Gastric Niche Research Articles

Helicobacter pylori chronic infection and mucosal inflammation switches the human gastric glycosylation pathways

Helicobacter pylori exploits host glycoconjugates to colonize the gastric niche. Infection can persist for decades promoting chronic inflammation, and in a subset of individuals lesions can silently progress to cancer. This study shows that H. pylori chronic infection and gastric tissue inflammation result in a remodeling of the gastric glycophenotype with increased expression of sialyl-Lewis a/x antigens due to transcriptional up-regulation of the B3GNT5, B3GALT5, and FUT3 genes. We observed that H. pylori infected individuals present a marked gastric local pro-inflammatory signature with significantly higher TNF-α levels and demonstrated that TNF-induced activation of the NF-kappaB pathway results in B3GNT5 transcriptional up-regulation. Furthermore, we show that this gastric glycosylation shift, characterized by increased sialylation patterns, favors SabA-mediated H. pylori attachment to human inflamed gastric mucosa. This study provides novel clinically relevant insights into the regulatory mechanisms underlying H. pylori modulation of host glycosylation machinery, and phenotypic alterations crucial for life-long infection. Moreover, the biosynthetic pathways here identified as responsible for gastric mucosa increased sialylation, in response to H. pylori infection, can be exploited as drug targets for hindering bacteria adhesion and counteract the infection chronicity.

The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient anti-recombination function of Helicobacter pylori MutS2.

Helicobacter pylori, a human pathogen, is a naturally and constitutively competent bacteria, displaying a high rate of intergenomic recombination. While recombination events are essential for evolution and adaptation of H. pylori to dynamic gastric niches and new hosts, such events should be regulated tightly to maintain genomic integrity. Here, we analyze the role of the nuclease activity of MutS2, a protein that limits recombination during transformation in H. pylori. In previously studied MutS2 proteins, the C-terminal Smr domain was mapped as the region responsible for its nuclease activity. We report here that deletion of Smr domain does not completely abolish the nuclease activity of HpMutS2. Using bioinformatics analysis and mutagenesis, we identified an additional and novel nuclease motif (LDLK) at the N-terminus of HpMutS2 unique to Helicobacter and related ε-proteobacterial species. A single point mutation (D30A) in the LDLK motif and the deletion of Smr domain resulted in ∼ 5-10-fold loss of DNA cleavage ability of HpMutS2. Interestingly, the mutant forms of HpMutS2 wherein the LDLK motif was mutated or the Smr domain was deleted were unable to complement the hyper-recombination phenotype of a mutS2(-) strain, suggesting that both nuclease sites are indispensable for an efficient anti-recombinase activity of HpMutS2.

Modification of Helicobacter pylori Peptidoglycan Enhances NOD1 Activation and Promotes Cancer of the Stomach.

Helicobacter pylori (H. pylori) is the strongest known risk factor for gastric carcinogenesis. One cancer-linked locus is the cag pathogenicity island, which translocates components of peptidoglycan into host cells. NOD1 is an intracellular immune receptor that senses peptidoglycan from Gram-negative bacteria and responds by inducing autophagy and activating NF-κB, leading to inflammation-mediated bacterial clearance; however chronic pathogens can evade NOD1-mediated clearance by altering peptidoglycan structure. We previously demonstrated that the H. pylori cag(+) strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. Using 2D-DIGE and mass spectrometry, we identified a novel mutation within the gene encoding the peptidoglycan deacetylase PgdA; therefore, we sought to define the role of H. pylori PgdA in NOD1-dependent activation of NF-κB, inflammation, and cancer. Coculture of H. pylori strain 7.13 or its pgdA(-) isogenic mutant with AGS gastric epithelial cells or HEK293 epithelial cells expressing a NF-κB reporter revealed that pgdA inactivation significantly decreased NOD1-dependent NF-κB activation and autophagy. Infection of Mongolian gerbils with an H. pylori pgdA(-) mutant strain led to significantly decreased levels of inflammation and malignant lesions in the stomach; however, preactivation of NOD1 before bacterial challenge reciprocally suppressed inflammation and cancer in response to wild-type H. pylori. Expression of NOD1 differs in human gastric cancer specimens compared with noncancer samples harvested from the same patients. These results indicate that peptidoglycan deacetylation plays an important role in modulating host inflammatory responses to H. pylori, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche.

697 Role of an Acid-Sensing Histidine Kinase in H. pylori Pathogenesis

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p<0.001) .We next infected new populations of gerbils with parental 7.13 and the two polar 7.13 Hp derivatives for up to 16 weeks. There were no differences in colonization among the groups, although all animals that developed adenocarcinoma harbored decreased bacterial burdens. Strain 7.131 induced premalignant lesions in 25%, 50%, 62.5%, and 75% at 4, 8, 12, and 16 weeks respectively. In contrast, the incidence of dysplasia and adenocarcinoma was only 25% and 12.5% with the 7.13 parental strain or the 7.13-2 derivative 16 weeks post-infection. In vivo-readapted strains from animals infected with parental 7.13 and 7.13-1 maintained their ability to induce high levels of NFκβ activation similar to the input strains, except for strains isolated from animals that developed cancer, which were attenuated in the ability to activate NF-κβ and translocate CagA. In contrast, readapted isolates from animals infected with strain 7.13-2 induced increased levels of NFκβ activation and CagA translocation in vitro, which was concordant with increased levels of inflammation in the corresponding gerbils. To define mechanisms that mediate these phenotypes, we examined cagY rearrangements by RFLP. We identified two distinct patterns, one specific for parental 7.13 and 7.131 and the other for strain 7.13-2. Readaptation revealed that a proportion of parental 7.13 (25%) and 7.13-1 (37.5%) isolate derivatives altered their cagY pattern, developing a 7.132-type pattern in the presence of cancer which was accompanied by decreases in NFκβ activation and CagA translocation. In contrast, 62.5% of Hp readapted isolates from animals infected with strain 7.13-2 changed their cagY pattern to one that mirrored the original 7.13 parental strain and those changes were linked to increases in inflammation, NFκβactivation and CagA translocation. Collectively, these results suggest Hp can modify function of the T4SS by alternating cagY genotype in response to the neoplastic landscape within gastric mucosa.

695 H. pylori cagY Rearrangements Modify Function of the cag Type 4 Secretion System in Response to Oncogenic Changes in the Gastric Microenvironment

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p<0.001) .We next infected new populations of gerbils with parental 7.13 and the two polar 7.13 Hp derivatives for up to 16 weeks. There were no differences in colonization among the groups, although all animals that developed adenocarcinoma harbored decreased bacterial burdens. Strain 7.131 induced premalignant lesions in 25%, 50%, 62.5%, and 75% at 4, 8, 12, and 16 weeks respectively. In contrast, the incidence of dysplasia and adenocarcinoma was only 25% and 12.5% with the 7.13 parental strain or the 7.13-2 derivative 16 weeks post-infection. In vivo-readapted strains from animals infected with parental 7.13 and 7.13-1 maintained their ability to induce high levels of NFκβ activation similar to the input strains, except for strains isolated from animals that developed cancer, which were attenuated in the ability to activate NF-κβ and translocate CagA. In contrast, readapted isolates from animals infected with strain 7.13-2 induced increased levels of NFκβ activation and CagA translocation in vitro, which was concordant with increased levels of inflammation in the corresponding gerbils. To define mechanisms that mediate these phenotypes, we examined cagY rearrangements by RFLP. We identified two distinct patterns, one specific for parental 7.13 and 7.131 and the other for strain 7.13-2. Readaptation revealed that a proportion of parental 7.13 (25%) and 7.13-1 (37.5%) isolate derivatives altered their cagY pattern, developing a 7.132-type pattern in the presence of cancer which was accompanied by decreases in NFκβ activation and CagA translocation. In contrast, 62.5% of Hp readapted isolates from animals infected with strain 7.13-2 changed their cagY pattern to one that mirrored the original 7.13 parental strain and those changes were linked to increases in inflammation, NFκβactivation and CagA translocation. Collectively, these results suggest Hp can modify function of the T4SS by alternating cagY genotype in response to the neoplastic landscape within gastric mucosa.

696 Whole-Genome Sequencing Detects Novel Virulence Variants of Helicobacter pylori cagA Gene and Its Relationship With Oncogenic cagA

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p<0.001) .We next infected new populations of gerbils with parental 7.13 and the two polar 7.13 Hp derivatives for up to 16 weeks. There were no differences in colonization among the groups, although all animals that developed adenocarcinoma harbored decreased bacterial burdens. Strain 7.131 induced premalignant lesions in 25%, 50%, 62.5%, and 75% at 4, 8, 12, and 16 weeks respectively. In contrast, the incidence of dysplasia and adenocarcinoma was only 25% and 12.5% with the 7.13 parental strain or the 7.13-2 derivative 16 weeks post-infection. In vivo-readapted strains from animals infected with parental 7.13 and 7.13-1 maintained their ability to induce high levels of NFκβ activation similar to the input strains, except for strains isolated from animals that developed cancer, which were attenuated in the ability to activate NF-κβ and translocate CagA. In contrast, readapted isolates from animals infected with strain 7.13-2 induced increased levels of NFκβ activation and CagA translocation in vitro, which was concordant with increased levels of inflammation in the corresponding gerbils. To define mechanisms that mediate these phenotypes, we examined cagY rearrangements by RFLP. We identified two distinct patterns, one specific for parental 7.13 and 7.131 and the other for strain 7.13-2. Readaptation revealed that a proportion of parental 7.13 (25%) and 7.13-1 (37.5%) isolate derivatives altered their cagY pattern, developing a 7.132-type pattern in the presence of cancer which was accompanied by decreases in NFκβ activation and CagA translocation. In contrast, 62.5% of Hp readapted isolates from animals infected with strain 7.13-2 changed their cagY pattern to one that mirrored the original 7.13 parental strain and those changes were linked to increases in inflammation, NFκβactivation and CagA translocation. Collectively, these results suggest Hp can modify function of the T4SS by alternating cagY genotype in response to the neoplastic landscape within gastric mucosa.

Helicobacter pylori: Genomic Insight into the Host-Pathogen Interaction.

The advent of genomic analyses has revolutionized the study of human health. Infectious disease research in particular has experienced an explosion of bacterial genomic, transcriptomic, and proteomic data complementing the phenotypic methods employed in traditional bacteriology. Together, these techniques have revealed novel virulence determinants in numerous pathogens and have provided information for potential chemotherapeutics. The bacterial pathogen, Helicobacter pylori, has been recognized as a class 1 carcinogen and contributes to chronic inflammation within the gastric niche. Genomic analyses have uncovered remarkable coevolution between the human host and H. pylori. Perturbation of this coevolution results in dysregulation of the host-pathogen interaction, leading to oncogenic effects. This review discusses the relationship of H. pylori with the human host and environment and the contribution of each of these factors to disease progression, with an emphasis on features that have been illuminated by genomic tools.

Purification and Characterization of Pseudomonas aeruginosa PAO1 Asparaginase

The prevalence of bacterial infection has been gradually increased for the last decades. This phenomenon is predicted as one of serious problems to human security in the next decades. Helicobactorpylorihas been clarified to be related to gastric inflammation and a variety of diseases including gastric cancer. Recently, it has been known that H. pylori asparaginase is necessary for the growth in host infection and might be involved in inhibition of lymphocyte function at gastric niche. On the other hand, Pseudomonas aeruginosa is known to cause a fence-sitting infectious disease. However, no studies on a physiological role of asparaginase in relation to pathogenicity of P. aeruginosahave been found. Also, there are no studies on its physiological role in relation to pathogenicity of P. aeruginosa have been found. Therefore, we focused on purification and characterization of P. aeruginosaPAO1 asparaginase in order to clarify its physiological roles in this study. Intracellular asparaginase was highly purified and characterized from P. aeruginosa PAO1. The profile of DEAE-cellufine A 500 column chromatography suggested that two types of asparaginases were produced in P. aeruginosaPAO1. The optimum pH and temperature of the one enzyme was 9.5 and 40°C, respectively while those of the another was 9.5 and 65°C, respectively. Those results indicated that P. aeruginosa PAO1 had two different types of asparaginases.

High resolution electron microscopy of the Helicobacter pylori Cag type IV secretion system pili produced in varying conditions of iron availability.

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the "pilus". Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface(. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

Genome sequencing analysis reveals virulence-related gene content of Ochrobactrum intermedium strain 229E, a urease-positive strain isolated from the human gastric niche.

We report draft genome sequence of Ochrobactrum intermedium strain 229E concurrent with Helicobacter pylori in urease positive gastric biopsy of non-ulcer dyspeptic individual from Southern part of India. Since the role of Ochrobactrum in human gastric environment is poorly understood, comprehensive pathological, microbiological, and genome level understanding are necessary to evaluate its association with H. pylori in the gastric niche. Comparative analysis of O. intermedium 299E strain revealed functional similarities with virulence related gene clusters present in H. pylori genomes, which probably might aid in its ability to persist in the human gastric mucosa. However, H.pylori specific vacuolating cytotoxin (vacA) involved in vacuolization, cytotoxicity, and T-cell inhibition was absent in the O. intermedium 229E genome. Taken together, O. intermedium 229E shared numerous features like secretion system, urease, and flagella with H.pylori genome sequence that might aid concurrence in the gastric niche.

Immunity, inflammation, and vaccines for Helicobacter pylori.

Helicobacter pylori colonizes mucosa, activates Toll-like and Nod-like receptors, and usually elicits a gastric T-helper 1/17 (Th1/Th17) type of immune response. Among several bacterial factors, the secreted peptidyl prolyl cis, trans-isomerase of H.pylori represents a key factor driving Th17 inflammation. A complex and fascinating balance between H.pylori and host factors takes part in the gastric niche and is responsible for the chronicity of the infection. Novel insights into the innate and adaptive responses against H.pylori, dealing with gastric epithelial cells, cytokines, and immune evasion have been elucidated over the past year and are discussed for the development of an effective vaccine.

Helicobacter pylori targets cancer-associated apical-junctional constituents in gastroids and gastric epithelial cells

ObjectiveHelicobacter pylori strains that express the oncoprotein CagA augment risk for gastric cancer. However, the precise mechanisms through which cag+ strains heighten cancer risk have not been fully delineated and...

Helicobacter pylori: expect the unexpected

Helicobacter pylori is one of the most common bacterial infections worldwide, and virtually all infected persons develop coexisting gastritis, a signature feature of which is the capacity to persist for decades. In support of its lifestyle, H. pylori has evolved to express an array of diverse phenotypes, including enzyme functional diversity, that help to subvert obstacles presented by the human host, which permits long-term microbial colonization. The versatility of the newly discovered enzyme LpxJ may allow H. pylori to quickly adapt to dynamic and hostile conditions present within its cognate gastric niche.

Helicobacter pylori: bacterial strategy for incipient stage and persistent colonization in human gastric niches.

Helicobacter pylori (H. pylori) undergoes decades long colonization of the gastric mucosa of half the population in the world to produce acute and chronic gastritis at the beginning of infection, progressing to more severe disorders, including peptic ulcer disease and gastric cancer. Prolonged carriage of H. pylori is the most crucial factor for the pathogenesis of gastric maladies. Bacterial persistence in the gastric mucosa depends on bacterial factors as well as host factors. Herein, the host and bacterial components responsible for the incipient stages of H. pylori infection are reviewed and discussed. Bacterial adhesion and adaptation is presented to explain the persistence of H. pylori colonization in the gastric mucosa, in which bacterial evasion of host defense systems and genomic diversity are included.

Special issue

Microbial species participate in the genesis of a substantial number of malignancies worldwide, and conservative estimates indicate that > 15% of all cancer cases are attributable to infectious agents. Helicobacter pylori is the most common bacterial infection worldwide, and colonization typically lasts for decades in the absence of targeted antimicrobial therapy. One biological cost to this long-term relationship is a marked increased risk for gastric adenocarcinoma, the second leading cause of cancer-related death in the world, prompting the World Health Organization to designate H. pylori as a class I carcinogen. Approximately 660,000 new cases of gastric cancer per year are attributable to H. pylori, making this pathogen the most common infectious agent linked to malignancy. Eradication of H. pylori significantly decreases the risk of developing gastric adenocarcinoma in infected individuals; however, only a percentage of colonized persons ever develop neoplasia. Disease risk involves well-choreographed interactions between pathogen and host, which are dependent upon strain-specific bacterial factors, host genotypic traits and/or environmental conditions. H. pylori strains are extremely diverse, freely recombining as panmictic populations. However, the use of broad-range 16S rRNA PCR coupled with high throughput sequencing has demonstrated that H. pylori does not simply exist as a monoculture within the human stomach, but instead is a resident of a distinct gastric microbial ecosystem. While H. pylori is the dominant species, the presence of other microorganisms provides a genetic repository which may facilitate the generation of novel traits that influence gastric carcinogenesis. Host polymorphisms within genes that regulate immunity and oncogenesis also heighten the risk for gastric cancer in conjunction with H. pylori strain-specific constituents. Further, environmental conditions such as iron deficiency and high salt intake can influence H. pylori phenotypes that lower the threshold for disease. Universal test and treat strategies for H. pylori are not feasible due to the high prevalence of infection as well as the expense and side effects of antibiotic therapy, and eradication rates are often less than 80% in regions of the world where gastric cancer is most prevalent. These observations, in conjunction with evidence that carriage of certain strains is inversely related to esophageal adenocarcinoma, atopic diseases and potentially inflammatory bowel disease, underscore the importance of identifying mechanisms that regulate interactions of H. pylori with its host, which promote carcinogenesis. Delineation of bacterial, host and environmental mediators that augment gastric cancer risk has profound ramifications for both physicians and biomedical researchers, as such findings will not only focus prevention approaches that target H. pylori-infected human populations at increased risk for stomach cancer, but will also provide mechanistic insights into inflammatory carcinomas that develop beyond the gastric niche. The articles contained in this special issue of Gut Microbes have been authored by world-class investigators and provide fresh insights into specific mediators of H. pylori-induced disease and, importantly, how such mediators may influence strategies for the prevention, diagnosis and treatment of oncogenic consequences stemming from infection. These are critical issues to address since many useful resources for analysis now exist including genome sequences (H. pylori and non-H. pylori constituents of the gastric microbiome; rodent and human), measurable phenotypes (CagA phosphorylation) and innovative humanized animal models. Such resources provide a unique opportunity to address key unanswered questions regarding mechanisms through which the host response to particular H. pylori strains, which may be modified by environmental factors, drives carcinogenesis.

Cloning, purification and preliminary crystallographic analysis of the complex of Helicobacter pylori α-carbonic anhydrase with acetazolamide.

Helicobacter pylori infection of the stomach can lead to severe gastroduodenal diseases such as gastritis, peptic ulcers and gastric cancers. Periplasmic H. pylori α-carbonic anhydrase (HpαCA) is essential for the acclimatization of the bacterium to the acidity of the stomach. Through the action of urease and carbonic anhydrases, the H. pylori periplasmic pH is maintained at around 6 in an environment with a pH as low as 2, which in turn facilitates the maintenance of a cytoplasmic pH close to neutral, allowing growth in the gastric niche. Crystals of HpαCA in complex with the inhibitor acetazolamide have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals have the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a = 37.0, b = 82.4, c = 150.8 Å. An X-ray diffraction data set was collected from a single crystal to 1.7 Å resolution. Calculation of the self-rotation function using this data and molecular replacement showed that the asymmetric unit contains an HpαCA dimer.

The impact of Helicobacter pylori infection on the gastric microbiota of the rhesus macaque.

Helicobacter pylori colonization is highly prevalent among humans and causes significant gastric disease in a subset of those infected. When present, this bacterium dominates the gastric microbiota of humans and induces antimicrobial responses in the host. Since the microbial context of H. pylori colonization influences the disease outcome in a mouse model, we sought to assess the impact of H. pylori challenge upon the pre-existing gastric microbial community members in the rhesus macaque model. Deep sequencing of the bacterial 16S rRNA gene identified a community profile of 221 phylotypes that was distinct from that of the rhesus macaque distal gut and mouth, although there were taxa in common. High proportions of both H. pylori and H. suis were observed in the post-challenge libraries, but at a given time, only one Helicobacter species was dominant. However, the relative abundance of non-Helicobacter taxa was not significantly different before and after challenge with H. pylori. These results suggest that while different gastric species may show competitive exclusion in the gastric niche, the rhesus gastric microbial community is largely stable despite immune and physiological changes due to H. pylori infection.

Helicobacter pyloridownregulates expression of human β-defensin 1 in the gastric mucosa in a type IV secretion-dependent fashion

Helicobacter pylori establishes a chronic lifelong infection in the human gastric mucosa, which may lead to peptic ulcer disease or gastric adenocarcinoma. The human beta-defensins (hβDs) are antimicrobial peptides, hβD1 being constitutively expressed in the human stomach. We hypothesized that H. pylori may persist, in part, by downregulating gastric hβD1 expression. We measured hβD1 and hβD2 expression in vivo in relation to the presence, density and severity of H. pylori infection, investigated differential effects of H. pylori virulence factors, and studied underlying signalling mechanisms in vitro. Significantly lower hβD1 and higher hβD2 mRNA and protein concentrations were present in gastric biopsies from infected patients. Those patients with higher-level bacterial colonization and inflammation had significantly lower hβD1 expression, but there were no differences in hβD2. H. pylori infection of human gastric epithelial cell lines also downregulated hβD1. Using wild-type strains and isogenic mutants, we showed that a functionalcag pathogenicity island-encoded type IV secretion system induced this downregulation. Treatment with chemical inhibitors or siRNA revealed that H. pylori usurped NF-κB signalling to modulate hβD1 expression. These data indicate that H. pylori downregulates hβD1 expression via NF-κB signalling, and suggest that this may promote bacterial survival and persistence in the gastric niche.

Helicobacter pylori Salvages Purines from Extracellular Host Cell DNA Utilizing the Outer Membrane-Associated Nuclease NucT

Helicobacter pylori is a bacterial pathogen that establishes life-long infections in humans, and its presence in the gastric epithelium is strongly associated with gastritis, peptic ulcer disease, and gastric cancer. Having evolved in this specific gastric niche for hundreds of thousands of years, this microbe has become dependent on its human host. Bioinformatic analysis reveals that H. pylori has lost several genes involved in the de novo synthesis of purine nucleotides, and without this pathway present, H. pylori must salvage purines from its environment in order to grow. While the presence and abundance of free purines in various mammalian tissues has been loosely quantified, the concentration of purines present within the gastric mucosa remains unknown. There is evidence, however, that a significant amount of extracellular DNA is present in the human gastric mucosal layer as a result of epithelial cell turnover, and this DNA has the potential to serve as an adequate purine source for gastric purine auxotrophs. In this study, we characterize the ability of H. pylori to grow utilizing only DNA as a purine source. We show that this ability is independent of the ComB DNA uptake system, and that H. pylori utilization of DNA as a purine source is largely influenced by the presence of an outer membrane-associated nuclease (NucT). A ΔnucT mutant exhibits significantly reduced extracellular nuclease activity and is deficient in growth when DNA is provided as the sole purine source in laboratory growth media. These growth defects are also evident when this nuclease mutant is grown in the presence of AGS cells or in purine-free tissue culture medium that has been conditioned by AGS cells in the absence of fetal bovine serum. Taken together, these results indicate that the salvage of purines from exogenous host cell DNA plays an important role in allowing H. pylori to meet its purine requirements for growth.

Helicobacter pyloriouter membrane protein HopQ identified as a novel T4SS-associated virulence factor

Helicobacter pylori is a bacterial pathogen that colonizes the gastric niche of ∼ 50% of the human population worldwide and is known to cause peptic ulceration and gastric cancer. Pathology of infection strongly depends on a cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). Here, we aimed to identify as yet unknown bacterial factors involved in cagPAI effector function and performed a large-scale screen of an H. pylori transposon mutant library using activation of the pro-inflammatory transcription factor NF-κB in human gastric epithelial cells as a measure of T4SS function. Analysis of ∼ 3000 H. pylori mutants revealed three non-cagPAI genes that affected NF-κB nuclear translocation. Of these, the outer membrane protein HopQ from H. pylori strain P12 was essential for CagA translocation and for CagA-mediated host cell responses such as formation of the hummingbird phenotype and cell scattering. Besides that, deletion of hopQ reduced T4SS-dependent activation of NF-κB, induction of MAPK signalling and secretion of interleukin 8 (IL-8) in the host cells, but did not affect motility or the quantity of bacteria attached to host cells. Hence, we identified HopQ as a non-cagPAI-encoded cofactor of T4SS function.