Dynamics of Lewis b Binding and Sequence Variation of the babA Adhesin Gene during Chronic Helicobacter pylori Infection in Humans

ABSTRACTHelicobacter pyloriundergoes rapid microevolution during chronic infection, but very little is known about how this affects host interaction factors. The best-studied adhesin ofH. pyloriis BabA, which mediates binding to the blood group antigen Lewis b [Le(b)]. To study the dynamics of Le(b) adherence during human infection, we analyzed pairedH. pyloriisolates obtained sequentially from chronically infected individuals. A complete loss or significant reduction of Le(b) binding was observed in strains from 5 out of 23 individuals, indicating that the Le(b) binding phenotype is quite stable during chronic human infection. Sequence comparisons ofbabAidentified differences due to mutation and/or recombination in 12 out of 16 strain pairs analyzed. Most amino acid changes were found in the putative N-terminal extracellular adhesion domain. One strain pair that had changed from a Le(b) binding to a nonbinding phenotype was used to study the role of distinct sequence changes in Le(b) binding. By transformations of the nonbinding strain with ababAgene amplified from the binding strain,H. pyloristrains with mosaicbabAgenes were generated. Recombinants were enriched for a gain of Le(b) binding by biopanning or for BabA expression on the bacterial surface by pulldown assay. With this approach, we identified several amino acid residues affecting the strength of Le(b) binding. Additionally, the data showed that the C terminus of BabA, which is predicted to encode an outer membrane β-barrel domain, plays an essential role in the biogenesis of this protein.IMPORTANCEHelicobacter pyloricauses a chronic infection of the human stomach that can lead to ulcers and cancer. The bacterium can bind to gastric epithelial cells with specialized outer membrane proteins. The best-studied protein is the BabA adhesin which binds to the Lewis b blood group antigen. SinceH. pyloriis a bacterium with very high genetic variability, we asked whetherbabAevolves during chronic infection and how mutations or recombination inbabAaffect binding. We found that BabA-mediated adherence was stable in most individuals but observed a complete loss of binding or reduced binding in 22% of individuals. One strain pair in which binding was lost was used to generatebabAsequences that were mosaics of a functional allele and a nonfunctional allele, and the mosaic sequences were used to identify amino acids critically involved in binding of BabA to Lewis b.

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The Helicobacter pylori Autotransporter ImaA (HP0289) Modulates the Immune Response and Contributes to Host Colonization

Infection and Immunity ◽

10.1128/iai.00312-12 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2286-2296 ◽

Cited By ~ 13

Author(s):

William E. Sause ◽

Andrea R. Castillo ◽

Karen M. Ottemann

Keyword(s):

Immune Response ◽

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Dependent Manner ◽

Wild Type ◽

Content Type ◽

H Pylori ◽

Murine Infections

ABSTRACTThe human pathogenHelicobacter pyloriemploys a diverse collection of outer membrane proteins to colonize, persist, and drive disease within the acidic gastric environment. In this study, we sought to elucidate the function of the host-induced geneHP0289, which encodes an uncharacterized outer membrane protein. We first generated an isogenicH. pylorimutant that lacksHP0289and found that the mutant has a colonization defect in single-strain infections and is greatly outcompeted in mouse coinfection experiments with wild-typeH. pylori. Furthermore, we used protease assays and biochemical fractionation coupled with an HP0289-targeted peptide antibody to verify that the HP0289 protein resides in the outer membrane. Our previous findings showed that theHP0289promoter is upregulated in the mouse stomach, and here we demonstrate thatHP0289expression is induced under acidic conditions in an ArsRS-dependent manner. Finally, we have shown that theHP0289mutant induces greater expression of the chemokine interleukin-8 (IL-8) and the cytokine tumor necrosis factor alpha (TNF-α) in gastric carcinoma cells (AGS). Similarly, transcription of the IL-8 homolog keratinocyte-derived chemokine (KC) is elevated in murine infections with the HP0289 mutant than in murine infections with wild-typeH. pylori. On the basis of this phenotype, we renamed HP0289 ImaA forimmunomodulatoryautotransporter protein. Our work has revealed that genes inducedin vivoplay an important role inH. pyloripathogenesis. Specifically, the outer membrane protein ImaA modulates a component of the host inflammatory response, and thus may allowH. pylorito fine tune the host immune response based on ImaA expression.

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Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

Applied and Environmental Microbiology ◽

10.1128/aem.02701-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7351-7359 ◽

Cited By ~ 8

Author(s):

Aleksandra W. Debowski ◽

Phebe Verbrugghe ◽

Miriam Sehnal ◽

Barry James Marshall ◽

Mohammed Benghezal

Keyword(s):

Helicobacter Pylori ◽

Animal Models ◽

Chronic Infection ◽

Fluorescent Protein ◽

Expression System ◽

Content Type ◽

Conditional Expression ◽

H Pylori ◽

Green Fluorescent

ABSTRACTDeletion mutants and animal models have been instrumental in the study ofHelicobacter pyloripathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement duringH. pyloricolonization and chronic infection. To achieve this goal, we adapted theEscherichia coliTn10-derived tetracycline-inducible expression system for use inH. pylori. TheureApromoter was modified by inserting one or twotetoperators to generate tetracycline-responsive promoters, nameduPtetO, and these promoters were then fused to the reportergfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboringtetRanduPtetO-GFPwas characterized by measuring GFP activity and by immunoblotting. The twotet-responsiveuPtetOpromoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of theuPtetO-GFPconstruct and the nature of the promoter driving expression oftetRinfluenced the strength of theuPtetOpromoters upon induction. Integration ofuPtetO-GFPandtetRconstructs at different genomic loci was stablein vivoand did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expressionin vivoduring chronic infection. These results open new experimental avenues for dissectingH. pyloripathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.

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A Conventional Beagle Dog Model for Acute and Chronic Infection with Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.67.6.3112-3120.1999 ◽

1999 ◽

Vol 67 (6) ◽

pp. 3112-3120 ◽

Cited By ~ 35

Author(s):

Giacomo Rossi ◽

Michela Rossi ◽

Claudia G. Vitali ◽

Damiano Fortuna ◽

Daniela Burroni ◽

...

Keyword(s):

Helicobacter Pylori ◽

Malt Lymphoma ◽

Chronic Infection ◽

Acute Infection ◽

Chronic Phase ◽

Human Infection ◽

Peptic Ulcers ◽

Treatment And Prevention ◽

History Of ◽

H Pylori

ABSTRACT Helicobacter pylori has been widely recognized as an important human pathogen responsible for chronic gastritis, peptic ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Little is known about the natural history of this infection since patients are usually recognized as having the infection only after years or decades of chronic disease. Several animal models ofH. pylori infection, including those with different species of rodents, nonhuman primates, and germ-free animals, have been developed. Here we describe a new animal model in which the clinical, pathological, microbiological, and immunological aspects of human acute and chronic infection are mimicked and which allows us to monitor these aspects of infection within the same individuals. Conventional Beagle dogs were infected orally with a mouse-adapted strain of H. pylori and monitored for up to 24 weeks. Acute infection caused vomiting and diarrhea. The acute phase was followed by polymorphonuclear cell infiltration, interleukin 8 induction, mononuclear cell recruitment, and the appearance of a specific antibody response against H. pylori. The chronic phase was characterized by gastritis, epithelial alterations, superficial erosions, and the appearance of the typical macroscopic follicles that in humans are considered possible precursors of MALT lymphoma. In conclusion, infection in this model mimics closely human infection and allows us to study those phases that cannot be studied in humans. This new model can be a unique tool for learning more about the disease and for developing strategies for treatment and prevention.

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Natural Competence Promotes Helicobacter pylori Chronic Infection

Infection and Immunity ◽

10.1128/iai.01042-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 209-215 ◽

Cited By ~ 31

Author(s):

Marion S. Dorer ◽

Ilana E. Cohen ◽

Tate H. Sessler ◽

Jutta Fero ◽

Nina R. Salama

Keyword(s):

Helicobacter Pylori ◽

Chronic Infection ◽

Acute Infection ◽

Host Cells ◽

High Rate ◽

Natural Competence ◽

Content Type ◽

Type Iv ◽

Competence Factor ◽

H Pylori

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with regard to organisms with restricted host ranges, such as the human stomach pathogenHelicobacter pylori. In most cases,H. pyloriinfects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2 months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection, thecagYcomponent of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population ofcagY+andcagYmutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common inH. pyloristrains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was normal. Thus, exchange of DNA between genetically heterogeneousH. pyloristrains may improve chronic colonization. The strains and methods described here will be important tools for defining both the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.

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Oral Immunization with Recombinant Mycobacterium smegmatis Expressing the Outer Membrane Protein 26-Kilodalton Antigen Confers Prophylactic Protection against Helicobacter pylori Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.05306-11 ◽

2011 ◽

Vol 18 (11) ◽

pp. 1957-1961 ◽

Cited By ~ 6

Author(s):

Lin Lü ◽

Han-qing Zeng ◽

Pi-long Wang ◽

Wei Shen ◽

Ting-xiu Xiang ◽

...

Keyword(s):

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Chronic Gastritis ◽

Outer Membrane Protein ◽

Mycobacterium Smegmatis ◽

Interleukin 2 ◽

Oral Immunization ◽

Content Type ◽

H Pylori

ABSTRACTHelicobacter pyloriinfection is prevalent worldwide and results in chronic gastritis, which may lead to gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. We have previously reported that oral immunization with recombinantMycobacterium smegmatisexpressing theH. pyloriouter membrane protein 26-kilodalton (Omp26) antigen affords therapeutic protection againstH. pyloriinfection in mice. In the present study, we investigated the prophylactic effects of this vaccine candidate onH. pylorichallenge in mice. We found that oral immunization with recombinantMycobacteriumOmp26 significantly reducedH. pyloricolonization in the stomach compared to inoculation with wild-typeM. smegmatisin control mice. Six of the recombinantMycobacterium-immunized mice (60%) were completely protected fromH. pyloriinfection. The severity ofH. pylori-associated chronic gastritis assessed histologically was significantly milder in mice vaccinated with recombinantMycobacteriumthan in control animals. Mice immunized with recombinantMycobacteriumshowed enhanced antigen-specific lymphocyte proliferation and antibody responses. Moreover, immunization with recombinantMycobacteriumresulted in an increased expression of interleukin-2 and gamma interferon in the stomach and spleen, as determined by reverse transcription-PCR analysis. Our results collectively suggest that vaccination with recombinantMycobacteriumOmp26 confers prophylactic protection againstH. pyloriinfection. The inhibition ofH. pyloricolonization is associated with the induction of antigen-specific humoral and cell-mediated immune responses.

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Crude Preparations of Helicobacter pylori Outer Membrane Vesicles Induce Upregulation of Heme Oxygenase-1 via Activating Akt-Nrf2 and mTOR–IκB Kinase–NF-κB Pathways in Dendritic Cells

Infection and Immunity ◽

10.1128/iai.00190-16 ◽

2016 ◽

Vol 84 (8) ◽

pp. 2162-2174 ◽

Cited By ~ 17

Author(s):

Su Hyuk Ko ◽

Da Jeong Rho ◽

Jong Ik Jeon ◽

Young-Jeon Kim ◽

Hyun Ae Woo ◽

...

Keyword(s):

Helicobacter Pylori ◽

Dendritic Cells ◽

Outer Membrane ◽

Heme Oxygenase ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Heme Oxygenase 1 ◽

Content Type ◽

Iκb Kinase ◽

H Pylori

Helicobacter pylorisheds outer membrane vesicles (OMVs) that contain many surface elements of bacteria. Dendritic cells (DCs) play a major role in directing the nature of adaptive immune responses againstH. pylori, and heme oxygenase-1 (HO-1) has been implicated in regulating function of DCs. In addition, HO-1 is important for adaptive immunity and the stress response. AlthoughH. pylori-derived OMVs may contribute to the pathogenesis ofH. pyloriinfection, responses of DCs to OMVs have not been elucidated. In the present study, we investigated the role ofH. pylori-derived crude OMVs in modulating the expression of HO-1 in DCs. Exposure of DCs to crudeH. pyloriOMVs upregulated HO-1 expression. Crude OMVs obtained from acagA-negative isogenic mutant strain induced less HO-1 expression than OMVs obtained from a wild-type strain. CrudeH. pyloriOMVs activated signals of transcription factors such as NF-κB, AP-1, and Nrf2. Suppression of NF-κB or Nrf2 resulted in significant attenuation of crude OMV-induced HO-1 expression. Crude OMVs increased the phosphorylation of Akt and downstream target molecules of mammalian target of rapamycin (mTOR), such as S6 kinase 1 (S6K1). Suppression of Akt resulted in inhibition of crude OMV-induced Nrf2-dependent HO-1 expression. Furthermore, suppression of mTOR was associated with inhibition of IκB kinase (IKK), NF-κB, and HO-1 expression in crude OMV-exposed DCs. These results suggest thatH. pylori-derived OMVs regulate HO-1 expression through two different pathways in DCs, Akt-Nrf2 and mTOR–IKK–NF-κB signaling. Following this induction, increased HO-1 expression in DCs may modulate inflammatory responses inH. pyloriinfection.

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In VivoSequence Variation in HopZ, a Phase-Variable Outer Membrane Protein of Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.00977-12 ◽

2012 ◽

Vol 80 (12) ◽

pp. 4364-4373 ◽

Cited By ~ 23

Author(s):

Lynn Kennemann ◽

Birgit Brenneke ◽

Sönke Andres ◽

Lars Engstrand ◽

Thomas F. Meyer ◽

...

Keyword(s):

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Human Migration ◽

Phase Variable ◽

Status Change ◽

Content Type ◽

H Pylori

ABSTRACTTheHelicobacter pyloriouter membrane protein HopZ is regulated by a phase-variable CT repeat and occurs in two distinct allelic variants. Whole-genome comparisons of isolates from one human volunteer recently provided evidence forin vivoselection for thehopZON status. We explored the frequency of sequence variation inhopZduring acute and chronic human infection and studied the association ofhopZwith the phylogeographic population structure ofH. pylori. hopZON variants were cultured from 24 out of 33 volunteers challenged with thehopZOFF strain BCS 100. Transmission ofH. pyloriwithin families was also frequently associated with a status change ofhopZ. In contrast,hopZsequences obtained from 26 sets of sequential isolates from chronically infected individuals showed no changes of status, suggesting that thehopZstatus selected during early infection is subsequently stable. Mutations leading to amino acid changes in HopZ occurred more frequently in ON than in OFF status isolates during chronic infection, indicating that sequence changes are more likely the result of positive selection in ON isolates than of a loss of negative selection pressure in OFF isolates. Analysis of 63 isolates from chronically infected individuals revealed no significant correlation ofhopZstatus with chronic atrophic gastritis.hopZsequences were obtained from a globally representative collection of 54H. pyloristrains. AllH. pyloripopulations containedhopZ-positive isolates. The data suggest thathopZhas been acquired and split into the two variants before the human migration out of Africa.

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Diversification of the AlpB Outer Membrane Protein of Helicobacter pylori Affects Biofilm Formation and Cellular Adhesion

Journal of Bacteriology ◽

10.1128/jb.00729-16 ◽

2016 ◽

Vol 199 (6) ◽

Cited By ~ 9

Author(s):

Hideo Yonezawa ◽

Takako Osaki ◽

Toshiyuki Fukutomi ◽

Tomoko Hanawa ◽

Satoshi Kurata ◽

...

Keyword(s):

Helicobacter Pylori ◽

Outer Membrane ◽

Mutant Strain ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Bacterial Biofilm ◽

Content Type ◽

Successful Infection ◽

H Pylori

ABSTRACT Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pylori. IMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends upon the specific sequence of alpB. This in turn was shown to be important in the ability to adhere to gastric cells. We anticipate that these results will provide new insight into the molecular mechanisms of H. pylori colonization.

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Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

International Journal of Molecular Sciences ◽

10.3390/ijms22094823 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4823

Author(s):

María Fernanda González ◽

Paula Díaz ◽

Alejandra Sandoval-Bórquez ◽

Daniela Herrera ◽

Andrew F. G. Quest

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Outer Membrane ◽

Extracellular Vesicles ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gastric Epithelium ◽

Infected Cells ◽

H Pylori

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.

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Diversity of 3′ variable region of cagA gene in Helicobacter pylori strains isolated from Chinese population

Gut Pathogens ◽

10.1186/s13099-021-00419-3 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Zhijing Xue ◽

Yuanhai You ◽

Lihua He ◽

Yanan Gong ◽

Lu Sun ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Amino Acid ◽

Chinese Population ◽

Statistical Difference ◽

Variable Region ◽

Repeat Region ◽

Geographical Regions ◽

H Pylori ◽

Epiya Motifs

Abstract Background The cytotoxin-associated gene A (cagA) is one of the most important virulence factors of Helicobacter pylori (H. pylori). There is a highly polymorphic Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region in the C-terminal of CagA protein. This repeat region is thought to play an important role in the pathogenesis of gastrointestinal diseases. The aim of this study was to investigate the diversity of cagA 3′ variable region and the amino acid polymorphisms in the EPIYA segments of the CagA C-terminal region of H. pylori, and their association with gastroduodenal diseases. Methods A total of 515 H. pylori strains from patients in 14 different geographical regions of China were collected. The genomic DNA from each strain was extracted and the cagA 3′ variable region was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and analyzed using MEGA 7.0 software. Results A total of 503 (97.7%) H. pylori strains were cagA-positive and 1,587 EPIYA motifs were identified, including 12 types of EPIYA or EPIYA-like sequences. In addition to the four reported major segments, several rare segments (e.g., B′, B″ and D′) were defined and 20 different sequence types (e.g., ABD, ABC) were found in our study. A total of 481 (95.6%) strains carried the East Asian type CagA, and the ABD subtypes were most prevalent (82.1%). Only 22 strains carried the Western type CagA, which included AC, ABC, ABCC and ABCCCC subtypes. The CagA-ABD subtype had statistical difference in different geographical regions (P = 0.006). There were seven amino acid polymorphisms in the sequences surrounding the EPIYA motifs, among which amino acids 893 and 894 had a statistical difference with gastric cancer (P = 0.004). Conclusions In this study, 503 CagA sequences were studied and analyzed in depth. In Chinese population, most H. pylori strains were of the CagA-ABD subtype and its presence was associated with gastroduodenal diseases. Amino acid polymorphisms at residues 893 and 894 flanking the EPIYA motifs had a statistically significant association with gastric cancer.

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