Modiﬁcationof Helicobacterpylori Peptidoglycan Enhances ... · pgdA was accomplished via insertion of a kanamycin cassette into pgdA as described (12). An H. pylori 7.13 pgdA comple-mented

Tumor and Stem Cell Biology

Modification ofHelicobacter pylori PeptidoglycanEnhances NOD1 Activation and Promotes Cancerof the StomachGiovanni Suarez1, Judith Romero-Gallo1, M. Blanca Piazuelo1, Ge Wang2, Robert J. Maier2,Lennart S. Forsberg3, Parastoo Azadi3, Martin A. Gomez4,5, Pelayo Correa1, andRichard M. Peek Jr1

Abstract

Helicobacter pylori (H. pylori) is the strongest known risk factorfor gastric carcinogenesis. One cancer-linked locus is the cagpathogenicity island, which translocates components of pep-tidoglycan into host cells. NOD1 is an intracellular immunereceptor that senses peptidoglycan from Gram-negative bacteriaand responds by inducing autophagy and activating NF-kB,leading to inflammation-mediated bacterial clearance; howeverchronic pathogens can evade NOD1-mediated clearance byaltering peptidoglycan structure. We previously demonstratedthat the H. pylori cagþ strain 7.13 rapidly induces gastric cancerin Mongolian gerbils. Using 2D-DIGE and mass spectrometry,we identified a novel mutation within the gene encoding thepeptidoglycan deacetylase PgdA; therefore, we sought to definethe role ofH. pylori PgdA in NOD1-dependent activation of NF-kB, inflammation, and cancer. Coculture of H. pylori strain 7.13

or its pgdA� isogenic mutant with AGS gastric epithelial cells orHEK293 epithelial cells expressing a NF-kB reporter revealed thatpgdA inactivation significantly decreased NOD1-dependent NF-kB activation and autophagy. Infection ofMongolian gerbils withan H. pylori pgdA� mutant strain led to significantly decreasedlevels of inflammation and malignant lesions in the stomach;however, preactivation of NOD1 before bacterial challenge recip-rocally suppressed inflammation and cancer in response to wild-typeH. pylori. Expression of NOD1differs in human gastric cancerspecimens compared with noncancer samples harvested from thesame patients. These results indicate that peptidoglycan deacety-lation plays an important role in modulating host inflammatoryresponses to H. pylori, allowing the bacteria to persist and inducecarcinogenic consequences in the gastric niche. Cancer Res; 75(8);1749–59. �2015 AACR.

IntroductionHelicobacter pylori (H. pylori) is the most common bacterial

infection worldwide and biologic costs incurred by chronic gas-tritis include an increased risk for gastric adenocarcinoma (1–4).However, only a percentage of colonized persons develop neo-plasia. One strain-specific virulence locus that augments cancerrisk is the cag pathogenicity island, which encodes a type IVsecretion system (T4SS) that translocates CagA into epithelialcells (5–13). However, most persons colonizedwith cagAþ strainsdo not develop cancer (1), suggesting that other H. pylori con-stituents affect disease risk.

In addition to CagA, the cag T4SS delivers peptidoglycan intohost cells where it is recognized by NOD1, an intracytoplasmic

sensor of peptidoglycan components (14–18). H. pylori can alsodeliver peptidoglycan into host cells via outer membrane vesicles(19). Most gastrointestinal epithelial cells express NOD1 andactivation of NOD1 by the muropeptide g-D-glutamyl-meso-dia-minopimelic acid (iE-DAP) leads to cytokine production as wellas induction of autophagy (20, 21). NOD1 activation is tightlyregulated by a negative autocrine feedback system, in whichNOD1-regulated effectors concomitantly suppress the down-stream effects of NOD1 activation (22–24).

Chronic pathogens can evade NOD1-mediated clearance byaltering peptidoglycan structure. NOD1 sensing of H. pylori pep-tidoglycan induces NF-kB activation and expression of type I IFNvia IFN-regulatory factor 7, MIP-2, and b-defensin (14, 15, 20, 22)andH. pylori colonizesNod1�/�-deficient mice more densely com-paredwithwild-typemice (14, 22). In humans, genetic variation inATG16L1, which encodes a key effector of NOD1-dependentautophagy and inflammation, alters susceptibility to H. pyloriinfection (25). The role of aberrant NOD1 activation by H. pyloriin gastric carcinogenesis, however, has not yet been investigated.

We previously demonstrated that in vivo adaptation aug-ments the ability of an H. pylori strain (7.13) to induce gastriccancer in Mongolian gerbils (12). Using mass spectrometry, wesubsequently identified a novel mutation within the geneencoding the peptidoglycan deacetylase, PgdA (HP0310homolog) in this carcinogenic strain (26). We now define therole of PgdA in NOD1-dependent activation of NF-kB, inflam-mation and inflammation-related cancer that develops inresponse to H. pylori.

1Departments of Cancer Biology and Medicine, Vanderbilt University,Nashville, Tennessee. 2Department of Microbiology, University ofGeorgia, Athens, Georgia. 3Complex Carbohydrate Research Center,University of Georgia, Athens, Georgia. 4Department of Medicine,National University of Colombia, Bogota, Colombia. 5Hospital El TunalUnit of Gastroenterology, Bogota, Colombia.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

CorrespondingAuthor:Richard Peek, Vanderbilt University, 1030CMRB IV, 2215Garland Avenue, Nashville, TN 37232-2279. Phone: 615-322-5200; Fax: 615-343-6229; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-2291

�2015 American Association for Cancer Research.

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Materials and MethodsBacterial strains

H. pylori wild-type strains or isogenic cagA� or cagE�

mutants have been described (12). Disruption of H. pyloripgdA was accomplished via insertion of a kanamycin cassetteinto pgdA as described (12). An H. pylori 7.13 pgdA comple-mented strain was generated by insertion of the pgdA gene intothe hp0203/0204 intergenic chromosomal region. Flankingsequences targeting hp0203 and hp0204 were cloned into thevector pBSC103. A chloramphenicol resistance cassette and theH. pylori 7.13 pgdA gene were then cloned into hp0203/hp0204that was previously inserted in pBSC103. H. pylori pgdA� wasnaturally transformed with pBSC103-pgdA and colonies select-ed for chloramphenicol and kanamycin resistance were testedby PCR and Western blot analysis to confirm re-expression ofpgdA (Supplementary Fig. S1).

Cell lines and cultureAGS cells (CRL-1739) and HEK 293 cells (CRL-1573) were

purchased from ATCC and tested for mycoplasma contamina-tion. AGS cells stably expressing a luciferase-based NF-kBreporter were generated by transfection of the plasmidpGL4.32[luc2P/NF-kB-RE/Hygro] (Promega), antibiotic selec-tion and cloning. AGS cells stably transfected with the NF-kBreporter were also transfected with a mix of shRNAs targetingNOD1. Colonies were selected using puromycin (10 mg/mL)and tested for NOD1 expression by real-time RT-PCR andWestern blot analysis.

Mass spectrometryAnalysis of H. pylori peptidoglycan by mass spectrometry was

performed as reported (27).H. pyloriwere harvested, washedwithice-cold 20 mmol/L sodium acetate (pH 5), centrifuged, resus-pended, and then added to boiling 4% SDS buffered with 20mmol/L sodiumacetate (pH5) for 30minutes. Sampleswere thencooled, and SDS-insoluble material was collected by centrifuga-tion. The pellet was resuspended in 5mL of 100mmol/L Tris-HCl(pH 7.5) and 10mmol/L NaCl. Of note, 10 mg/mL DNase and 50mg/mL of RNase (Sigma) were added and incubated for 2 hours.Of note, 50 mg/mL proteinase K (Invitrogen) was added to thereaction and incubated overnight. The SDS-insoluble materialwas reextracted by boiling in 1% SDS and collected by centrifu-gation. The peptidoglycan pellet was resuspended in distilledwater, lyophilized, and suspended in 200 mL of 20 mmol/Lsodium phosphate buffer (pH 4.8). Following sonication, sus-pensions were digested with 50 mg/50 mL of muramidase for 18hours at 37�C. Enzyme incubations were placed in a 100�C bathfor 3 minutes, cooled, and then centrifuged to remove insolublematerial. Supernatants were then filtered and concentrated formass spectrometry analysis.

Portions of the reaction mixtures resulting from lysozymedigestion were desalted by dialysis (500molecular weight cutoff)for 2 days. The retained solutions, containing muramyl peptides,were concentrated and analyzed by MALDI-TOF mass spectrom-etry as described (27). Chemical N-acetylation of peptidoglycanwas performed as described (27).

Cell viabilityCell viability was measured using the vibrant MTT cell assay kit

following the manufacturer's instructions (Molecular probes).Control and NOD1-overexpressing (pUNO1-hNOD1, Invivo-

gen) HEK293 cells were plated in a 96-well plate at 4 � 104

cells/well. The following day, media were replaced with 100 mL ofDMEM supplemented with 10% FBS and 1.2mmol/L ofMTT andincubated for 4 hours at 37�C.

Adhesion assaysCocultures of AGS cells and H. pylori (MOI 10) were harvested

after 30 minutes of incubation. AGS cells were washed to removeunbound bacteria, lysed with distilled water, serial dilutions wereplated on TSA-blood agar plates and colony-forming units (CFU)were determined as described (12).

Luciferase assayAGS cells stably transfected with the NF-kB reporter were

cocultured with H. pylori and lysed using 500 mL of luciferasereporter lysis buffer (Promega). Twenty microliters of cell lysatewas then mixed with 100 mL of luciferase assay substrate (Pro-mega) and luciferase activity was measured.

Gentamycin protection assaysCocultures of AGS cells and H. pylori (MOI 10) were incu-

bated for 3 hours, supernatants were removed, and cells wereincubated for an additional 3 hours with gentamycin (250mg/mL). Cells were lysed with sterile water and serial dilutionswere plated.

Real-time RT-PCRMongolian gerbil primers and probes were designed based on

gerbilmRNA sequences deposited in theNCBI database. RNAwasisolated using Qiagen RNeasy Kit and gDNA was removed bydigestion with RNAse free-DNAse I (Promega). cDNA was syn-thesized using the High Capacity cDNA Reverse Transcription Kit(Applied Biosystems). qPCR was performed using a TaqManUniversal PCR Master Mix or SYBR Green Universal PCR MasterMix (Applied Biosystems) in a 7300 Real Time PCR System(Applied Biosystems). All samples were normalized to expressionof GAPDH.

Western blot analysisAGS cells cocultured with H. pylori were lysed, centrifuged and

proteins were separated using 12% SDS-PAGE mini gels, trans-ferred to PVDF membranes and membranes were blocked with1% BSA. For detection of LC3B, membranes were incubated for 1hour with rabbit anti-LC3B (dilution 1:500; Novus Biologicals).An anti-rabbit HRP-conjugated (Santa Cruz Biotechnology) sec-ondary antibody was then incubated with membranes at a1:5,000 dilution for 1 hour.

Transmission electron microscopyCocultures of AGS cells and H. pylori were fixed in 2.5%

gluteraldehyde in 0.1 mol/L cacodylate buffer, transferred to 4�C,and left overnight. Samples were then washed in 0.1 mol/Lcacodylate buffer, incubated for 1 hour in 1%osmium tetraoxide,and washed with 0.1 mol/L cacodylate buffer. Samples weredehydrated using a graded ethanol series and incubated for 5minutes in 100% ethanol and propylene oxide (PO) followed bytwo exchanges of pure PO. Samples were infused with 25% Epon812 resin and 75% PO, infiltrated with 50% Epon 812 resin and50% PO, exchanged with new 50% Epon 812 resin and 50% PO,and incubated overnight. Samples were then subjected to a75%:25% (resin: PO) exchange, and exchanged into pure epoxy

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Cancer Res; 75(8) April 15, 2015 Cancer Research1750




resin overnight. The resin was exchanged using freshly made pureepoxy resin, incubated for 3 hours, and embedded in epoxy resinand polymerized at 60�C for 48 hours.

Of note, 500 nm to 1-micron thick sections were generatedusing a Leica Ultracut microtome. Thick sections were contraststained with 1% toluidine blue and imaged with a Nikon AZ100microscope. Ultra-thin sections (78 nm)were then cut and placedonto 300-mesh copper grids, post-section stained with 2% uranylacetate (aqueous) for 15minutes, and thenwith lead citrate for 15minutes. Samples were imaged on a Philips/FEI Tecnai T12electron microscope.

Confocal microscopyAGS cells were plated (1 � 105 cells/well) in 4-well slides

(Nunc) for 24 hours, and cocultured with H. pylori for 6 hours.Live staining with Image-IT lysosomal and nuclear labeling kit(Life technologies) was performed per themanufacturer's instruc-tions. AGS cells expressing the fusion protein GFP-LC3 werecocultured with H. pylori, fixed with cytofix/cytoperm (BectonDickinson), andmounted. Imageswere acquired using a LSM710META inverted confocal microscope (Zeiss).

Rodent infectionsAll procedures were approved by the Animal Care Committee

of Vanderbilt University (Nashville, TN). Gerbils were treatedwith or without C12-iE-DAP (Invivogen) at doses of 5, 20, or 40mg/animal in 1mLof PBS for 6 days via gavage (day 1–6). Animalswere challenged with 2 � 109 H. pylori 7.13 wild-type or pgdA�

mutant strains at two timepoints (days 3 and 5) as previouslydescribed (12). Serum samples and gastric tissue were harvestedand a single pathologist scored indices of inflammation andcancer as described (12). For quantitative culture, serial dilutionsof homogenized tissue were plated on selective antibiotic TSA-blood agar plates as described (12).

ELISAH. pyloriwild-type strain 7.13 (1�109 cells)was lysed in 200mL

of IP Lysis/wash buffer (Thermo). ELISA plates were coated with100 mL of lysate diluted 1:20 in coating buffer (0.1 M Sodiumcarbonate, pH9.5) at 4�Covernight.Wellswere thenblockedwith250 mL of BSA for 1 hour, and 100 mL of serumdiluted in PBS-BSA0.1% 1:20 was incubated for 2 hours followed by a 1-hourincubation with protein G-conjugated with HRP (2.5 mg/mL).Tetramethylbenzidine was used as a substrate, and the colorimet-ric density at wavelength 450 nm was measured.

IHC and real-time RT-PCR on tissueThe Institutional Review Board of the "Hospital El Tunal" in

Bogota, Colombia approved this protocol. Biopsy samplesfrom 34 patients with moderately differentiated intestinal-typegastric adenocarcinoma, obtained via endoscopy from foci ofcancer or unaffected regions were used for IHC (n ¼ 10; 4 male,mean-age 65.1 years/6 female, mean-age 63.8 years) or real-time RT-PCR (n ¼ 24; 16 male, mean-age 65.5 years/8 female,mean-age 66 years). Samples were stained with a monoclonalanti-NOD1 antibody (1:300, R&D Biosciences) or an isotypecontrol antibody. A single pathologist (M.B. Piazuelo) scoredNOD1 staining by assessing the percentage of NOD1þ epithe-lial cells and grading the intensity of NOD1 staining in epi-thelial cells semiquantitatively. For RT-PCR, gastric tissue washomogenized and RNA extracted (Qiagen), according to the

manufacturer's instructions. Reverse-transcriptase PCR andqPCR were performed, according to the manufacturer's instruc-tions, to determine relative differences in expression levels ofNOD1 (Hs00196075_m1), CDX2 (Hs01078080_m1), SOX2(Hs01053049_s1), and TRAF3 (Hs00936781_m1) normalizedto levels of GAPDH (4326317E; Applied Biosystems).

Statistical analysisThe Mann–Whitney test was used to compare two groups, and

one-way ANOVA with a Newman–Keuls post-test was used tocompare three or more groups. Data were plotted and analyzedusing Prism V. 5b (GraphPad software Inc).

ResultsInactivation of H. pylori pgdA affects peptidoglycan acetylationand attenuates NOD1-dependent NF-kB activation induced byH. pylori in vitro

Our previous publication (26) reported that levels of PgdAinitially appeared to be different in the derivativeH. pylori strain7.13 compared with its progenitor strain B128. However,detailed inspection of the 2D-DIGE gels revealed that apparentdifferences in protein levels resulted from the presence ofdifferent charged isoforms, rather than an abundance changein PgdA, between the two strains. This difference was confirmedby genomic sequencing of pgdA in strains B128 and 7.13, whichdemonstrated a single point mutation that converted a cysteineat amino acid position 34 in strain B128 to an arginine in strain7.13 (26). We therefore quantified levels of expression of PgdAin wild-type strains B128 and 7.13, an isogenic 7.13 pgdA�

mutant, and a 7.13 pgdA complemented strain. There were nodifferences in expression of PgdA between the H. pylori 7.13wild-type strain, the B128 wild-type strain, or the 7.13 pgdAcomplemented mutant and, as expected, no PgdA expressionwas present in the 7.13 pgdA� isogenic mutant (SupplementaryFig. S1A).

We next sought to determine whether PgdA functions as apeptidoglycandeacetylase in our prototype strain of interest, 7.13.Therefore, we analyzed the muropeptide composition of pepti-doglycan purified from wild-type H. pylori strain 7.13, the 7.13isogenic pgdA� mutant, and the 7.13 pgdA complemented strain.During preparation of the muropeptides, an important initialobservation was that the three peptidoglycan samples respondedvery differently to muramidase treatment. Although peptidogly-can from the pgdA�mutant was completely digested by lysozyme,peptidoglycan from the WT and the pgdA complemented strainswere highly resistant to lysozyme treatment, with more than halfof the peptidoglycan from the latter two strains remaining insol-uble (data not shown). This result suggested that the comple-mented pgdA strain was similar to the WT strain in terms of itspeptidoglycan structure.

After using more intense conditions for digestion, the digestedpeptidoglycan samples (muropeptide fragments) were subjectedtoMALDI-TOF analysis. A distinct set of muropeptides (MP) with42 mass unit (acetyl group) differences (MP1470 and MP1512)were identified that readily distinguished theWT strain 7.13 fromthe pgdA� mutant strain (Fig. 1A). The proposed structure forMP1512 is GlcNAc-MurNAc-GlcNAc-MurNAc, which harbors alinked side chain of amino acids Ala-Glu-Dap-Ala-Ala. When oneof the GlcNAc moieties is deacetylated (GlcNH2), MP1512becomes MP1470 (Supplementary Fig. S1B). Wild-type strain

H. pylori, NOD1, Gastric Cancer

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7.13 contained relatively equal amounts ofMP1470 andMP1512(ratio deacetylated/acetylated ¼ 1.1); however, the extent ofdeacetylation was greatly diminished in the pgdA� mutant strain(ratio deacetylated/acetylated ¼ 0.49; Fig. 1A). In the pgdA com-plemented strain, the ratio of intensities of these two MPs reap-proached equality (ratio deacetylated/acetylated¼ 0.84), suggest-ing that deacetylation activitywas nearly restored. To demonstratethis more definitively, chemical N-acetylation of the muropep-tides was performed. Both MPs (1470 and 1512) became 1554(þ1 or 2 acetyl groups) after chemical N-acetylation (data notshown). These data are concordantwith results from the lysozymesensitivity studies, indicating that PgdA functions as a peptido-glycan deacetylase in H. pylori strain 7.13.

To determine whether endogenous NOD1 was functionallyresponsive to carcinogenic H. pylori cagþ strain 7.13, AGS cellswere stably transfected with a NF-kB reporter. Treatment oftransfected cells with the NOD1-selective agonist C12-iE-DAP

significantly increased luciferase activity (Fig. 1B). TransfectedAGS cells were then infected with wild-type (WT) strain 7.13,which also significantly increased luciferase activity comparedwith uninfected cells (Fig. 1B). Isogenic inactivation of pgdAsignificantly attenuated NF-kB activation compared with thewild-type strain; however, activation was nearly completelyrestored when AGS cells were infected with the H. pylori pgdAcomplemented mutant (Fig. 1B).

We then tested a cagE mutant, which lacks a functional cagT4SS, to define the role of the T4SS in NOD1-dependent NF-kBactivation. Loss of cagE significantly reduced the ability of H.pylori to activate NF-kB (Fig. 1B). The cag island delivers notonly peptidoglycan, but also CagA into host cells, and CagA canactivate NF-kB (Fig. 1B; ref. 28). Deletion of cagA in strain 7.13partially, but not completely, reduced NF-kB activation (Fig.1B). These results suggest that a component of NF-kB activationthat develops in response to H. pylori may be mediated by a

Figure 1.Inactivation of pgdA affectspeptidoglycan acetylation andattenuates NOD1-dependent NF-kBactivation induced by H. pyloriin vitro. A, peptidoglycan from WTstrain 7.13, a pgdA� mutant, or acomplemented pgdA (pgdA/c)strain was analyzed by massspectrometry and ratios betweenm/z 1470 (deacetylated) and m/z1512 (acetylated) were calculated.AGS cells transfected with a NF-kBluciferase reporter (B), cotransfectedwith a dominant negative NOD1(DN NOD1; C), or transfected withshRNA targeting NOD1 or NOD2 (D)were cocultured with WT strain7.13, a pgdA� mutant, apgdA-complemented mutant(pgdA/c), a cagA� mutant, or acagE� mutant for 4 hours. C12-iE-DAPand PMA were used as NOD1-dependent and NOD1-independentactivators of NF-kB, respectively.(�� , P � 0.001; �� , P � 0.01;� , P � 0.05; þ, P � 0.05 vs.uninfected control). Data representmean � SEM from at least threeexperiments.

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different effector translocated by the cag type IV secretionsystem (such as peptidoglycan).

Toestablish specificity,AGScellswere stably cotransfectedwithaNF-kB reporter and either a truncated form of NOD1, which lacksthe CARD4 domain and exerts a dominant negative effect (DNNOD1, Fig. 1C), or shRNA targeting NOD1 or NOD2 (Fig. 1D).NOD1 activation in response to treatment with C12-iE-DAP orH.pylori, but not the NOD1-independent NF-kB activator PMA, wassignificantly attenuated in cells transfected with DN NOD1 orNOD1-specific, but not NOD2-specific, shRNA, indicating thatactivation of NOD1 by H. pylori is specific (Fig. 1C and D).NOD1-specific shRNA suppressed endogenous NOD1 by approx-imately 60% (Supplemental Fig. 2). Thus, NOD1 is functionallyactive in gastric epithelial cells infectedwith a carcinogenicH. pyloricagþ strain, and this is dependent on peptidoglycan deacetylaseand a functional cag T4SS.

Inactivation of pgdA attenuates H. pylori-induced autophagyHaving demonstrated that H. pylori can activate NOD1, we

next examined the effects of PgdA on autophagy (21, 29). Wefirst compared binding of wild-type strain 7.13 or a 7.13 pgdA�

mutant to AGS cells; no differences in adherence were foundbetween the strains (Fig. 2A). However, there were strikingdifferences in intracellular survival as the number of viablepgdA� mutants recovered was significantly reduced comparedwith the wild-type strain (Fig. 2B). Transmission electronmicroscopy and Lysotracker staining revealed that the numberof lysosomeswas significantly increased in cells infectedwith theisogenic 7.13 pgdA� mutant compared with the wild-type strain(Fig. 2C and D).

The formation of lysosomes can represent one step in theautophagy pathway. In addition to autophagy, however, lyso-somes also represent a cellular constituent that can degradeendocytic substrates (30). To determine this more definitively,we next defined the role of PgdA on autophagy by quantifying asine qua non autophagic response, conversion of LC3-I to LC3-II.Confocal microscopy using AGS cells transfected with a GFP-LC3fusion protein to ascertain the presence of intracellular vesiclescontaining LC3 demonstrated that loss of pgdA attenuated theincrease in levels of LC3 inducedbywild-type strain 7.13 (Fig. 2E).These results were subsequently confirmed as levels of LC3-II weresignificantly reduced in cells infected with the pgdA� mutantcompared with cells infected with wild-type H. pylori (Fig. 2F).These functional studies support our earlier results focused onNOD1 activation, and indicate that PgdA deacetylase plays a rolein regulating NOD1-dependent cellular responses, includingautophagy. Furthermore, these data suggest that increased for-mation of lysosomes induced by the pgdA� mutant reflects anincrease in endocytotic trafficking to lysosomes that is indepen-dent of autophagy.

Colonizationofwild-typeH. pylorior pgdA�mutants in a rodentmodel of gastric cancer

We next sought to determine whether loss of microbialconstituents required for NOD1 activation altered pathologicresponses in a rodent model of H. pylori-induced gastric carci-nogenesis. There was a significant difference in colonizationefficiency between Mongolian gerbils infected with the wild-type strain (100%) and the isogenic pgdA� mutant (60%), at 2and 12 weeks combined (Fig. 3A). Colonization density levels

Figure 2.Inactivation of pgdA sensitizes H. pylori to intracellular degradation butnot autophagy. A, cocultures of H. pylori and AGS cells were lysed andplated to quantify adherent bacteria. Data represent mean � SEM fromat least three experiments. NS, nonsignificant. B, gentamycin protectionassays were performed to quantify viable intracellular H. pylori. (�� , P �0.001). Data represent mean � SEM from at least three experiments.C, transmission electron microscopy of WT strain 7.13 or a pgdA� mutantcocultured with AGS cells for 4 hours. Arrows, bacteria; arrowheads,phagolysosomal vesicles. D, confocal images of AGS cells coculturedwith WT 7.13 strain or a pgdA� mutant for 4 hours. Cells werestained with LysoTracker DND-99 (red) and nuclear stain Hoechst 33342(white). E, confocal images of AGS cells expressing a GFP-LC3 fusionprotein (green) cocultured with WT strain 7.13 or its pgdA� mutantfor 6 hours. Cells were fixed and mounted in media containing DAPI(blue). F, Western blot analysis of AGS cells cocultured with WTstrain 7.13 or its pgdA� mutant using anti-LC3 and anti-actinantibodies, and subsequent densitometry analysis from threereplicates (� , P � 0.05).






were also significantly decreased in gerbils successfully infectedwith the pgdA� mutant versus the wild-type strain (Fig. 3B andC). Levels of anti-H. pylori antibodies were significantly loweramong gerbils infected with the 7.13 pgdA� mutant versus thewild-type strain 12 weeks postchallenge (Fig. 3D). We nextcompared bacterial colonization density and levels of anti-H.pylori antibodies. For gerbils infected with wild-type strain 7.13,there was a significant inverse relationship between coloniza-tion density and antibody levels (correlation coefficient, r2 ¼0.71, P ¼ 0.0006, data not shown). In contrast, there was asignificant concordant relationship between antibody levelsand colonization density in gerbils infected with the pgdA�

mutant (correlation coefficient, r2 ¼ 0.63, P ¼ 0.002, data notshown).

Gerbils infected with wild-type H. pylori developed signifi-cantly higher inflammatory scores compared with gerbils suc-cessfully infected with the pgdA� mutant strain (Fig. 4B). Wethen compared bacterial colonization density and severity ofinflammation in gerbils infected with the wild-type strainversus the pgdA� mutant strain. In gerbils infected with eitherWT strain 7.13 or the pgdA� mutant, there was an inverserelationship between colonization density and levels of inflam-mation (data not shown). However, in gerbils infected with WTH. pylori (n ¼ 3) or the pgdA� mutant (n ¼ 5), and that hadsimilar levels of colonization density (range 1 � 106 to 2 � 106

CFU/gram stomach tissue), inflammation scores were very

different. All five of the pgdA�-mutant infected gerbils hadinflammation scores of <1; in contrast, two of the three WT-infected gerbils had inflammation scores ranging between 2and 3. Consistent with increased severity of inflammation,gastric adenocarcinoma was observed more frequently inwild-type 7.13-infected gerbils (50%) compared with gerbilsinfected with the 7.13 pgdA�-mutant strain (0%; Fig. 4C).Collectively, these data indicate that PgdA plays a critical rolein H. pylori-induced gastric carcinogenesis.

Preactivation of NOD1 suppresses H. pylori-induced signalingand injury in vivo and in vitro

The effects of NOD1 activation are tightly regulated andNOD1-induced constituents can inhibit the subsequent con-sequences of NOD1 activation (22–24). We sought to deter-mine whether activation of NOD1 before H. pylori infectioncould modify injury within the gastric niche. Gerbils weretreated with the NOD1-specific agonist C12-iE-DAP for 6 daysvia gavage. To determine the efficacy of agonist treatment forNOD1-mediated NF-kB activation, the NF-kB target KC (Cxcl1)was quantified in gastric tissue by real-time RT-PCR following a6-day treatment. Levels of KC in gerbils treated with the NOD1agonist increased in a dose-dependent fashion compared withvehicle control (Fig. 5A). These results indicate that C12-iE-DAP can successfully activate NOD1-dependent signaling inthe stomach.

Figure 3.Loss of H. pylori pgdA decreasescolonization in Mongolian gerbils. A,colonization efficiency of H. pyloristrains in gerbils. Two and 12 weekspostchallenge results are combined (�,P � 0.05). B, silver-stain imagesfrom gerbils infected with WT orpgdA� mutant strains, 12 weekspost-challenge. C, colonizationdensity of WT or pgdA� strains 12weeks postchallenge (� , P � 0.05). D,H. pylori-specific antibodies werequantified by ELISA in sera harvestedfrom gerbils challenged with WT orpgdA� mutant 7.13 strains for12 weeks (� , P � 0.05).

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We then examined the effects of NOD1 activation before H.pylori infection. Following 2 days of C12-iE-DAP treatment, ger-bils were challenged with or without strain 7.13 and evaluated 12weeks postchallenge. Preactivation of NOD1 led to a significantreduction in levels of colonization, inflammation, and the devel-opment of cancer (Fig. 5B–D). We also normalized for coloni-zation density in these experiments and found that densities werenot significantly different among infected gerbils developingcancer that were pretreated with C12-iE-DAP versus vehicle con-trol (C12-iE-DAP-treated, n ¼ 5, mean density 1.2 � 105 CFU/g;vehicle-treated, n ¼ 7, mean density 2.4 � 106 CFU/g; P ¼ 0.11).Although these numbers are small, they suggest that the severity ofdisease ismore dependent onNOD1activation than colonizationdensity per se.

We next sought to investigate these in vivo observations ingreater depth using manipulatable in vitro systems. HEK293 cellscarrying a NF-kB alkaline phosphatase-linked reporter andcotransfected with a human NOD1 overexpression vector(NOD1) or a control construct (Control) were purchased and

cocultured with H. pylori strain 7.13 for 24 hours and NF-kBactivity was quantified. Levels of NF-kB activation were signifi-cantly reduced in H. pylori-infected NOD1-overexpressing cellscompared with infected control cells (Fig. 6A). To extend theseresults, Null or NOD1-overexpressing cells were infected withanotherH. pylori cagþ strain, J166. Similar to results obtainedwithstrain 7.13, overexpression of NOD1 attenuatedNF-kB activationinduced by strain J166, findings consistent with our in vivo data(Fig. 5). We also examined cell viability in NOD1-overexpressingcells thatwere either coculturedwithH. pylori, or treatedwithC12-iE-DAPor PMA. Therewere nodifferences in viability between anyof the groups, indicating that the combination of NOD1 over-expression andH. pylori infection does not induce increased levelsof cell death (Supplementary Fig. S3).

We then used a complementary strategy to examine the effectsof NOD1 activation on H. pylori-induced cellular responses byinhibiting NOD1 activation in vitro for extended periods of timebefore exposure to H. pylori. For these studies, AGS cells weretreated with either NOD1-targeting shRNA or ML-130, a specificinhibitor of NOD1 activation, 72 hours before infection. Extend-ed inhibition ofNOD1before infection reciprocally increasedNF-kB activation in response to H. pylori, indicating that manipula-tion of NOD1 can affect cellular responses to this pathogen (Fig.6B and C).

Our results above indicate that preactivation of NOD1 sup-pressed H. pylori-induced signaling, raising the possibility of aNOD1-dependent negative feedback loop. To explore theseobservations in greater depth, we focused on a specific

Figure 5.Preactivation of NOD1 in Mongolian gerbils before H. pylori infectionattenuates disease. A, gerbilswere treatedwith different doses of C12-iE-DAPfor 6 days. Expression of KC (Cxcl1) at day 7 was measured by RT-PCR as anindicator of NOD1 activation. Data represent mean � SEM. Colonizationdensity (B), inflammation (C), and incidence of adenocarcinoma (D) in gerbilstreated with C12-iE-DAP (40 mg/animal/day) for 3 days before and 3 daysafter challenge with WT strain 7.13. Animals were sacrificed 12 weekspostchallenge. (�� , P � 0.01; � , P � 0.05).

Figure 4.Inactivation of PgdA alters H. pylori-induced inflammation and diseaseoutcome in Mongolian gerbils. A, H&E-stained gastric tissue from gerbilschallenged with WT strain 7.13 or a pgdA� mutant strain for 12 weeks. UI,uninfected. Bottom left, severe gastritis (arrow); bottom right, invasiveadenocarcinoma (arrow). B, inflammatory scores in gerbils infectedwith H. pylori 12 weeks postinfection (�� , P � 0.001); data representmean � SEM. C, disease outcome in gerbils challenged with 7.13 WT orits pgdA� mutant, 12 weeks postchallenge.






downstream target of NOD1, TRAF3 (22), which can also beregulated by AP-1. Coculture of AGS cells with wild-type strain7.13 increased expression of TRAF3 in AGS cells but this wasattenuated by inhibition of AP-1; further, inhibition of AP-1 orTRAF3 led to a reciprocal increase in NF-kB activity within thecontext of H. pylori infection (Fig. 6D–F). TRAF3-specific shRNAsuppressed endogenous TRAF3 by approximately 50% (Supple-mentary Fig. S2). Thus, AP-1 and TRAF3 are components of anegative feedback loop activated by H. pylori that can ultimatelysuppress NF-kB signaling.

NOD1 expression in human gastric tissueWe next extended our studies and examined NOD1 in gastric

tissue harvested from different gastric sites using a population ofH. pylori-infected patients from Colombia with intestinal-typegastric adenocarcinoma. Each patient served as their own controland samples were isolated from tumoral tissue and unaffectedregions of the stomach.

The vast majority of NOD1 staining was restricted to gastricepithelial cells and there was no staining using an isotypecontrol antibody (Fig. 7A). Epithelial staining intensity and

Figure 6.Overexpression or inhibition of NOD1before H. pylori infection alters NF-kBactivation. HEK293 cells carrying a NF-kB alkaline phosphatase-linkedreporter and cotransfected with ahuman NOD1-overexpression vector(pUNO1-hNOD1) or a control construct(Control) were purchased andcocultured with strains 7.13 or J166 for24 hours. Supernatantswere harvestedand alkaline phosphatase activity wasquantified (A). AGS cells were stablytransfected with a NF-kB-Luciferaselinked reporter and either nontargetingshRNA or shRNA specific for NOD1 (B)or TRAF3 (F) or pretreated for 3 dayswith ML130, a specific inhibitor of NOD1activation (C), or SR11302, a specificinhibitor of AP-1 (D). Luciferase activitywas measured after 4 hours ofcoculture with H. pylori. TRAF3 mRNAexpression was measured by real-timeRT-PCR in AGS cells pretreated withSR11302 for 3 days and then challengedwithWT strain 7.13 for 4 hours (E). Datarepresent mean � SEM from at leastthree experiments. (�� , P � 0.001;�� , P � 0.01; � , P � 0.05; þ, P � 0.05compared with uninfected cells).

Suarez et al.





Figure 7.Expression of NOD1 is diminished inhuman malignant tissue. A, IHC forNOD1 in tumoral (red) and non-tumoral epithelia (middle) comparedwith isotype control (bottom). B,NOD1 staining intensity in epithelialcells (top) and percentage of NOD1-positive epithelial cells (bottom). Datarepresent mean � SEM. (�, P � 0.05).C, qRT-PCR expression for NOD1 andrelated genes in gastric biopsies fromtumoral and nontumoral tissue(�� , P � 0.001; �� , P � 0.01;� ,P�0.05). Results expressed as ratioof target gene/GAPDH mRNA incancer or noncancer samples relativeto the mean ratio in nontumor tissue.Data represent mean � SEM fromthree replicates/sample. D, workingmodel of NOD1 activation anddownstream consequences within thecontext of H. pylori infection.






the number of gastric epithelial cells expressing detectableNOD1 were significantly higher in noncancer compared withcancer samples (Fig. 7A and B). These results were subsequentlyconfirmed in an independent set of samples using real-time RT-PCR (Fig. 7C).

CDX2 is a trans-differentiation factor that can influence thedevelopment of intestinal-type gastric adenocarcinoma (31).Previous studies have demonstrated that NF-kB activation leadsto increased production of CDX2 via suppression of SOX2(31–34). Our in vitro results in Fig. 6 indicate that suppressinglevels of NOD1 for prolonged periods of time lead to increasedNF-kB expression within the context of H. pylori infection. There-fore, we next quantified levels of CDX2 and SOX2 expression inintestinal-type gastric cancer and nonmalignant gastric tissue.Levels of CDX2 expression were significantly increased but levelsof SOX2were significantly decreased inmalignant comparedwithnonmalignant tissue (Fig. 7C). Furthermore, the NOD1 down-stream target TRAF3 was also reduced in cancer versus noncancertissue, similar to levels ofNOD1 (Fig. 7C).GAPDHwas used as thecontrol gene to normalize results for target genes and expressionlevels for all genes testedwerewithin the limits of detection. Thus,during the late stages of gastric carcinogenesis, NOD1 expressionis reduced in intestinal-type gastric adenocarcinomas comparedwith uninvolved gastric tissue, which is accompanied by anincrease in CDX2 expression.

DiscussionH. pylori cagþ strains deliver components of peptidoglycan into

epithelial cells via the cag secretion system, leading to NOD1-dependent signaling (14) and our laboratory has shown that H.pylori peptidoglycan can lead to decreased apoptosis, increasedproliferation, and increased cell migration (35). However, therelationship between NOD1 activation and H. pylori-inducedgastric carcinogenesis remains unclear. Our current results havenow demonstrated that NOD1 has an important role in gastriccarcinogenesis and further that H. pylori-induced injury can besignificantly decreased by preactivation of this receptor, whichmay, over longperiods of infection, lead to reductions in the levelsof NOD1 expression and its target genes (Fig. 7D).

Our in vivo studies demonstrate that loss of PgdA significantlyattenuated the development of inflammation and cancer in Mon-golian gerbils infected with H. pylori, implicating this microbialconstituent in the cascade to carcinogenesis. These data mirrorresults derived from amousemodel ofH. pylori infection inwhichloss of PgdA led to a significant colonization defect (36). We alsofound in a Colombian population of patients with intestinal-typegastric cancer, that levels of NOD1 were significantly lower incancer versus noncancer specimens, and this was accompanied byan increase in expression of the intestinal-specific transcriptionfactor CDX2. This is of interest as Allison and colleagues founddiffering results (37). Specifically, their results from patients

residing in Australia indicated that mRNA expression levels ofNOD1 were increased in cancer versus nontumor samples (37).

Another difference in relation to the in vitro experiments is thatprevious studies have shown that deacetylation of H. pylori pep-tidoglycan can lead to decreased NOD1 activation (38). Ourcurrent results indicate that isogenic inactivation ofH. pylori pgdAin strain 7.13 attenuated NF-kB activation compared with levelsinduced by the wild-type strain; further analysis, however, dem-onstrated that preactivation of NOD1 actually suppressed H.pylori-induced signaling. We speculate that, in addition to timingof NOD1 activation, differences in the histologic types of gastricadenocarcinoma studied in conjunction with different H. pyloristrain and human ancestry, and possibly in vitro growth condi-tions, may account for these findings and further work is neededto understand these differences in greater depth. Collectively,these results suggest that manipulation of NOD1 may representa novel strategy to prevent or treat pathologic outcomes inducedby H. pylori infection.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G. Suarez, R. Maier, R.M. PeekDevelopment of methodology: G. Suarez, R.M. PeekAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Suarez, J. Romero-Gallo, M.B. Piazuelo, R. Maier,L.S. Forsberg, M.A. Gomez, P. CorreaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):G. Suarez, G. Wang, R. Maier, L.S. Forsberg, P. Azadi,R.M. PeekWriting, review, and/or revision of the manuscript: G. Suarez, G. Wang,R. Maier, L.S. Forsberg, P. Azadi, M.A. Gomez, P. Correa, R.M. PeekAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Suarez, J. Romero-Gallo, R. Maier, L.S.Forsberg, R.M. PeekStudy supervision: R.M. Peek

AcknowledgmentsThe authors thank Drs. Laura Greenfield and Nicola Jones for the GFP-LC3

fusion protein, and Drs. Janice Williams and Mary Dawes for their expertise inimaging.

Grant SupportThis work was supported by NIH R01-DK58587, R01-CA77955, P01-

CA116087, P30-DK058404, P01-CA028842, University of Georgia ResearchFoundation, Complex Carbohydrate Research Center, Chemical Sciences,Geosciences and Biosciences Division, Office of Basic Energy Sciences, andU.S. DOE grant DE-FG02-93ER20097.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 4, 2014; revised February 19, 2015; accepted February 20,2015; published OnlineFirst March 2, 2015.

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2015;75:1749-1759. Published OnlineFirst March 2, 2015.Cancer Res Giovanni Suarez, Judith Romero-Gallo, M. Blanca Piazuelo, et al. Activation and Promotes Cancer of the Stomach

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Modiﬁcationof Helicobacterpylori Peptidoglycan Enhances ... · pgdA was accomplished via insertion of a kanamycin cassette into pgdA as described (12). An H. pylori 7.13 pgdA comple-mented

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