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Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori–induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from l-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori. However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori. Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori–induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.

Helicobacter pylori (H. pylori) is the strongest known risk for gastric cancer. The H. pylori cag type IV secretion system is an oncogenic locus that translocates peptidoglycan into host cells, where it is recognized by NOD1, an innate immune receptor. Beyond this, the role of NOD1 in H. pylori–induced cancer remains undefined. To address this knowledge gap, we infected two genetic models of Nod1 deficiency with the H. pylori cag+ strain PMSS1: C57BL/6 mice, which rarely develop cancer, and INS-GAS FVB/N mice, which commonly develop cancer. Infected C57BL/6Nod1–/− and INS-GASNod1−/− mice acutely developed more severe gastritis, and INS-GASNod1−/− mice developed gastric dysplasia more frequently compared with Nod1+/+ mice. Because Nod1 genotype status did not alter microbial phenotypes of in vivo–adapted H. pylori, we investigated host immunologic responses. H. pylori infection of Nod1−/− mice led to significantly increased gastric mucosal levels of Th1, Th17, and Th2 cytokines compared with Nod1 wild-type (WT) mice. To define the role of specific innate immune cells, we quantified cytokine secretion from H. pylori–infected primary gastric organoids generated from WT or Nod1−/− mice that were cocultured with or without WT or Nod1−/− macrophages. Infection increased cytokine production from gastric epithelial cells and macrophages and elevations were significantly increased with Nod1 deficiency. Furthermore, H. pylori infection altered the polarization status of Nod1−/− macrophages compared with Nod1+/+ macrophages. Collectively, these studies demonstrate that loss of Nod1 augments inflammatory and injury responses to H. pylori. Nod1 may exert its restrictive role by altering macrophage polarization, leading to immune evasion and microbial persistence.Significance:These findings suggest that manipulation of NOD1 may represent a novel strategy to prevent or treat pathologic outcomes induced by H. pylori infection.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma, yet only a minority of infected persons ever develop this malignancy. One cancer-linked locus is the cag type 4 secretion system (cagT4SS), which translocates an oncoprotein into host cells. A structural component of the cagT4SS is CagY, which becomes rapidly altered during in vivo adaptation in mice and rhesus monkeys, rendering the cagT4SS nonfunctional; however, these models rarely develop gastric cancer. We previously demonstrated that the H. pylori cag+ strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. We now use this model, in conjunction with samples from patients with premalignant lesions, to define the effects of a carcinogenic host environment on the virulence phenotype of H. pylori to understand how only a subset of infected individuals develop cancer. H. pylori cagY sequence differences and cagT4SS function were directly related to the severity of inflammation in human gastric mucosa in either a synchronous or metachronous manner. Serial infections of Mongolian gerbils with H. pylori strain 7.13 identified an oscillating pattern of cagT4SS function. The development of dysplasia or cancer selected for attenuated virulence phenotypes, but robust cagT4SS function could be restored upon infection of new hosts. Changes in the genetic composition of cagY mirrored cagT4SS function, although the mechanisms of cagY alterations differed in human isolates (mutations) versus gerbil isolates (addition/deletion of motifs). These results indicate that host carcinogenic phenotypes modify cagT4SS function via altering cagY, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche. Cancer Res; 77(9); 2401–12. ©2017 AACR.

Primer sequences used for real time RT-PCR quantification.

PCR and sequencing primer sequences

Supplemental Western blot analysis, cagY and cagPAI RFLPs, cagPAI sequence alignment, and strains nomenclature.

Detailed Materials and Methods

RT2 profiler PCR array analysis for NF-kB gene targets in primary gastric epithelial cell organoids induced by strain 7.13

Chemokine and cytokine expression in C57BL/6 mice at 2, 20, and 90 days post-infection with H. pylori PMSS1

Chemokine and cytokine expression in INS-GAS mice at 2, 20, 40, and 90 days post-infection with H. pylori PMSS1.

Mouse NF-kB RT2 profiler PCR array of gastroids following infection with either H. pylori strain PMSS1 or 7.13.

Helicobacter pylori infection is the main risk factor for the development of gastric cancer, the third leading cause of cancer death worldwide. H. pylori colonizes the human gastric mucosa and persists for decades. The inflammatory response is ineffective in clearing the infection, leading to disease progression that may result in gastric adenocarcinoma. We have shown that polyamines are regulators of the host response to H. pylori, and that spermine oxidase (SMOX), which metabolizes the polyamine spermine into spermidine plus H2O2, is associated with increased human gastric cancer risk. We now used a molecular approach to directly address the role of SMOX, and demonstrate that Smox-deficient mice exhibit significant reductions of gastric spermidine levels and H. pylori-induced inflammation. Proteomic analysis revealed that cancer was the most significantly altered functional pathway in Smox-/- gastric organoids. Moreover, there was also less DNA damage and β-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and β-catenin activation was confirmed in human gastric organoids that were treated with a novel SMOX inhibitor. These findings indicate that SMOX promotes H. pylori-induced carcinogenesis by causing inflammation, DNA damage, and activation of β-catenin signaling.

BACKGROUND & AIMS Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined if Spd treatment reduces colitis and carcinogenesis. METHODS SMOX was quantified in human colitis and associated dysplasia using RT-qPCR and immunohistochemistry. We used wild-type (WT) and Smox–/– C57BL/6 mice treated with dextran sulfate sodium (DSS) or azoxymethane (AOM)-DSS as models of colitis and CAC, respectively. Mice with epithelial-specific deletion of Apc were used as a model of sporadic colon cancer. Animals were supplemented or not with Spd in the drinking water. Colonic polyamines, inflammation, tumorigenesis, transcriptomes, and microbiomes were assessed. RESULTS SMOX mRNA levels were decreased in human ulcerative colitis tissues and inversely correlated with disease activity, and SMOX protein was reduced in colitis-associated dysplasia. DSS colitis and AOM-DSS-induced dysplasia and tumorigenesis were worsened in Smox–/– versus WT mice and improved in both genotypes with Spd. Tumor development caused by Apc deletion was also reduced by Spd. Smox deletion and AOM-DSS treatment were both strongly associated with increased expression of alpha-defensins, which was reduced by Spd. A shift in the microbiome, with reduced abundance of Prevotella and increased Proteobacteria and Deferribacteres, occurred in Smox–/– mice and was reversed with Spd. CONCLUSIONS Loss of SMOX is associated with exacerbated colitis and CAC, increased alpha-defensin expression, and dysbiosis of the microbiome. Spd supplementation reverses these phenotypes, indicating that it has potential as an adjunctive treatment for colitis and chemopreventive for colon carcinogenesis.

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Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.

SUMMARY Innate responses of myeloid cells defend against pathogenic bacteria via inducible effectors. Deoxyhypusine synthase (DHPS) catalyzes the transfer of the N-moiety of spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form the amino acid hypusine. Hypusinated EIF5A (EIF5AHyp) transports specific mRNAs to ribosomes for translation. We show that DHPS is induced in macrophages by two gastrointestinal pathogens, Helicobacter pylori and Citrobacter rodentium, resulting in enhanced hypusination of EIF5A. EIF5AHyp was also increased in gastric macrophages from patients with H. pylori gastritis. Furthermore, we identify the bacteria-induced immune effectors regulated by hypusination. This set of proteins includes essential constituents of antimicrobial response and autophagy. Mice with myeloid cell-specific deletion of Dhps exhibit reduced EIF5AHyp in macrophages and increased bacterial burden and inflammation. Thus, regulation of translation through hypusination is a critical hallmark of the defense of eukaryotic hosts against pathogenic bacteria., Graphical Abstract, In Brief Gobert et al. demonstrate that hypusination, a specific mechanism regulating translation, is induced in macrophages by bacteria. Hypusination is required for the translation of inducible antimicrobial effectors. Mice that specifically lack hypusination in macrophages are highly susceptible to Helicobacter pylori and Citrobacter rodentium, two pathogens of the gastrointestinal tract.

ObjectiveGastric cancer is the third leading cause of cancer death worldwide and infection byHelicobacter pyloriis the strongest risk factor. We have reported increased epidermal growth factor receptor (EGFR) phosphorylation in theH. pylori-induced human carcinogenesis cascade, and association with DNA damage. Our goal was to determine the role of EGFR activation in gastric carcinogenesis.DesignWe evaluated gefitinib, a specific EGFR inhibitor, in chemoprevention ofH. pylori-induced gastric inflammation and cancer development. Mice with genetically targeted epithelial cell-specific deletion ofEgfr(EfgrΔepimice) were also used.ResultsIn C57BL/6 mice, gefitinib decreasedCxcl1andCxcl2expression by gastric epithelial cells, myeloperoxidase-positive inflammatory cells in the mucosa and epithelial DNA damage induced byH. pyloriinfection. Similar reductions in chemokines, inflammatory cells and DNA damage occurred in infectedEgfrΔepiversusEgfrfl/flcontrol mice. InH. pylori-infected transgenic insulin-gastrin (INS-GAS) mice and gerbils, gefitinib treatment markedly reduced dysplasia and carcinoma. Gefitinib blockedH. pylori-induced activation of mitogen-activated protein kinase 1/3 (MAPK1/3) and activator protein 1 in gastric epithelial cells, resulting in inhibition of chemokine synthesis. MAPK1/3 phosphorylation and JUN activation was reduced in gastric tissues from infected wild-type and INS-GAS mice treated with gefitinib and in primary epithelial cells fromEfgrΔepiversusEgfrfl/flmice. Epithelial EGFR activation persisted in humans and mice afterH. pylorieradication, and gefitinib reduced gastric carcinoma in INS-GAS mice treated with antibiotics.ConclusionsThese findings suggest that epithelial EGFR inhibition represents a potential strategy to prevent development of gastric carcinoma inH. pylori-infected individuals.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma, yet only a minority of infected persons ever develop this malignancy. One cancer-linked locus is the cag type 4 secretion system (cagT4SS), which translocates an oncoprotein into host cells. A structural component of the cagT4SS is CagY, which becomes rapidly altered during in vivo adaptation in mice and rhesus monkeys, rendering the cagT4SS nonfunctional; however, these models rarely develop gastric cancer. We previously demonstrated that the H. pylori cag+ strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. We now use this model, in conjunction with samples from patients with premalignant lesions, to define the effects of a carcinogenic host environment on the virulence phenotype of H. pylori to understand how only a subset of infected individuals develop cancer. H. pylori cagY sequence differences and cagT4SS function were directly related to the severity of inflammation in human gastric mucosa in either a synchronous or metachronous manner. Serial infections of Mongolian gerbils with H. pylori strain 7.13 identified an oscillating pattern of cagT4SS function. The development of dysplasia or cancer selected for attenuated virulence phenotypes, but robust cagT4SS function could be restored upon infection of new hosts. Changes in the genetic composition of cagY mirrored cagT4SS function, although the mechanisms of cagY alterations differed in human isolates (mutations) versus gerbil isolates (addition/deletion of motifs). These results indicate that host carcinogenic phenotypes modify cagT4SS function via altering cagY, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche. Cancer Res; 77(9); 2401–12. ©2017 AACR.

Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.

Helicobacter pylori (H. pylori) is the strongest known risk for gastric cancer. The H. pylori cag type IV secretion system is an oncogenic locus that translocates peptidoglycan into host cells, where it is recognized by NOD1, an innate immune receptor. Beyond this, the role of NOD1 in H. pylori–induced cancer remains undefined. To address this knowledge gap, we infected two genetic models of Nod1 deficiency with the H. pylori cag+ strain PMSS1: C57BL/6 mice, which rarely develop cancer, and INS-GAS FVB/N mice, which commonly develop cancer. Infected C57BL/6Nod1–/− and INS-GASNod1−/− mice acutely developed more severe gastritis, and INS-GASNod1−/− mice developed gastric dysplasia more frequently compared with Nod1+/+ mice. Because Nod1 genotype status did not alter microbial phenotypes of in vivo–adapted H. pylori, we investigated host immunologic responses. H. pylori infection of Nod1−/− mice led to significantly increased gastric mucosal levels of Th1, Th17, and Th2 cytokines compared with Nod1 wild-type (WT) mice. To define the role of specific innate immune cells, we quantified cytokine secretion from H. pylori–infected primary gastric organoids generated from WT or Nod1−/− mice that were cocultured with or without WT or Nod1−/− macrophages. Infection increased cytokine production from gastric epithelial cells and macrophages and elevations were significantly increased with Nod1 deficiency. Furthermore, H. pylori infection altered the polarization status of Nod1−/− macrophages compared with Nod1+/+ macrophages. Collectively, these studies demonstrate that loss of Nod1 augments inflammatory and injury responses to H. pylori. Nod1 may exert its restrictive role by altering macrophage polarization, leading to immune evasion and microbial persistence. Significance: These findings suggest that manipulation of NOD1 may represent a novel strategy to prevent or treat pathologic outcomes induced by H. pylori infection.

Background & Aims Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis. Methods The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori–induced carcinogenesis and in C57BL/6 mice treated with azoxymethane–dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori–infected INS-GAS mice was assessed by whole-exome sequencing. Results We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane–dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity. Conclusions Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.

Helicobacter pylori (H. pylori) is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. An H. pylori constituent that augments cancer risk is the strain-specific cag pathogenicity island, which encodes a type IV secretion system (T4SS) that translocates a pro-inflammatory and oncogenic protein, CagA, into epithelial cells. However, the majority of persons colonized with CagA+ H. pylori strains do not develop cancer, suggesting that other microbial effectors also have a role in carcinogenesis. Toll-like receptor 9 (TLR9) is an endosome bound, innate immune receptor that detects and responds to hypo-methylated CpG DNA motifs that are most commonly found in microbial genomes. High-expression tlr9 polymorphisms have been linked to the development of premalignant lesions in the stomach. We now demonstrate that levels of H. pylori-mediated TLR9 activation and expression are directly related to gastric cancer risk in human populations. Mechanistically, we show for the first time that the H. pylori cancer-associated cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Activation of TLR9 occurs through a contact-dependent mechanism between pathogen and host, and involves transfer of microbial DNA that is both protected as well as exposed during transport. These results indicate that TLR9 activation via the cag island may modify the risk for malignancy within the context of H. pylori infection and provide an important framework for future studies investigating the microbial-epithelial interface in gastric carcinogenesis.

Introduction Hypusine is an amino acid found only in the eukaryotic translation initiation factor 5A (EIF5A). Hypusine is synthesized by the enzyme deoxyhypusine synthase (DHPS) that transfers the N-moiety of spermidine to a lysine residue in EIF5A. This post-translational modification (hypusination) is essential to binding specific mRNAs. The EIF5AHyp/mRNA complex is translocated to the cytoplasm, reaches ribosomes, and allows for translation. We analyzed the role of hypusination in the innate response of macrophages. Results The gastric pathogen Helicobacter pylori stimulated DHPS expression and hypusination in murine bone marrow-derived macrophages (BMmacs). We generated myeloid-specific Dhps knockout mice (DhpsΔmye) by crossing Dhpsfl/fl mice to Lyz2-cre mice. By iTRAQ proteomics, 29 of the proteins induced by H. pylori in BMmacs from Dhpsfl/fl mice were significantly less expressed in infected DhpsΔmye mice. These included antibacterial effectors (IRG1, NOS2), cytokines (TNF-α, IL-1β), and mediators of autophagy (SQSTM, IRGM1). These findings were confirmed by Western blot. DhpsΔmye mice infected with H. pylori exhibited increased colonization (p Conclusions Hypusination in myeloid cells is critical for host innate immunity to limit the infectivity of pathogenic bacteria.

Chronic infection of the gastric mucosa by Helicobacter pylori is associated with an increased risk of developing gastric cancer; however, the vast majority of infected individuals never develop this disease. One H. pylori virulence factor that increases gastric cancer risk is the cag pathogenicity island, which encodes a bacterial type IV secretion system. Cyclooxygenase-2 (COX-2) expression is induced by proinflammatory stimuli, leading to increased prostaglandin E2 (PGE2) secretion by gastric epithelial cells. COX-2 expression is increased in gastric tissue from H. pylori -infected persons. H. pylori also activates the epidermal growth factor receptor (EGFR) in gastric epithelial cells. We now demonstrate that H. pylori -induced activation of COX-2 in gastric cells is dependent upon EGFR activation, and that a functional cag type IV secretion system and direct bacterial contact are necessary for full induction of COX-2 by gastric epithelial cells. PGE2 secretion is increased in cells infected with H. pylori , and this induction is dependent on a functional EGFR. Increased apoptosis in response to H. pylori occurs in cells treated with a COX-2 inhibitor, as well as COX-2−/− cells, indicating that COX-2 expression promotes cell survival. In vivo, COX-2 induction by H. pylori is significantly reduced in mice deficient for EGFR activation compared with wild-type mice with a fully functional receptor. Collectively, these findings indicate that aberrant activation of the EGFR-COX-2 axis may lower the threshold for carcinogenesis associated with chronic H. pylori infection.

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1 − strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1 − mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1 − CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

Epidermal growth factor receptor (EGFR) signaling is a known mediator of colorectal carcinogenesis. Studies have focused on the role of EGFR signaling in epithelial cells, although the exact nature of the role of EGFR in colorectal carcinogenesis remains a topic of debate. Here, we present evidence that EGFR signaling in myeloid cells, specifically macrophages, is critical for colon tumorigenesis in the azoxymethane-dextran sodium sulfate (AOM-DSS) model of colitis-associated carcinogenesis (CAC). In a human tissue microarray, colonic macrophages demonstrated robust EGFR activation in the pre-cancerous stages of colitis and dysplasia. Utilizing the AOM-DSS model, mice with a myeloid-specific deletion of Egfr had significantly decreased tumor multiplicity and burden, protection from high-grade dysplasia and significantly reduced colitis. Intriguingly, mice with gastrointestinal epithelial cell-specific Egfr deletion demonstrated no differences in tumorigenesis in the AOM-DSS model. The alterations in tumorigenesis in myeloid-specific Egfr knockout mice were accompanied by decreased macrophage, neutrophil and T-cell infiltration. Pro-tumorigenic M2 macrophage activation was diminished in myeloid-specific Egfr-deficient mice, as marked by decreased Arg1 and Il10 mRNA expression and decreased interleukin (IL)-4, IL10 and IL-13 protein levels. Surprisingly, diminished M1 macrophage activation was also detectable, as marked by significantly reduced Nos2 and Il1b mRNA levels and decreased interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-1β protein levels. The alterations in M1 and M2 macrophage activation were confirmed in bone marrow-derived macrophages from mice with the myeloid-specific Egfr knockout. The combined effect of restrained M1 and M2 macrophage activation resulted in decreased production of pro-angiogenic factors, CXCL1 and vascular endothelial growth factor (VEGF), and reduced CD31+ blood vessels, which likely contributed to protection from tumorigenesis. These data reveal that EGFR signaling in macrophages, but not in colonic epithelial cells, has a significant role in CAC. EGFR signaling in macrophages may prove to be an effective biomarker of CAC or target for chemoprevention in patients with inflammatory bowel disease.

We recently delineated the importance of a type VI secretion system (T6SS) gene cluster in the virulence of diarrheal isolate SSU of Aeromonas hydrophila and showed that VasH, a σ 54 activator and T6SS component, was involved in the production of its associated effectors, e.g., hemolysin-coregulated protein. To identify additional T6SS effectors and/or secreted proteins, we subjected culture supernatants from deletion mutants of A. hydrophila , namely, a Δ act mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a Δ act Δ vasH mutant, to 2-dimensional gel electrophoresis and mass spectrometric analysis. Based on these approaches, we identified a member of the VgrG protein family, VgrG1, that contained a vegetative insecticidal protein (VIP-2) domain at its carboxyl-terminal end. Consequently, the vgrG1 gene was cloned in pBI-EGFP and pET-30a vectors to be expressed in HeLa Tet-Off cells and Escherichia coli , respectively. We assessed the ADP-ribosyltransferase (ADPRT) activity of various domains of purified recombinant VgrG1 (rVgrG1) and provided evidence that only the full-length VgrG1, as well as its carboxyl-terminal domain encoding the VIP-2 domain, showed ADPRT activity. Importantly, bacterium-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells, and we demonstrated translocation of VgrG1. Furthermore, our data indicated that expression of the genes encoding the full-length VgrG1 and its carboxyl-terminal domain in HeLa Tet-Off cells disrupted the actin cytoskeleton, which was followed by a decrease in cell viability and an increase in apoptosis. Taken together, these findings demonstrated for the first time that VgrG1 of A. hydrophila possessed actin ADPRT activity associated with its VIP-2 domain and that this domain alone was able to induce a rounded phenotype in HeLa Tet-Off cells, followed by apoptosis mediated by caspase 9 activation.

Recently, we reported that the type 6 secretion system (T6SS) of Aeromonas hydrophila SSU plays an important role in bacterial virulence in a mouse model, and immunization of animals with the T6SS effector haemolysin co-regulated protein (Hcp) protected them against lethal infections with wild-type bacteria. Additionally, we showed that the mutant bacteria deleted for the vasH gene within the T6SS gene cluster did not express the hcp gene, while the vasK mutant could express and translocate Hcp, but was unable to secrete it into the extracellular milieu. Both of these A. hydrophila SSU mutants were readily phagocytosed by murine macrophages, pointing to the possible role of the secreted form of Hcp in the evasion of the host innate immunity. By using the ΔvasH mutant of A. hydrophila, our in vitro data showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by increased bacterial virulence when rHcp was added along with the ΔvasH mutant in a septicaemic mouse model of infection. Analysis of the cytokine profiling in the intraperitoneal lavage as well as activation of host cells after 4 h of infection with the ΔvasH mutant supplemented with rHcp indicated that this T6SS effector inhibited production of pro-inflammatory cytokines and induced immunosuppressive cytokines, such as interleukin-10 and transforming growth factor-β, which could circumvent macrophage activation and maturation. This mechanism of innate immune evasion by Hcp possibly inhibited the recruitment of cellular immune components, which allowed bacterial multiplication and dissemination in animals, thereby leading to their mortality.

In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout ΔahyRI mutant. Protease production was substantially reduced in the ΔahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the ΔahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the ΔahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the ΔahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the ΔahyRI/ahyR+ complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.

Phospholipase A(2) (PLA(2))-activating protein (PLAA) is a novel signaling molecule that regulates the production of prostaglandins (PGE(2)) and tumor necrosis factor (TNF)-alpha. To characterize the function of native PLAA in situ, we generated HeLa (Tet-off) cells overexpressing plaa (plaa(high)) and control (plaa(low)) cells, with the plaa gene in opposite orientation in the latter construct. The plaa(high) cells produced significantly more PGE(2) and interleukin (IL)-6 compared to plaa(low) cells in response to TNF-alpha. There was an increased activation and/or expression of cytosolic PLA(2), cyclooxgenase-2, and NF-kappaB after induction of plaa(high) cells with TNF-alpha compared to the respective plaa(low) cells. Microarray analysis of plaa(high) cells followed by functional assays revealed increased production of proinflammatory cytokine IL-32 and a decrease in the production of annexin A4 and clusterin compared to plaa(low) cells. We demonstrated the role of annexin A4 as an inhibitor of PLA(2) and showed that addition of exogeneous clusterin limited the production of PGE(2) from plaa(high) cells. To understand regulation of plaa gene expression, we used a luciferase reporter system in HeLa cells and identified one stimulatory element, with Sp1 binding sites, and one inhibitory element, in exon 1 of the plaa gene. By using decoy DNA oligonucleotides to Sp1 and competitive binding assays, we showed that Sp1 maintains basal expression of the plaa gene and binds to the above-mentioned stimulatory element. We demonstrated for the first time that the induction of native PLAA by TNF-alpha can perpetuate inflammation by enhancing activation of PLA(2) and NF-kappaB.

We recently identified a novel type III secretion system (T3SS) effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, and demonstrated that mice infected with the DeltaaexU mutant were significantly protected from mortality. Although the NH(2)-terminal domain of this toxin exhibits homology to AexT of A. salmonicida, a fish pathogen, and ExoT/S of Pseudomonas aeruginosa, the COOH-terminal domain of AexU is unique, with no homology to any known proteins in the NCBI database. In this study, we purified the full-length AexU and its NH(2)-terminal (amino acid residues 1-231) and COOH-terminal (amino acid residues 232-512) domains after expression of their corresponding genes in Escherichia coli as histidine-tag fusion proteins using the bacteriophage T7 RNA polymerase/promoter-based pET-30a vector system. The full-length and NH(2)- and COOH-terminal domains of AexU exhibited ADP-ribosyltransferase activity, with the former two exhibiting much higher activity than the latter. These different forms of AexU were also successfully expressed and produced in the HeLa Tet-Off cell system using a pBI-EGFP vector, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and intracellular staining of the toxin using flow cytometric analysis. Production of AexU in HeLa cells resulted in possible actin reorganization and cell rounding, as determined by phalloidin staining and confocal microscopy. Based on electron microscopy, the toxin also caused chromatin condensation, which is indicative of apoptosis. Apoptosis of HeLa cells expressing and producing AexU was confirmed by 7-amino actinomycin D (7-AAD) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assays, by detection of cytoplasmic histone-associated DNA fragments, and by activation of caspases 3 and 9. These effects were much more pronounced in host cells that expressed and produced the full-length or NH(2)-terminal domain of AexU, compared to those that expressed and produced the COOH-terminal domain or the vector alone. This study represents the first characterization of this novel T3SS effector.

A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.

Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H2O2 production by >50% within 24 hours. Reporter assays indicated that direct interaction of miR-124 with the 3′-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 prior to infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer.

The pathogenesis associated withHelicobacter pyloriinfection is the result of both bacterial factors and the host response. We have previously shown thatH. pyloribinds to CD74 on gastric epithelial cells. In this study, we sought to identify the bacterial protein responsible for this interaction.H. pyloriurease from a pool of bacterial surface proteins was found to coprecipitate with CD74. To determine how urease binds to CD74, we used recombinant urease A and B subunits. Recombinant urease B was found to bind directly to CD74 in immunoprecipitation and flow cytometry studies. By utilizing both recombinant urease subunits and urease B knockout bacteria, the urease B-CD74 interaction was shown to induce NF-κB activation and interleukin-8 (IL-8) production. This response was decreased by blocking CD74 with monoclonal antibodies. Further confirmation of the interaction of urease B with CD74 was obtained using a fibroblast cell line transfected with CD74 that also responded with NF-κB activation and IL-8 production. The binding of theH. pyloriurease B subunit to CD74 expressed on gastric epithelial cells presents a novel insight into a previously unrecognizedH. pyloriinteraction that may contribute to the proinflammatory immune response seen during infection.

CD74 is known as the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) that regulates the cell biology and functions of MHC class II molecules. Class II MHC and Ii expression was believed to be restricted to classical antigen-presenting cells (APC); however, during inflammation, other cell types, including mucosal epithelial cells, have also been reported to express class II MHC molecules. Given the importance of Ii in the biology of class II MHC, we sought to examine the expression of Ii by gastric epithelial cells (GEC) to determine whether class II MHC molecules in these nonconventional APC cells were under the control of Ii and to further support the role that these cells may play in local immune and inflammatory responses during Helicobacter pylori infection. Thus we examined the expression of Ii on GEC from human biopsy samples and then confirmed this observation using independent methods on several GEC lines. The mRNA for Ii was detected by RT-PCR, and the various protein isoforms were also detected. Interestingly, these cells have a high level expression of surface Ii, which is polarized to the apical surface. These studies are the first to demonstrate the constitutive expression of Ii by human GEC.

Helicobacter pylori infects approximately half of the world's population and the bacterium is associated with gastric cancer and peptic and duodenal ulcers. In this study, Surface Enhanced Laser Desorption /Ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to identify the biomarkers from H. pylori infected gastric epithelial cells (GEC) to understand key mechanisms associated with pathogenesis. Using different chip surfaces, differential protein expression profile of GEC was obtained and several upregulated or downregulated biomarkers were detected on GEC, following H. pylori infection. Four different H. pylori infected GECs were compared based on their expression of MHC class II, a receptor reported to trigger apoptosis. One biomarker was identified in H. pylori infected GEC as Annexin A2 (Annexin II) from the flow through of the anion-exchange resin. The increased expression of Annexin II in GEC following H. pylori infection was further confirmed by Western Blot analyses and indicates its involvement in H. pylori pathogenesis.

Helicobacter pylori infection causes gastric cancer, the third leading cause of cancer death worldwide. More than half of the world’s population is infected, making universal eradication impractical. Clinical trials suggest that antibiotic treatment only reduces gastric cancer risk in patients with non-atrophic gastritis (NAG), and is ineffective once preneoplastic lesions of multifocal atrophic gastritis (MAG) and intestinal metaplasia (IM) have occurred. Therefore, additional strategies for risk stratification and chemoprevention of gastric cancer are needed. We have implicated polyamines, generated by the rate-limiting enzyme ornithine decarboxylase (ODC), in gastric carcinogenesis. During H. pylori infection, the enzyme spermine oxidase (SMOX) is induced, which generates hydrogen peroxide from the catabolism of the polyamine spermine. Herein, we assessed the role of SMOX in the increased gastric cancer risk in Colombia associated with the Andean mountain region when compared with the low-risk region on the Pacific coast. When cocultured with gastric epithelial cells, clinical strains of H. pylori from the high-risk region induced more SMOX expression and oxidative DNA damage, and less apoptosis than low-risk strains. These findings were not attributable to differences in the cytotoxin-associated gene A oncoprotein. Gastric tissues from subjects from the high-risk region exhibited greater levels of SMOX and oxidative DNA damage by immunohistochemistry and flow cytometry, and this occurred in NAG, MAG and IM. In Mongolian gerbils, a prototype colonizing strain from the high-risk region induced more SMOX, DNA damage, dysplasia and adenocarcinoma than a colonizing strain from the low-risk region. Treatment of gerbils with either α-difluoromethylornithine, an inhibitor of ODC, or MDL 72527 (N[superscript 1,]N[superscript 4]-Di(buta-2,3-dien-1-yl)butane-1,4-diamine dihydrochloride), an inhibitor of SMOX, reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with DNA damage. These data indicate that aberrant activation of polyamine-driven oxidative stress is a marker of gastric cancer risk and a target for chemoprevention., National Institutes of Health (U.S.) (Grant P01CA028842), Vanderbilt Digestive Disease Center (Grant P30DK058404)

Background & Aims The gastric cancer–causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress–induced apoptosis and DNA damage. A subpopulation of SMOX high cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. Methods SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H pylori –infected Egfr wa5 mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. Results SMOX expression and DNA damage were decreased, and apoptosis increased in H pylori –infected Egfr wa5 mice. H pylori –infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage high apoptosis low cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR–ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage high apoptosis low cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR–ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR–ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. Conclusions In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.