Muramylpeptide shedding modulates cell sensing ofShigella flexneri

Giulia Nigro,1 Luigi Lembo Fazio,1

Maria Celeste Martino,1 Giacomo Rossi,2

Ivan Tattoli,1† Valeria Liparoti,3 Cristina De Castro,3

Antonio Molinaro,3 Dana J. Philpott,4† andMaria Lina Bernardini1,5*1Dipartimento di Biologia Cellulare e dello Sviluppo,Sapienza-Università di Roma, Via dei Sardi 70,00185 Roma, Italy.2Facoltà di Medicina Veterinaria, Università di Camerino,62024 Matelica, MC, Italy.3Dipartimento di Chimica Organica e Biochimica,Università di Napoli Federico II, I-80126, Napoli, Italy.4Groupe d’Immunité Innée et Signalisation,Institut Pasteur, 75724 Paris Cedex 15, France.5Istituto Pasteur-Fondazione Cenci Bolognetti,Sapienza-Università di Roma, Piazzale Aldo Moro 5,00185 Roma, Italy.

Summary

Bacterial infections trigger the activation of innateimmunity through the interaction of pathogen-associated molecular patterns (PAMPs) with patternrecognition molecules (PRMs). The nucleotide-binding oligomerization domain (Nod) proteins areintracellular PRMs that recognize muramylpeptidescontained in peptidoglycan (PGN) of bacteria. It is stillunclear how Nod1 physically interacts with PGN,a structure internal to the Gram-negative bacterialenvelope. To contribute to the understanding of thisprocess, we demonstrate that, like Escherichia coli,Bordetella pertussis and Neisseria gonorrheae, theGram-negative pathogen Shigella spontaneouslyreleases PGN fragments and that this process canbe increased by inactivating either ampG or mppA,genes involved in PGN recycling. Both Shigellamutants, but especially the strain carrying the mppAdeletion, trigger Nod1-mediated NF-kB activation to agreater extent than the wild-type strain. Likewise,muramylpeptides spontaneously shed by Shigella areable per se to trigger a Nod1-mediated response con-sistent with the relative amount. Finally, we found that

qualitative changes in muramylpeptide shedding canalter in vivo host responses to Shigella infection.Our findings support the idea that muramylpeptidesreleased by pathogens during infection could modu-late the immune response through Nod proteins andthereby influence the outcome of disease.

Introduction

A certain number of invariant bacterial structures,so-called pathogen-associated molecular patterns(PAMPs), are selectively recognized by the host viapattern recognition molecules (PRMs), like Toll-like re-ceptors (TLRs) and nucleotide-binding oligomerizationdomain (Nod) molecules (Medzhitov, 2001; Inohara andNunez, 2003). Upon interaction between a specific PAMPand its cognate PRM, the innate immune system isalerted essentially through NF-kB activation and subse-quent cytokine production (Janssens and Beyaert, 2003).

Nod proteins are intracellular PRMs that recognizepathogens in the cytosol through sensing peptidoglycan(PGN) motifs. In particular, Nod1 recognizes the coredipeptide structure, g-D-glutamyl-meso-diaminopimelicacid (iE-DAP), contained in the PGN of Gram-negativebacteria (Chamaillard et al., 2003; Girardin et al., 2003a).Nod2, on the other hand, recognizes muramyldipeptide(MurNAc-L-Ala-D-isoGln) present on PGN of both Gram-negative and Gram-positive bacteria (Girardin et al.,2003b; Inohara et al., 2003). Genetic variants of Nod pro-teins are associated with inflammatory disorders (Fritzet al., 2006), thus reinforcing the link between bacterialsensing and inflammation.

Dramatic colonic inflammation is the feature of shigello-sis, a human infectious disease caused by the infection ofas few as 100 bacteria of the enteroinvasive pathogenShigella spp. (DuPont et al., 1989). Shigellae penetratethe baso-lateral pole of intestinal epithelial cells (IEC)through injection of Ipa proteins via a type III secretionsystem (TTSS) (Tran Van Nhieu et al., 2000). In IEC,shigellae stimulate NF-kB activation (Philpott et al., 2000)upon recognition of iE-DAP by Nod1 (Girardin et al., 2001;2003a). Following this step, inflammation mounts via theproduction of pro-inflammatory cytokines, primarily IL-8that stimulates polymorphonuclear leukocyte (PMN)recruitment (Philpott et al., 2000). Simultaneously, theTTSS secreted protein OspF acts to dephosphorylate

Received 18 September, 2007; revised 4 October, 2007; accepted 4October, 2007. *For correspondence. E-mail marialina.bernardini@uniroma1.it; Tel. (+39) 6 49917850; Fax (+39) 6 49917594. †Presentaddress: University of Toronto, Toronto, Canada.

Cellular Microbiology (2008) 10(3), 682–695 doi:10.1111/j.1462-5822.2007.01075.xFirst published online 27 November 2007

Journal compilation © 2007 Blackwell Publishing LtdNo claim to original US government works

mitogen-activated protein kinases in the host nucleus,thus preventing activation of a subset of NF-kB-responsive genes (Arbibe et al., 2007). Likewise, anotherTTSS effector, OspG, antagonizes degradation of theinhibitor IkBa by blocking its ubiquitinylation thus interfer-ing with NF-kB activation (Kim et al., 2005).

Therefore, specific virulence factors and likely hostcell responses mediated by PAMP–PRM interactionscould account for colonic inflammatory response followinginfection with Shigella.

However, despite the great number of studies focusedon the interaction of PRMs with PAMPs, it is still unclearhow in epithelial cells, which are not normally committedto ingest and digest pathogens, Nod1 could physicallyinteract in vivo with PGN, a structure internal to Gram-negative bacteria envelope.

Several studies have detailed that following PGNremodelling (Goodell and Schwarz, 1975; 1985; Cloud-Hansen et al., 2006) around 40–50% of PGN (Park,1993) is released during each bacterial generation andapproximately 90% of this material accumulates in theperiplasm, from where it is re-imported into the cyto-plasm for recycling (Goodell, 1985). This processleading to the release of minimal PGN products mightcontribute to pathogen recognition by Nod1 duringnatural infection.

In order to assess the biological role of these PGNderivatives we have rationally mutagenized Shigella flex-neri 5 in two genes, ampG and mppA, in the attempt toimpair PGN recycling and to augment the release ofmuropeptides by shigellae in host cells and tissues. Ouraim was to analyse whether and how these releasedmuramylpeptides might influence host cell sensing byPRMs and ultimately Shigella virulence.

Results

Muramylpeptide identification in sterile culturesupernatants of M90T, M90T DampG and M90T DmppA

In Escherichia coli, AmpG is a transmembrane proteinthat acts as a specific permease for intact muropeptides(tri- or tetra-) (Lindquist et al., 1993; Cheng and Park,2002) whereas the periplasmic binding protein MppAbinds murein tripeptides and utilizes general oligopeptidepermease (Opp) to transfer its bound ligand into the cyto-plasm where tripeptides are recycled (Park et al., 1998).

The ampG and mppA genes were deleted in S. flexneri5 wild-type strain, M90T, and in BS176 (Sansonetti et al.,1982), a non-invasive variant of M90T, lacking the viru-lence plasmid pWR100 (Buchrieser et al., 2000).

The mutants M90T DampG, M90T DmppA, BS176DampG and BS176 DmppA had the same shape and grewat the same rate as the parental strains (data not shown).

We investigated if parental and mutated strainsreleased PGN-related compounds in growth medium.Muropeptide fractions from sterile culture supernatants ofwild-type strains and mutants were purified by gel perme-ation chromatography and reverse-phase high-pressureliquid chromatography (HPLC) as previously reported(Garcia-Bustos and Dougherty, 1987; Jacobs et al., 1994;Folkesson et al., 2005). The amount of muramylpeptidesshed by the ampG and mppA mutants was 250 mg in 2.5 lof sterile culture medium, i.e. about 10-fold higher thanthat obtained by M90T or BS176. The purified muropep-tide fractions were analysed and identified by mass spec-trometry (MS).

The accumulated products in sterile culture super-natants of M90T DampG and BS176 DampG were allidentified as muropeptides containing the MurNAc(N-acetylmuramyl) moiety (Fig. 1A) while those recoveredfrom M90T DmppA and BS176 DmppA cultures wereessentially composed by peptide molecules (tri- topenta-) lacking the carbohydrate moiety. In addition, athigh molecular masses, the only MurNAc-containingspecies was anhydro-MurNAc-Ala-Glu-meso-DAP-Ala-Ala (Fig. 1B).

The yield of muropeptide fractions in sterile culturesupernatants of M90T and BS176 growth media was verylow compared with that of the ampG and mppA mutants,and contained the same compounds as the ampGmutants. In fact, the MS spectrum of the parental strains,M90T and BS176, showed the same blend of muropep-tides as the ampG mutants (details on MS profiles areprovided in Appendix S1).

Nod1, Nod2 and TLR2 activation by theShigella mutants

M90T DampG and M90T DmppA were first analysed invitro to evaluate their ability to invade, proliferate anddisseminate in HeLa cell monolayers. The results demon-strated that M90T DampG and M90T DmppA do notdisplay any defect in invasion or intracellular proliferationor intra or intercellular spreading (Fig. S1). Likewise, theabsence of MppA did not prevent M90T DmppA fromgrowing with haemin as the iron source as recently shownfor an E. coli double mutant harbouring the mppA anddppA deletions (Létoffé et al., 2006) (data not shown).

As NF-kB activation in epithelial cells infected with Shi-gella relies on Nod1-PGN sensing, we next evaluated theability of M90T DampG or M90T DmppA to induce NF-kBactivation in HEK293 cell monolayers expressing ectopicNod1 (Philpott et al., 2000; Girardin et al., 2003a).HEK293 cells were invaded with Shigella at multiplicity ofinfection (moi) of 50 and NF-kB activity and IL-8 produc-tion were evaluated after 3 and 5 h post infection (p.i.).After 3 h p.i. with M90T, M90T DampG or M90T DmppA

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NF-kB was activated 4-, 6- and 16-fold over the controlcells expressing Nod1 respectively (Fig. 2A). These datalargely remained unaltered after 5 h p.i. IL-8 secretionfollowed the same trend.

Transfection of HEK293 cell monolayers expressingectopic Nod1 with a plasmid encoding small interferingRNA (siRNA) for Nod1 prevented the three strains fromactivating NF-kB, and eliciting IL-8 production.

To verify whether the activation of Nod1 was exclusivelydue to the presence of the intracellular mutants, werepeated the full experimental scheme with the non-invasive variants of M90T, i.e. BS176, BS176 DampG andBS176 DmppA. HEK293 cells were exposed to non-invasive bacteria for 3 and 5 h, as above. Neither NF-kBactivation nor IL-8 production was observed under theseconditions (Fig. 2A), confirming that intracellular locationof bacteria was a prerequisite for activation of Nod1 byM90T and its variants.

We then evaluated whether the PRM Nod2 could beactivated by parental strains and the invasive and non-invasive variants. We applied the same experimental plandescribed for Nod1 in HEK293 cells expressing Nod2.After 3 h p.i. M90T stimulated Nod2 around threefold overthe control, while both M90T DampG and M90T DmppAstimulated Nod2 around fourfold more than the uninfectedcells expressing Nod2. At 5 h p.i. all three strains induceda fivefold NF-kB activation with respect to uninfectedcells. IL-8 values reflected this trend. As expected, inHEK293 cells transfected with a plasmid expressingsiRNA for Nod2 NF-kB activation and IL-8 release wasfully abrogated. HEK293 cells expressing Nod2 were thenexposed to non-invasive bacteria BS176, BS176 DampG

and BS176 DmppA for 3 and 5 h, as above. Neither NF-kBactivation nor IL-8 production was detected. Results areshown in Fig. 2B.

The TLR2/TLR1 heterodimer recognizes triacyl lipopro-teins contained in Gram-negative bacteria. To assesswhether the inactivation of ampG and mppA in Shigellacould affect the recognition of this pathogen by TLR2, weanalysed the ability of M90T and its mutants to activate thisPRM in HEK293 cells expressing TLR2. As TLR2 is a PRMlocated on cell membranes and not within the cytoplasm,like Nod1 and Nod2, HEK293 cell monolayers stably trans-fected with TLR2 and co-transfected with a plasmidexpressing TLR1 were exposed to bacteria [2 ¥ 107

colony-forming units (cfu) of each strain] killed with gen-tamicin and potentially releasing lipoproteins. Luciferaseactivity and IL-8 production were evaluated after 5 h ofcontact. We found that M90T, M90T DampG or M90TDmppA stimulated these PRMs around 23-, 30- and 32-foldover the control uninfected cells expressing TLR2/TLR1respectively. IL-8 production followed the trend observedwith NF-kB. In HEK293 cells expressing TLR2/TLR1 anddepleted of TLR2 by siRNA interference, the activity ofNF-kB and release of IL-8 were not observed (Fig. 2C).

Activation of Nod1, Nod2 by sterile supernatants ofM90T and BS176 ampG and mppA mutants

To gain further insights into the role of muramylpeptidesshed by the mutants on NF-kB activation, we used thesterile purified supernatants (SPS) of M90T, M90TDampG or M90T DmppA to measure NF-kB activity andIL-8 production in HEK293 cells expressing either Nod1 or

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Fig. 1. Mass spectrum of muramylpeptides in sterile culture supernatants of Shigella mutants. Positive ion matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum of muramylpeptides present in sterile culture supernatants of M90T DampG and BS176DampG (A) and M90T DmppA and BS176 DmppA (B). Each muropeptide species is represented by a pair of ion peaks differing by Dm/z 22[M+ H and M+ Na]+ except for ion at m/z 413 [M+ Na]+ that lacks the ion species [M+ H]+ at m/z 393 (see Appendix S1). Abbreviation: An,anhydro; DAP, meso-diaminopimelate; MurNAc, N-acetylmuramyl; GlcNAc, N-acetylglucosamine.

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Nod2, as above. Two and a half microlitres of SPS corre-sponding to 1 ¥ 107 cfu were transfected into HEK293cells as specified in Experimental procedures. NF-kBactivity and IL-8 production were evaluated 12 h later.

As shown in Fig. 3A, SPS of M90T, M90T DampG andM90T DmppA stimulated Nod1 4-, 7- and 12-fold overthe control uninfected cells expressing Nod1 respectively(P < 0.0001 for both M90T DampG and M90T DmppA

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Fig. 2. NF-kB activity and IL-8 production of HEK293 cells expressing either Nod1 or Nod2 or TLR2 and infected with invasive ornon-invasive strains.A. HEK293 cells transfected with a plasmid expressing hNod1 or hNod1 in association with Nod1 psiRNA were infected either with theinvasive strains M90T, M90T DampG and M90T DmppA, or with the non-invasive strains BS176, BS176 DampG and BS176 DmppA, for 3 hand 5 h p.i. NF-kB activation (left) and IL-8 production (right) were evaluated. At 3 h p.i. P = 0.01 for M90T DampG versus M90T andP < 0.0001 for M90T DmppA versus M90T.B. HEK293 cells transfected with a plasmid expressing hNod2 or hNod2 in association with Nod2 psiRNA were infected as above. At 3 h p.i.P = 0.0045 for M90T DampG versus M90T and P = 0.0092 for M90T DmppA versus M90T. At 5 h p.i. P = 0.09 for M90T DampG versus M90Tand P = 0.35 for M90T DmppA versus M90T.C. HEK293 cells stably expressing hTLR2 and transiently transfected with a plasmid encoding hTLR1 or hTLR1 in association with TLR2psiRNA were exposed during 5 h to M90T, M90T DampG and M90T DmppA killed by gentamicin (60 mg ml-1) as detailed in Experimentalprocedures. NF-kB activation (left) and IL-8 production (right) were evaluated. P = 0.965 for M90T DampG versus M90T and P = 1 for M90TDmppA versus M90T.Data are expressed as mean � SD of three independent experiments in triplicate. NF-kB values are reported as fold induction of relativeluciferase units (RLU) over unstimulated cells. Relative luciferase activity is calculated as the ratio between the value of the NF-kB-inducibleFirefly luciferase and that of the constitutive Renilla luciferase reporter. The asterisk indicates a NF-kB value extremely significant, P < 0.0001.Abbreviation: TriDAP, L-Ala-D-Glu-meso-DAP (1 mg ml-1); MDP, MurNAc-L-Ala-D-isoGln (1 mg ml-1); Pam3, Pam3CysSerLys4 (500 ng ml-1).

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versus M90T). IL-8 production confirmed this differenceamong the strains.

SPS of BS176, BS176 DampG and BS176 DmppA gaveresults similar to those of M90T and its variants (Fig. 3A),excluding any possible implication of other molecules –such as those produced by the TTSS machinery – inthe Nod1-mediated NF-kB activity and IL-8 productionunder our experimental conditions. In HEK293 depletedof Nod1, NF-kB activity and IL-8 release were notdetected.

We next analysed the contribution of Nod2 to the NF-kBinduction of the SPS of all mutants in HEK293 cellsexpressing Nod2. With SPS of M90T, Nod2 was not acti-vated, while with SPS of M90T DampG and M90T DmppANod2 was stimulated around two- and threefold over thecontrol uninfected cells expressing Nod2. IL-8 releasewas low under these conditions. As above, SPS of BS176,BS176 DampG and BS176 DmppA were analysed in par-allel with supernatants of M90T and M90T variants. The

results obtained were consistent with those obtained withM90T and its variants (Fig. 3B). In the presence of siRNAfor Nod2 NF-kB activity was abolished with SPS of allstrains tested.

Finally, we assessed whether the results obtained withSPS could be influenced by the presence of residuallipopolysaccharide (LPS) by evaluating NF-kB activity andIL-8 release in HEK293 cells expressing TLR4, MD2 andCD14. In this case SPS were added from ‘outside’ toHEK293 cells expressing this PRM and cofactors. NF-kBactivity was evaluated after 5 h of contact. Neither activa-tion of NF-kB nor IL-8 production was observed underthese conditions. On the contrary Shigella LPS addedfrom outside elicited both NF-kB activation and IL-8production. Likewise, in HEK293 cells expressing TLR2/TLR1 the exposure of SPS of either of the strains elicitedneither NF-kB stimulation nor IL-8 release in the presenceof a positive response given by Pam3CSK4 used as acontrol (data not shown).

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Fig. 3. NF-kB activity and IL-8 production in HEK293 cells expressing either Nod1 or Nod2 following stimulation of SPS of either M90T, M90TDampG and M90T DmppA or BS176, BS176 DampG and BS176 DmppA.A. HEK293 cells transfected with a plasmid expressing hNod1 or hNod1 in association with Nod1 psiRNA were transfected with SPS of eitherM90T, M90T DampG and M90T DmppA or BS176, BS176 DampG and BS176 DmppA as specified in Experimental procedures. NF-kBactivation (left) and IL-8 production (right) were evaluated after 12 h of stimulation. P < 0.0001 for M90T DampG and M90T DmppA versusM90T and BS176 DampG and BS176 DmppA versus BS176.B. HEK293 cells transfected with a plasmid expressing hNod2 or hNod2 in association with Nod2 psiRNA were transfected with SPS as aboveand NF-kB activation (left) and IL-8 production (right) were evaluated after 12 h of stimulation. P = 0.016 for M90T DampG versus M90T,P = 0.019 for M90T DmppA versus M90T, P = 0.024 for BS176 DampG versus BS176 and P = 0.007 BS176 DmppA versus BS176.Data are expressed as mean � SD of three independent experiments in triplicate. NF-kB values are reported as in Fig. 2. The asteriskindicates a NF-kB value extremely significant, P < 0.0001. Abbreviations are as in Fig. 2; Medium: sterile M9 minimal medium.

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Activation of NF-kB by either M90T or BS176 mutantsor SPS in HeLa cells

To verify the biological activity of these strains in condi-tions more close to those occurring in host tissues, werepeated the experimental scheme described above onHeLa cells naturally expressing the majority of PRMs(Uehara et al., 2007). Initially we measured NF-kB andIL-8 production upon either cell infection with M90T, M90TDampG and M90T DmppA (internal stimulation) or expo-sure to the non-invasive BS176, BS176 DampG andBS176 DmppA (external stimulation). After 3 h p.i. with theinvasive strains NF-kB was activated around ninefold withM90T, 14-fold with M90T DampG and 10-fold with M90TDmppA with respect to uninfected cells (P < 0.0001 M90TDampG versus M90T). At 5 h p.i. the NF-kB values elicitedby all strains were roughly double those observed at 3 hp.i. with these same strains (P < 0.0001 for M90T DampG

versus M90T and P = 0.009 for M90T DmppA versusM90T). Production of IL-8 followed the same trend. Onthe contrary, exposure of HeLa cells to BS176, BS176DampG and BS176 DmppA did not stimulate theactivation of NF-kB nor IL-8 production. Under condi-tions of Nod1 depletion, NF-kB and IL-8 values weredrastically reduced at 3 h p.i. and abrogated at 5 h p.i.(Fig. 4A).

We proceeded to analyse the possible contribution ofNod2 to NF-kB activation in this cell line infected withM90T and its variants. Depletion of this PRM by Nod2interference did not change significantly the resultsobtained with either of the strains in wild-type HeLa cells.

Several studies highlighted that antagonism andsynergy among the different PRMs play a crucial role indetermining final NF-kB activation (van Heel et al., 2005;Uehara et al., 2005). Therefore, we examined the roleplayed by TLR2 in this cell line by depleting the cells of the

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Fig. 4. NF-kB activity and IL-8 production in HeLa cells upon either infection with invasive or non-invasive strains or transfection with SPS.A. HeLa cells transfected with plasmid vector or with either hNod1 psiRNA or with hNod2 psiRNA or with hTLR2 psiRNA were infected withinvasive strains M90T, M90T DampG and M90T DmppA or non-invasive strains BS176, BS176 DampG and BS176 DmppA during 3 h and 5 hp.i. NF-kB activation (left) and IL-8 production (right) were assessed. At 3 h p.i. P < 0.0001 for M90T DampG versus M90T and P = 0.105 forM90T DmppA versus M90T. At 5 h p.i. P = 0.0003 for M90T DampG versus M90T and P = 0.0086 for M90T DmppA versus M90T. TriDAP andMDP were transfected into cells as described and used as positive controls for Nod1 and Nod2 respectively. Pam3 were exposed to HeLacells as specified and used as a control for TLR2.B. HeLa cells transfected with plasmid vector or with either hNod1 psiRNA or hNod2 psiRNA or hTLR2 psiRNA were transfected with SPS ofM90T, M90T DampG and M90T DmppA or of BS176, BS176 DampG and BS176 DmppA as detailed in Experimental procedures. NF-kBactivation (left) and IL-8 production (right) were observed after 6 h of stimulation. P < 0.0001 for M90T DampG and M90T DmppA versus M90Tand BS176 DampG and BS176 DmppA versus BS176. Positive controls were as above.Data are expressed as mean � SD of three independent experiments in triplicate. NF-kB values are reported as in Fig. 3. The asteriskindicates a NF-kB value extremely significant, P < 0.0001. Abbreviations are as in Fig. 2; Medium: sterile M9 minimal medium.

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expression of this PRM. At 3 h p.i. in HeLa cells infectedwith either of the strains NF-kB activity was unaffected.However, at 5 h p.i. under these conditions luciferasevalues were lower than those obtained in wild-type HeLacells although this decrease was not statisticallysignificant. We reasoned that external bacteria killed bygentamicin could contribute to NF-kB activation at latetimes of infection as shown in HEK293 cells expressingTLR2/TLR1. To analyse this hypothesis we repeated thesame experimental scheme applied on HEK293 express-ing TLR2/TLR1 in HeLa cells naturally expressing TLR2.Results confirmed that exposure of HeLa cells to shigellaekilled by gentamicin could stimulate NF-kB activation andthat TLR2 depletion significantly reduced this activity(Fig. S2).

Finally, we continued to analyse the biological activity ofSPS in this cell line. We adapted to HeLa cells the pro-cedure described for the experiments exploiting HEK293cells. Briefly, SPS were transfected into HeLa cells andNF-kB activation and IL-8 production were measured afterdifferent times. We found that in this cell line maximalstimulation was achieved around after 6 h of stimulationwhereas at 12 h no signal (including that given by PRMagonists) was observed. Therefore, we show only findingsobtained after 6 h of stimulation.

M90T DampG SPS elicited NF-kB values ninefold higherwith respect to controls while those of M90T DmppA andM90T resulted eight- and fourfold higher levels respec-tively (P < 0.0001 for both M90T DampG and M90T DmppAversus M90T and P = 0.07 M90T DampG versus M90TDmppA). IL-8 production followed the same trend. Like-wise, NF-kB and IL-8 values obtained by transfection ofSPS of BS176, BS176 DampG or BS176 DmppA reflectedthose seen with SPS of M90T and its mutants.Again, in thepresence of Nod1 siRNA both NF-kB activation and IL-8levels were significantly lower. In contrast to these results,the luciferase values remained roughly unaltered followingsilencing of either Nod2 or TLR2 (Fig. 4B).

In vivo virulence assessment

M90T, M90T DampG and M90T DmppA were assessedin mice by using two models of infection, the intranasal(i.n.) infection (Voino-Yasenetsky and Voino-Yasenetskaya, 1962) and the intravenous (i.v.) infection(Martino et al., 2005a). Following i.n. infection with a doseof 108 cfu (LD50) of Shigella mice develop a severe pneu-monia and die. Likewise, after i.v. injection of a dose of107 cfu (LD70) mice develop fulminant hepatitis and die. Inthe analysis of M90T, M90T DampG and M90T DmppA,survival of animals, bacterial counts in relevant organs,immunohistopathological features of the infected tissuesand cytokine expression were examined.

While M90T DampG displayed virulence levels similarto those of M90T, M90T DmppA was markedly attenuatedin both models. Following i.n. inoculation of 108 cfu (LD50)of M90T DmppA only 5% of the animals died. Likewise,only 8% of mice died after i.v. injection of 107 cfu (LD70)of this same strain. In accordance with mortality data,the number of cfu in the lung and liver, following i.n. andi.v. infection, respectively, was significantly reduced. Asalready described (Cersini et al., 2003), after 72 h in lungsof mice infected i.n. with the wild-type strain we foundapproximately 106 cfu; a similar value was calculated withM90T DampG while only 104 cfu were recovered in lungsinfected with M90T DmppA.

At 48 h after i.v. infection with either M90T or M90TDampG the livers of infected animals contained approxi-mately 106 cfu of Shigella (Martino et al., 2005a); thisvalue was only 102 cfu in animals infected i.v. with M90TDmppA.

Following i.v. infection LD50 of M90T DmppA was1 ¥ 108 cfu (versus 5 ¥ 106 with M90T) while when admin-istered i.n. LD50 was 8 ¥ 108 cfu. M90T DmppA harbouringthe wild-type copy of mppA carried by pSTBlue-1-mppAwas restored to full virulence in both infection models.Data are summarized in Table 1.

Table 1. Assessment of virulence of M90T, M90T DampG and M90T DmppA in the pulmonary (i.n.) and systemic (i.v.) models of shigellosis.

Strain

Murine pulmonary model Murine systemic model

Mortality (%)a cfu per lung (¥104)b Mortality (%)c cfu per liver (¥104)d cfu per spleen (¥104)

M90T 50 465.42 � 34 70 873.15 � 74.31 9.88 � 0.34M90T DampG 47 234.95 � 11 67 970.02 � 85.71 2.66 � 0.63M90T DmppA 5 5.65 � 0.3e 8 0.02 � 0.01e 0.004 � 0.002M90T DmppA pSTblue-1-mppA 56 165.00 � 72 75 1247 � 543 5.76 � 0.59

a. n = 24 per strain. The animals were infected intranasally (i.n.) with a dose of 1 ¥ 108 cfu and the LD50 was obtained at 72 h p.i.b. Surviving animals were sacrificed at 72 h p.i. and lungs were removed and tested for bacterial counts.c. n = 18 per strain. The animals were infected intravenously (i.v.) with a dose of 1 ¥ 107 cfu and the LD70 was obtained at 48 h p.i.d. Surviving animals were sacrificed at 48 h p.i. and liver and spleen were removed and tested for bacterial counts.e. Colony-forming units (cfu) in either the lung or liver of animals infected with M90T versus cfu in the lung or liver of mice infected with M90TDmppA, P < 0.0001.

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Histopathology, immunohistochemistry of and cytokineproduction in lungs of mice infected i.n.

Histopathological analysis was performed on tissue sec-tions of lungs removed 72 h after i.n. infection. Alterationsin pulmonary tissues were quantified and recorded as themean of different scores related to inflammation as previ-ously reported (Cersini et al., 2003; Martino et al., 2005b)and are summarized in Appendix S2.

Haematoxylin-eosin (HE)-stained lung sections wereexamined to assess the degree of: (i) thickening ofalveolar septum, infiltration and diffusion of neutrophilsin alveolar spaces, (ii) bronchiolar inflammation and epi-thelial damage and (iii) bronchiolar-associated lymphoidtissue (BALT) activation. Lung sections were alsotreated with monoclonal antibodies (mAbs) to evaluatethe expression and distribution of IL-6 and ShigellaLPS.

Wild-type strain and M90T DampG caused maximallesions due to large areas of severe suppurative bron-chopneumonia in which bronchioles were filled with neu-trophils and sometimes with a mixture of cell debris,mucus and macrophages (Fig. 5A). This severe inflam-mation was accompanied with a strong expression of IL-6and with intense LPS staining. In M90T-infected tissues,LPS was associated with inflammatory cells and free inthe mucus while in lungs infected with M90T DampG, LPSwas strongly stained in mononuclear cells (MNs) withinBALT (Fig. 5A and Appendix S2). In lungs infected withM90T DmppA the tissue architecture was not significantlyaltered and septa and airways remained substantiallyunaffected. A strong reaction of BALT was observedaccompanied with the development of characteristicperivascular cuffings. IL-6 expression was high and LPSstaining moderate, especially located inside MNs associ-ated with BALT (Fig. 5A and Appendix S2). In infectedlungs, the production of IL-6 assessed at 12, 24 and 72 hp.i. was similar with all strains. In contrast, IFN-g yield wassignificantly higher with M90T DmppA at all time points(Fig. 5B).

Histopathology and immunohistochemistry of livers ofmice infected i.v.

Histopathological analysis of tissue sections of livers wasperformed on mice sacrificed 48 h after i.v. infection asalready reported (Martino et al., 2005a). Livers infectedi.v. with M90T showed severe degeneration and cloudyswelling of hepatocytes (Fig. 5C). Necrotic areas wereassociated with the presence of microgranulomas. Theseaggregates, consisting of focal leukocyte reaction tohepatic cell death, showed a central area of mild necrosis.Immunohistochemical analysis revealed that LPS ofM90T and M90T DampG seemed to be associated with

some MNs within microgranulomas as well as present inthe extracellular areas (Fig. 5C). In contrast with theseresults, the liver of M90T DmppA-infected animals did notdisplay degenerative alterations; nevertheless, large andwell-defined microgranulomatous lesions were presentwith an enlarged necrotic central area, which werestrongly stained by the anti-Shigella LPS mAb (Fig. 5C).Apoptotic cells are a hallmark of Shigella infection in theliver and apoptosis plays a crucial role in worsening thelesions in the hepatic tissue (Martino et al., 2005a,b). WithM90T, a great number of apoptotic cells were observedwithin microgranulomas and throughout the hepaticparenchyma, while with M90T DmppA the number of apo-ptotic cells was low within microgranulomas and in thesurrounding areas (Fig. 5C and Appendix S2).

Tissue alterations were quantified as previouslyreported (Cersini et al., 2003; Martino et al., 2005a) andsummarized in Appendix S2.

Discussion

In this study we analysed the contribution of PGN sensingto the overall cellular response to Shigella infection. First,we investigated whether Shigella releases PGN frag-ments during PGN remodelling as originally shown forE. coli (Goodell and Schwarz, 1985) and for two humanpathogens, Bordetella pertussis (Goldman et al., 1982;Cookson et al., 1989) and Neisseria gonorrhoeae (Sinhaand Rosenthal, 1980). Then, we evaluated whether andhow the PGN derivatives shed by Shigella could stimulatethe innate immune response. Finally, we analysedwhether Shigella virulence could be modulated by alteringthe release of these PGN fragments.

Our results demonstrate that Shigella spontaneouslyreleases PGN fragments and that this process can begreatly increased by inactivating either ampG or mppAgenes, both involved in PGN recycling, as first shown byJacobs through the deletion of ampG in E. coli (Jacobset al., 1994; Cloud-Hansen et al., 2006). We found thatthe increase of muramylpeptide shedding corresponds tosignificant Nod1 activation in epithelial cells. In fact, theShigella ampG and mppA mutants trigger Nod1-mediatedNF-kB activation to a greater extent than the wild-typestrain. Likewise, SPS added from ‘inside’ to host cells areable to stimulate Nod1 to varying degrees, depending ontheir relative amount and composition. In particular,muramylpeptides shed by M90T DmppA whose composi-tion differs significantly from that of M90T and M90TDampG were especially efficient in mediating Nod1 stimu-lation in HEK293 cells expressing ectopic Nod1.

In contrast, Nod2 is not activated by the PGN mixturereleased by wild-type bacteria and is only weakly acti-vated by muramylpeptides released by either of themutants.

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Biological activity of muramylpeptides has beenalready reported in the context of N. gonorrhoeae (Sinhaand Rosenthal, 1980) and B. pertussis pathogenesis(Goldman et al., 1982) as the PGN fragments released bythese microorganisms cause damage to ciliated cellssimilar to the effects provoked by these pathogens duringnatural infection (Melly et al., 1984; Flak and Goldman,1999). Furthermore, in a recent study Hasegawa et al.(2006) demonstrated that a higher level of Nod1-stimulatory activity was found in culture supernatants ofvarious bacteria and not in extracts from whole bacterialcells. Our findings together with these observationssupport the idea that muramylpeptides released by patho-gens during natural infection could modulate the immuneresponse through qualitative and quantitative differencesin Nod protein activation.

In HeLa cells infected with Shigella, Nod1 drives bac-terial recognition (Girardin et al., 2001). Our data confirmthis as, in this cell line, the absence of Nod1 dramaticallydecreases the activation of NF-kB, especially at late timesof infection. In contrast, depletion of Nod2 does not alterthe ability of Shigella to activate NF-kB, while depletion ofTLR2 slightly reduces the NF-kB activation, suggesting apotential involvement of this PRM to the response of hosttissues to Shigella.

In contrast with results obtained in HEK293 cultures, inHeLa cells infected with either M90T DampG and M90TDmppA, NF-kB was roughly equally stimulated. Thesedifferences between the two cell lines might be explainedby the fact that NF-kB activation in treated HEK293 cells

depends on Nod1 overexpression while in HeLa cells, it isthe endogenous Nod1 that triggers the response. Ectopicexpression of Nod1 could emphasize differences in Nod1sensing between the two mutants that would be otherwisenot detectable. Furthermore, differences in PRM expres-sion between the two cell lines could influence the signaltransduction as reported for TLR (Kurt-Jones et al., 2004).Finally, in HeLa cells, living shigellae might activate othernot yet identified PRMs thus affecting the immuneresponse initially triggered by Nod1.

In vivo, the virulence capacity of Shigella DampG wascomparable to that of the wild-type strain, which is inaccordance with the absence of virulence attenuation fora Salmonella enterica serovar Typhimurium DampG strain(Folkesson et al., 2005). In contrast to this result, ShigellaDmppA was strongly attenuated. In lungs of mice infectedwith M90T DmppA, the IL-6 production was comparable tothat induced by wild-type bacteria while the level of IFN-gwas higher. IFN-g could contribute to pathogen clearanceas shown during natural and experimental shigellosis(Way et al., 1998). Similarly, the liver of animals infectedi.v. with M90T DmppA contained larger microgranulomas(Martino et al., 2005a,b), than those induced by M90T, inwhich a significant presence of IFN-g expressing cellsmight facilitate Shigella clearance. We are currently inves-tigating the molecular mechanisms underlying the attenu-ation of M90T DmppA and how the reduction of virulencecould rely on the peculiar ability of this strain and itssupernatant to activate Nod1. An attractive hypothesiscould be that M90T DmppA is particularly able to elicit the

Fig. 5. Lung and liver reactions following intranasal infection or intravenous infection with M90T, M90T DampG or M90T DmppA.A. Histochemical and immunohistochemical (IH) analysis of lungs infected with M90T, M90T DampG or M90T DmppA. The animals weresacrificed at 72 h p.i. From top to bottom: HE staining of infected lung sections and avidin-biotin immunoperoxidase labelling of IL-6, andS. flexneri 5 LPS.HE: In uninfected control the absence of inflammatory infiltrates and BALT activation are evident; samples infected with either M90T or M90TDampG show a suppurative bronchopneumonia and a poor BALT activation; with M90T DmppA alveolar spaces and airways are free frominflammatory cells while areas of BALT activation are associated with mild phlogosis. Original magnification ¥200, bars 50 mm.IL-6: In sections of lungs infected with either M90T or M90T DampG the amount and distribution of cells expressing IL-6 are similar. With M90TDmppA IL-6 production is mainly localized in MNs of BALT. Arrows point to areas showing IL-6 staining. Original magnification ¥200, bars 50 mm.Shigella flexneri 5 LPS: With M90T LPS is mainly present extracellularly in bronchial lumen (arrows); with M90T DampG LPS is found in someMNs (arrowheads) and free in bronchial lumen (arrows); with M90T DmppA LPS immunostaining is weak and restricted to MNs (arrowheads).Original magnification ¥1000, bars 250 mm.B. Production of IL-6 and IFN-g in lungs of animals infected with M90T, M90T DampG and M90T DmppA (n = 3 per group). Uninfected controlsrepresent four mice. Lungs were removed at 12, 24 and 48 h p.i., homogenized in PBS and supernatants served as samples for IL-6 ELISA.The asterisk indicates a statistically significant difference in value compared with wild-type strain (P < 0.005).C. Histochemical and IH analysis of livers infected with M90T, M90T DampG or M90T DmppA. The mice infected i.v. were sacrificed 48 h p.i.From top to bottom: HE staining, avidin-biotin immunoperoxidase labelling of S. flexneri 5 LPS, colorimetric TUNEL analysis of infected liversection.HE: With M90T a diffuse hepatocyte swelling with necrotic areas is accompanied with the presence of a microgranuloma (dashed line); withM90T DampG a microgranuloma is observable (dashed line) surrounded by a degenerate parenchyma; in the M90T DmppA-infected sample anextended microgranuloma (dashed line) displays a large central area of necrosis associated with the absence of hepatocyte degeneration.Shigella flexneri 5 LPS immunostaining: With either M90T or M90T DampG, S. flexneri LPS is located extracellularly in a large area in the centralportion of a microgranuloma (arrowheads) or in a few MNs outside this area (arrows); with M90T DmppA LPS is exclusively localized in thecentral area of a microgranuloma (arrowheads), associated with necrotic material or expressed inside a certain number of MNs.TUNEL colorimetric assay: Some TUNEL-positive cells are present in sections of the liver of the uninfected control mouse; in samples infectedwith either M90T or M90T DampG, a great number of TUNEL-positive nuclei of hepatocytes and lymphoid cells within (arrowheads) and outside(arrows) the microgranulomatous areas confirms a high apoptotic index; with M90T DmppA a small number of TUNEL-positive cells are presentinside (arrowheads) as well as outside (arrows) the microgranulomatous area.Original magnification of all panels ¥400, bars 100 mm.

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Nod1-dependent activation of antibacterial factors likeantimicrobial peptides (AMPs) in the liver and in the res-piratory tract in early time of infection. Several bacterialfactors including muramyl dipeptide have been demon-strated to stimulate AMPs expression and secretion(Ayabe et al., 2000).

Recent studies have stressed the role of bacterial PGNcomposition in immune evasion of various pathogens(Chaput et al., 2006; 2007; Boneca et al., 2007) and haveunveiled bacterial strategies to modulate the PGN struc-ture or to inject muramylpeptides into host cells (Bartole-schi et al., 2002; Viala et al., 2004). Here we show that theimmunopotential of Shigella is significantly influenced byquantitative and qualitative alterations in muramylpeptideshedding, thus suggesting that the regulation of PGNrelease could represent a further sophisticated bacterialstrategy to survive in host tissues and to evade immunedefences.

Experimental procedures

Bacterial strains, plasmids and growth conditions

The S. flexneri 5 strains used are M90T streptomycin-resistant(SmR) and its non-invasive derivative BS176 (Martino et al.,2005b). Bacteria were grown in Trypticase soy broth (TSB) (BBL,Becton Dickinson and Co., Cockeysville, MD) or agar (TSA).Minimal medium was prepared with M9 salts, glucose (0.2%) andnicotinic acid (10 mg ml-1). When necessary, kanamycin, ampicil-lin, streptomycin or chloramphenicol was added to cultures at 50,100, 100 or 20 mg ml-1 respectively.

Bacterial strains’ construction

DNA manipulations were carried out by standard methods follow-ing manufacturer’s suggestions. To obtain M90T DampG twoDNA fragments from M90T ampG were amplified by PCR withthe following primers: ampGF1 (5′-GCTCTAGAGTTCAGCCATATTGCTGATCCTGG-3′) and ampGR1 (5′-TGCACTGCAGTTCCAGCGTTTTGGGCACAGG-3′) for fragment 1 (575 bp); ampGF2(5′-TGCACTGCAGTCGGGTTTGATGCGGGTGAAGTA-3′) andampGR2 (5′-CGGAATTCATCCAGCAAACCACCAAGCACGAC-3′) for fragment 2 (636 bp). The two fragments were ligated witha 829 bp PstI cassette encoding chloramphenicol resistancefrom pGEM-CAT (a gift from A. Covacci, Chiron Vaccines, Siena,Italy), cloned into pGP704 and introduced into M90T and BS176to replace the wild-type ampG through allelic exchange. Like-wise, to yield M90T DmppA and BS176 DmppA two DNA frag-ments, of 684 bp and 498 bp from M90T mppA, were amplifiedwith the primers: mppAF1 (5′-GTCCTAGAGGTGCGCCACATAAAGATGAG CC-3′), mppAR1 (5′-TGCACTGCAGAATATCCTTCAACAGCTTCTG-3′), mppAF2 (5′-TGCACTGCAGTTACGCCGGAACCTTCGCCGTTTG-3′) and mppAR2 (5′-CGGAATTCCGCCACATCTTCAGGATTATTAATG-3′), ligated with a 1.2 kb PstIcassette encoding kanamycin resistance from pUK4K and thenused to replace the wild-type mppA in M90T and BS176 (SF1841in S. flexneri 2a 301 genome corresponds to E. coli mppA). Theentire M90T ampG gene was amplified by using the following

primers: ampGCF (5′-ATG TCCAGTCAATATTTACG-3′) andampGCR (5′-TACGTCAGATGCGTTTTTCG-3′). The 1475 bpfragment was then cloned into pSTBlue-1 (Perfectly BluntTM

Cloning kit, Novagen).Likewise, a sequence (2034 bp) containing the entire M90T

mppA gene was cloned into pSTBlue-1 by using the followingprimers: mppACF (5′-ACTGGTTTGAACGTGCGAAG-3′) andmppACR (5′-TACCAAAACCACGGTATGGG- 3′).

Analysis of muramylpeptides in sterile bacterialculture supernatants

In order to analyse muramylpeptides present in sterile culturesupernatants M90T, BS176, M90T DampG, M90T DmppA,BS176 DampG and BS176 DmppA were cultured overnight in M9medium up to OD600 = 1.5. Sterile culture medium (2.5 l) of M90T,M90T DampG and M90T DmppA was then lyophilized, redis-solved in water, extensively desalted by successive gel filtrationchromatography, reduced by sodium borohydride, purified byHPLC and analysed by MS (Garcia-Bustos and Dougherty, 1987;Jacobs et al., 1994; Folkesson et al., 2005). All supernatants ofbacterial cultures (20 ml), lyophilized and used in in vitro assayswere formerly analysed for contamination by either lipoprotein orLPS by Gas-Chromatography/Mass Spectrometry of fatty acids(Wollenweber and Rietschel, 1990).

Cell cultures and infections

HeLa and HEK293 cells were maintained in D-MEM (Cambrex BioScience, Walkersville) supplemented with 10% FBS (Cambrex BioScience, Walkersville). HEK293 huTLR2 (gDhTLR2), kind gift ofArturo Zychlinsky, were grown in complete medium as abovesupplemented with geneticin (Gibco BRL) 500 mg ml-1. Stabletransfected cell line 293-hTLR4/MD2-CD14 (InvivoGen) werecultured in DMEM with 10% FBS and 10 mg ml-1 Blasticidin-S(InvivoGen) and 50 mg ml-1 HygroGold® (InvivoGen).

HEK293 and HeLa cell infections with invasive and non-invasive strains were performed as previously reported (Philpottet al., 2000; Cersini et al., 2003). Incubation of the infected cellsin the presence of gentamicin was prolonged to 3 or 5 h. Toevaluate TLR2 activation bacterial cultures (2 ¥ 107 cfu) wereadded with gentamicin (60 mg ml-1) and then exposed to HEK293cells expressing TLR2/TLR1 as specified above.

Transfections

HEK293 and HeLa cells were transiently transfected with Poly-Fect Transfection Reagent (Qiagen) according to the manufac-turer’s instructions. Briefly, the cells were transfected overnightwith a reaction mix composed by 5 ml of Polyfect TrasfectionReagent, 25 ng of Firefly luciferase reporter constructs, pGL3.ELAM.tk, and 2.5 ng of Renilla luciferase reporter plasmid,pRLTK. Additionally, HEK293 cells were transfected with 250 ngof hNod1 (pcDNA3-Nod1-FLAG) or hNod2 (pUNO-hNOD2a –InvivoGen) while HEK293 huTLR2 were transfected overnightwith 250 ng of hTLR1 (pUNO-hTLR1 – InvivoGen). Vector plas-mids (pcDNA3) were used as controls in all transfectionexperiments.

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Stimulation with sterile purified supernatants

The day before transfection, the cells were seeded into 24-wellplates to obtain a semi-confluent monolayer. At the same time ofthe transfection 2.5 ml of SPS corresponding to 1 ¥ 107 cfu wereadded to the cell culture medium. NF-kB-dependent luciferaseactivation and IL-8 production were measured the following dayfor HEK293 cells and 6 h after transfection for HeLa cells.

Luciferase assays

The cells were transiently transfected in 24-well plates. Luciferaseassays were carried out using the Dual Luciferase kit (Promega).Firefly luciferase reporter constructs, containing five NF-kB ele-ments at the promoter region, were transfected together with theRenilla luciferase reporter plasmid as an internal control. Cellswere lysed in 100 ml of passive lysis buffer, and 20 ml of lysate wasassayed for Firefly and Renilla activity according to the manufac-turer’s instructions. The data shown represent the mean values �

SD of three separate experiments performed in triplicate. Resultsare reported as fold induction of relative luciferase units (RLU)over unstimulated cells. Relative luciferase activity is calculated asthe ratio between the value of the NF-kB-inducible Fireflyluciferase and that of the constitutive Renilla luciferase reporter.

gTriDAP (L-Ala-D-Glu-meso-DAP), chemically synthesized byAnaSpec (San Jose, CA), MDP (MurNAc-L-Ala-D-isoGln) andPam3CSK4 (N-Palmitoyl-S-[2,3- bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl- [S]-seryl- [S]- lysyl- [S]- lysyl- [S]- lysyl- [S]- lysine ¥3 HCl), purchased from InvivoGen, were used as positive con-trols for Nod1, Nod2 and TLR2/TLR1 activation respectively.

siRNA assays

siRNA inhibition of Nod1, Nod2 or TLR2 was carried out by using,respectively, psiRNAh-Nod1, psiRNAh-Nod2 and psiRNAh-TLR2constructs, all from InvivoGen.

For HEK293 cells, 25 ng of plasmid expressing psiRNA orvector control (containing a scramble sequence) was transfectedtogether the plasmid expressing the corresponding PRM.

Nod1, Nod2 and TLR2 depletion was verified by Westernblot analysis with mouse mAbs anti-DYKDDDK (Clone 2EL-1B11, Chemicon International), anti-Nod2 (2D9, Cayman Chemi-cal Company, Ann Arbor, MI) and anti-hTLR2 (eBiosciences,cloneTL2.1) respectively. A monoclonal anti-b-tubulin (Clone TUB2.1, Sigma) antibody was used as a control.

In HeLa cells siRNA inhibition of Nod1, Nod2 and TLR2 wasperformed as follows: cells were first transfected with the siRNAexpressing plasmid using the procedure described above.Seventy-two hours after the first transfection the cells werere-transfected with pGL3.ELAM.tk and pRLTK to evaluate NF-kBactivation and IL-8 production as detailed above.

In HeLa cells Nod1 inhibition was evaluated through reversetranscription polymerase chain reaction (RT-PCR) and Real-Timeas previously described (Welter-Stahl et al., 2006). Nod2 andTLR2 depletion was analysed as described for HEK293 cells.

Intranasal infection of mice

Five-week-old Balb/C female mice (Charles River, Italy) were

infected i.n. with a dose of 108 cfu. After 72 h mice were sacrificedand lungs were removed and processed for histopathologicalstudies, bacterial counts and RT-PCR analysis as reported(Cersini et al., 2003). Alternatively, the mice were kept undersurvey during 7 days after infection.

For bacterial counts and enzyme-linked immunosorbent assay(ELISA) studies the samples were treated as described (n = 8 foreach condition). Each experiment was repeated at least threetimes.

Intravenous infection of mice

Mice were challenged by injection of the caudal vein with 200 mlof sterile saline solution (SSS) of bacterial suspension, containing107 cfu (LD70) as already described (Martino et al., 2005a). After48 h the surviving animals were sacrificed and liver and spleenremoved and prepared for histopathology and counts of viablebacteria, as reported. Alternatively, the mice otherwise infectedwere kept under survey during 7 days after infection.

Uninfected mice having received 200 ml of saline solutionthrough the caudal vein were used as controls in each experi-ment (n = 8 per experimental group). Each experiment wasrepeated at least three times.

Histopathology

For morphological and immunohistochemical (IH) examination,tissue samples were fixed in 10% buffered formalin for 48 h,processed and embedded in paraffin. Sections of 3 mm thick werestained with HE or processed for IH. For IH, tissue’s sections weretreated as described (Martino et al., 2005a,b). Primary antibodiesare: anti-S. flexneri 5a LPS (dimeric IgA, 6 mg ml-1) (Martinoet al., 2005a) and anti-mouse mAb IL-6 (Serotec, Oxford, UK).

A microgranuloma was defined as a well-circumscribed cellaggregation composed of five or more mononuclear phagocytes.Pro-apoptotic effect of S. flexneri was highlighted through aTUNEL colorimetric staining (DeadEnd, Promega) according tothe manufacturer’s instructions.

ELISA

Homogenized lungs were microfuged at top speed for 5 min andthe supernatants were used for cytokine analysis. Murine IL-6and IFN-g levels in tissues were assayed by using ELISA kits(R&D Systems, Minneapolis, USA) according to the manufactur-er’s instructions. Human IL-8 levels in cell culture supernatantswere measured using ELISA kits (R&D Systems, Minneapolis,USA).

Statistical analysis

Two-tailed t-test was used to compare cytokine titres andluciferase data. Mortality experiments were assessed using Fish-er’s exact test.

Data were presented as mean � SD, and the numbers ofindependent experiments were indicated in each legend of thefigures. A P-value < 0.05 was considered statistically significant.A P-value < 0.0001 was considered extremely significant.

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Acknowledgements

We thank M. Rescigno for helpful discussion and technicalsupport, C. Tang for critical reading of the manuscript, I. Martini,G. Levi and M. Esposito for technical help, G. Bertini, P. Piccoliand G. Cortese for care of the mice. This work was supported bygrants from the Italian Ministero dell’Istruzione, Università eRicerca (PRIN 2006) and WHO (V27/181/184).

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Supplementary material

The following supplementary material is available for this articleonline:Fig. S1. Intracellular growth kinetics of M90T, M90T DampG andM90T DmppA in HeLa cell monolayer. HeLa cells were infectedwith the Shigella strains and intracellular growth kinetics evalu-ated during incubation for 9 h p.i. Means � SD of the numbers ofbacteria calculated in three separate invasion assays.Fig. S2. TLR2 activation in HeLa cells exposed to Shigella killedby gentamicin. Bacterial cultures (2 ¥ 107 cfu) were added withgentamicin (60 mg ml-1) and then exposed to HeLa cells pre-treated with gentamicin at the concentration of 60 mg ml-1 for30 min. Incubation was prolonged for 5 h.Appendix S1. Muramylpeptide analysis.Appendix S2. Histopathological scores in lungs and livers ofmice infected with Shigella flexneri 5 M90T or M90T DampG orM90T DmppA.

This material is available as part of the online article from:http://www.blackwell-synergy.com/doi/abs/10.1111/j.1462-5822.2007.01075.x

Please note: Blackwell Publishing is not responsible for thecontent or functionality of any supplementary materials suppliedby the authors. Any queries (other than missing material) shouldbe directed to the corresponding author for the article.

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