (sero-vars 4a and 4c) had an amicwa region consisting of about 2.2 kbcontaining four DNA repeats, whereas members of subgroupIIb (serovars 4b, 4d, and 4e) had an amicwa region consisting ofabout 1.7 kb containing only three repeats. The sequence sim-ilarity was very high within each group (97.1 to 100% nucleo-tide identity in group I and 89.3 to 100% nucleotide identity ingroup II) but very low between the two groups (37.3 to 46.2%nucleotide identity) (Table 2).

Analysis of deduced amino acid sequences yielded the same

FIG. 5. Phylogenetic trees based on the nucleotide (A) and amino acid (B) sequences of the Ami CWA domains from the 12 L. monocytogenesserovars. The trees were constructed with the NJplot program. The bar above each tree indicates genetic distance.

TABLE 2. Sequence similarity for group I and group II Ami CWA domains

Serovar

% Identitya

Group I Group II

Serovar1/2a

Serovar1/2b

Serovar1/2c

Serovar3a

Serovar3b

Serovar3c

Serovar7

Serovar4a

Serovar4b

Serovar4c

Serovar4d

Serovar4e

1/2a 98.9 99.4 99.4 98.8 99.4 98.8 54.0 54.4 43.9 54.4 54.41/2b 97.2 98.6 98.9 99.8 98.6 99.8 43.9 54.4 43.9 54.4 54.41/2c 99.2 97.2 99.4 98.5 100 98.5 54.1 54.4 43.9 54.4 54.43a 99.0 97.3 99.0 98.8 99.4 98.8 43.9 54.0 43.75 54.0 54.03b 97.1 99.8 97.1 97.3 98.5 99.7 43.75 54.2 43.75 54.2 54.23c 99.2 97.2 100 99 97.1 98.5 54.1 54.4 43.9 54.4 54.47 97.1 99.9 97.2 97.3 99.8 97.2 54.0 54.4 43.9 54.4 54.44a 46.1 38.0 46.2 46.2 38.0 46.2 38.0 95.1 98.1 95.1 94.94b 43.2 43.1 43.0 46.1 43.1 43.0 43.1 90.4 94.1 100 99.84c 37.3 37.5 37.3 37.4 37.6 37.3 37.6 96.8 89.6 94.1 93.94d 43.2 43.1 43.0 43.1 43.1 43.0 43.1 90.4 100 89.5 99.84e 43.3 43.1 43.0 43.3 43.1 43.0 43.2 90.1 99.5 89.3 99.5

a The levels of amino acid and nucleotide identity are shown on the upper right and the lower left, respectively.

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division into group I (serogroups 1/2 and 3 and serovar 7) andgroup II (serogroup 4) and the same subdivision of group IIinto subgroup IIa (serovars 4a and 4c) and subgroup IIb (se-rovars 4b, 4d, and 4e) (Fig. 5B). The levels of amino acididentity ranged from 98.5 to 100% within group I and from93.9 to 100% within group II but did not exceed 54.4% whencomparisons were made between the two groups (Table 2).Figure 6A shows the structural organization of the CWA do-mains within each group. The Ami CWA domain is 656 aminoacids long and contains four 160-amino-acid repeats (eightGW modules) in group I, is 674 amino acids long and contains

four 165-amino-acid repeats (eight GW modules) in subgroupIIa, and is 509 amino acids long and contains three 165-amino-acid repeats (six GW modules) in subgroup IIb. These dataneed to be confirmed with a larger number of strains. How-ever, they are fully consistent with a recent study showing thatAmi has an apparent molecular mass of �80 kDa in serovar 4band �98 kDa in serovars 1/2a, 1/2b, 1/2c, 3c, and 7 (18).Sequence alignments revealed that the distribution of chargedand hydrophobic amino acid residues is remarkably conserved(Fig. 6B). The analysis also revealed a short sequence of vari-able length located just upstream of the GW dipeptide of b

FIG. 6. Ami CWA domains from the 12 serovars of L. monocytogenes. (A) General organization of the Ami CWA domains in the variousserovars. (B) Sequence alignments. We defined a consensus sequence (threshold, 96%) from the repeat region of each group. Dots indicateresidues that are completely conserved in all repeats; positions are shaded if the chemical nature of the residues was conserved in all the sequenceswithin the repeats, as follows: h, aliphatic residues (ILV); , aromatic residues (YFWH) (of the hydrophobic residues); �, basic residues (KHR);and -, acidic residues (ED) (of the polar residues). Secondary structure predictions are shown below the sequences; �-strands are represented byarrows.

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modules, which presumably corresponds to a loop. Finally, thecomparison of secondary structures predicted from consensussequences suggested that Ami proteins produced by the vari-ous serovars have a common structure (Fig. 6B).

DISCUSSION

A number of previous studies have attempted to identify acorrelation between virulence gene allelic diversity and sero-vars. However, analyses of polymorphism of hly (also calledhlyA or lisA previously), mpl, plcA, inlA, and inlB have failed tofind factors involved in L. monocytogenes pathogenicity thatare serovar dependent (6, 33, 41, 42). We provide here the firstexample of allelic polymorphism correlated with the somaticantigen in a gene involved in the pathogenicity of L. monocy-togenes. Our results indicate that O antigens I, II, IV, XII, andXIII occur exclusively in group I of Ami sequences (serogroups1/2 and 3 and serovar 7) and O antigens V, VI, VII, VIII, IX,and X occur exclusively in group II (serogroup 4), while Hantigens A, B, C, and D occur in both group I and group II.This clearly shows that the sequence of the CWA domain ofAmi molecules correlates with the somatic antigen and notwith the flagellar antigen.

These data suggest that the anchoring of Ami involves abacterial factor that varies with the somatic antigen. This factormight be the cell wall TA as these molecules are major com-ponents of the somatic antigens (8). TA consist of polyribitol-phosphate chains that may be decorated with various sugar orN-acetylamino sugar residues. In serogroups 1/2 and 3 andserovar 7, the polyribitolphosphate chains are replaced by rham-nose and/or N-acetylglucosamine at C-2 and/or C-4 of ribitol-phosphate. In serogroup 4 (and serogroups 5 and 6 in thegenus Listeria), N-acetylglucosamine is integrated into thepolymer chains and attached to the C-2 or C-4 hydroxyl groupof ribitolphosphate; replacement by glucosyl or galactosyl res-idues may occur at C-3 and/or C-6 of N-acetylglucosamine.Given the differences between the Ami CWA domains ingroups I and II and the strong similarities of these domainswithin each group, it is more likely that the structure of thepolyribitolphosphate backbone plays an important role in theanchoring of Ami rather than TA-associated decorating sug-ars. Similar observations have been made with L. monocyto-genes phage endolysins Ply118 and Ply500 (23), which bind tothe surfaces of serogroup 1/2 and 3 and serovar 7 Listeria cellsand to serogroup 4 and 6 and serovar 5cells, respectively, viathe C-terminal binding domain (24).

The supposed role of bacterial factors correlated with thesomatic antigen for the targeting of Ami molecules may ex-plain why, when added exogenously to bacterial cells, the CWAdomains of Ami 4b and Ami 1/2a bind more efficiently tobacterial cells belonging to the same serovar. This is also con-sistent with recent experiments comparing expression of theCWA domains of Ami 1/2a and Ami 4b in Listeria innocuaserovar 6a, which displays TA similar to serogroup 4 TA; inthis bacterial host, the Ami 4b CWA domain is expressed muchbetter than the Ami 1/2a CWA domain at the bacterialsurface (Milohanic, unpublished data). These data are rem-iniscent of a previous study in which the workers comparedproduction of an InlB-Ami hybrid comprising the first 398amino acids of InlB and the eight GW modules of Ami 1/2a

in L. monocytogenes EGD (serovar 1/2a) and L. innocuaBUG 499 (serovar 6a) (3, 19). This study showed that thehybrid protein was entirely surface associated when it wasproduced in L. monocytogenes and was mostly secreted intothe supernatant in L. innocua. Thus, the Ami CWA domainclearly needs molecules that are linked to or part of theautologous somatic antigen to efficiently target Ami to thebacterial surface.

It was recently shown that Ami contributes to the adhesionof L. monocytogenes serovar 1/2a to mammalian cells via itsCWA domain (29). We now present evidence that the Amimolecules produced by serovar 1/2a and 4b strains displaydistinct adhesion patterns in two cell models widely used toevaluate the interaction of Listeria with human cells, the hepa-tocytic Hep-G2 model (5) and the enterocytic Caco-2 model(10, 11); while the two CWA domains bind to Caco-2 humanenterocytic cells similarly, the Ami 1/2a CWA domains bind toHep-G2 human hepatocytic cells about two times more effi-ciently. The molecular basis of the different adhesion patternsof the CWA domains of Ami 1/2a and Ami 4b remains to beelucidated. It is possible that Ami 4b binds to cells less effi-ciently because it contains fewer GW modules. Adhesion ac-tivity is strong with the unprocessed form of the staphylococcalautolysin-adhesin AtlE from Staphylococcus epidermidis, whichhas six GW modules, and is weaker with the amidase- andglucosaminidase-processing products, which only have fourand two GW modules, respectively (16). Similar observationshave been made with the autolysin-adhesin Aas from Staphy-lococcus saprophyticus (17).

Whether the differences in the adhesion patterns of Ami1/2a and Ami 4b have a significant impact in terms of patho-genicity remains an open question. Ami is not per se a majoractor in the interaction between Listeria and host cells. The lossof adhesion resulting from inactivation of ami is significantonly in inlA and/or inlB mutants, probably because InlA andInlB largely overcome the defect in the Ami cell adhesionfunction (28, 29). However, we believe that Ami contributesto the fine-tuning of the molecular events that occur in theListeria-cell interaction process. It was shown previouslythat overexpression of Ami CWA severely inhibited theentry of L. monocytogenes into Hep-G2 cells but not theentry into Caco-2 cells (29). This surprising result suggeststhat if the association between bacteria and the cell surfaceis too strong, as is the case with Hep-G2 cells, the entryprocess may be hindered. Alternatively, since the CWA do-mains of Ami 1/2a and InlB both have a strong affinity forglycosaminoglycans, Ami 1/2a may hinder the binding ofInlB to its glycosylated receptor, the Met receptor tyrosinekinase (20, 37). Ami 1/2a may thus interfere with the entryprocess mediated by InlB, which is more important for theinvasion of Hep-G2 cells than for the invasion of Caco-2cells (3). Whatever the underlying mechanism involved, theoverall weaker adhesion of Ami 4b may be an advantage forthe invasion of certain host cells. This may result in a some-what different behavior of L. monocytogenes serovar 4b inthe living host. To approach this question, it is necessary toevaluate the pathogenicity of ami null mutants constructedin serovar 1/2a and 4b backgrounds.

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ACKNOWLEDGMENTS

We thank H. Bierne for kindly providing pET28.a-5, P. Velge for thegift of strains, and C. Buch and I. Senegas for critical reading of themanuscript.

This work was supported by the Institut Pasteur, the Paris V Uni-versity, the Ministere de l’Education Nationale, de la Recherche et dela Technologie, the EU (grant BMH4CT96 0659/RA03813), the Fon-dation pour la Recherche Medicale, GlaxoWellcome, and SmithKlineBeecham. P.C. is an international investigator of the Howard HughesMedical Institute.

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