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The duodenal ulcer promoting gene (dupA), located in the plasticity region of Helicobacter pylori (H. pylori), is predicted to form a type IV secretory system (T4SS) with vir genes around dupA. In the study, we investigated the association between the dupA cluster status and the virulence of H. pylori in a littoral region of Northeast China. Two hundred and sixty-two H. pylori strains isolated from the chronic gastritis were examined to evaluate the dupA cluster status, cag PAI genes and vacA genotype using PCR and Western blot. Histopathologic evaluations of biopsy specimens were performed to analysis the association between the dupA cluster and the inflammatory response. IL-8 productions in gastric mucosa and from GES-1 cells co-cultured with H. pylori were measured, respectively, to analysis the association between the dupA cluster status and IL-8 production. We found that gastric mucosal inflammatory cell infiltration was significantly higher in patients with dupA-positive H. pylori, including H. pylori with complete dupA cluster (2.71 ± 0.79) and incomplete dupA cluster (2.09 ± 0.61) than in patients with dupA-negative strain (1.73 ± 0.60, p < 0.01), whereas no significant difference in the gastric mucosal atrophy was found according to the status of dupA cluster. Gastric mucosal IL-8 levels were higher in the complete dupA cluster group than in other groups (p < 0.01), and IL-8 production from GES-1 cells was also significantly higher in strains with a complete dupA cluster (1527.9 ± 180.0 pg/ml) than in those with an incomplete dupA cluster (1229.4 ± 75.3 pg/ml, p < 0.01) or those with dupA negative (1201.9 ± 92.3 pg/ml, p < 0.01). In conclusion, the complete dupA cluster in H. pylori is associated with inflammatory cell infiltration and IL-8 secretion, and H. pylori strain with a complete dupA cluster seems to be more virulent than other strains with the incomplete dupA cluster or dupA negative.

The function of intact long-type DupA protein in Helicobacter pylori was analyzed using immunoblotting and molecular biology techniques in the study. After cloning, expression and purification, ATPase activity of DupA protein was detected. Antibody was produced for localization and interaction proteins analysis. The dupA-deleted mutant was generated for adhesion and CagA protein translocation assay, susceptibility to different pH, IL-8 secretion assay, cytotoxicity to MKN-45 cells and proteins-involved apoptosis analysis. DupA protein exhibited an ATPase activity (129.5±17.8 U/mgprot) and located in bacterial membrane, while it did not involve the adhesion and CagA protein delivery of H. pylori. DupA protein involved the urease secretion as the interaction proteins. The wild type strain had a stronger growth in low pH than the dupA-deleted mutant (p < 0.001). IL-8 productions from GES-1 cells infected with the wild type strain were significantly higher than from those with the mutant (p < 0.001). The amounts of vital MKN-45 cells were decreased and the numbers of apoptotic cells were increased with the wild type strain, compared to those with the mutant after 12 h (p < 0.05). The increase of cleaved Caspase-3 and Bax was significantly higher and the decrease of Bcl-2 was more obvious in MKN-45 cells exposed to the wild type strain than that exposed to the mutant after 6 h. We demonstrate that intact long-type DupA protein located in membrane as ATPase is a true virulence factor associated with duodenal ulcer development involving the IL-8 induction and urease secretion, while it inhibits gastric cancer cell growth in vitro by activating the mitochondria-mediated apoptotic pathway., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Abstract Background: Helicobacter pylori (H. pylori) is a major human pathogen that is responsible for various gastroduodenal diseases. We investigated the prevalence of H. pylori virulence markers in a region at high risk of gastric cancer., Methods: One hundred and sixteen H. pylori strains were isolated from patients with gastroduodenal diseases. cagA, the cagA 3' variable region, cagPAI genes, vacA, and dupA genotypes were determined by PCR, and some amplicons of the cagA 3' variable region, cagPAI genes and dupA were sequenced., Results: cagA was detected in all strains. The cagA 3' variable region of 85 strains (73.3%) was amplified, and the sequences of 24 strains were obtained including 22 strains possessing the East Asian-type. The partial cagPAI presented at a higher frequency in chronic gastritis (44.4%) than that of the severe clinical outcomes (9.7%, p < 0.001). The most prevalent vacA genotypes were s1a/m2 (48.3%) and s1c/m2 (13.8%). Thirty-six strains (31.0%) possessed dupA and sequencing of dupA revealed an ORF of 2449-bp. The prevalence of dupA was significantly higher in strains from patients with the severe clinical outcomes (40.3%) than that from chronic gastritis (20.4%, p = 0.02)., Conclusion: The high rate of East Asian-type cagA, intact cagPAI, virulent vacA genotypes, and the intact long-type dupA may underlie the high risk of gastric cancer in the region., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: To review the research progress on Type IV secretion system (T4SS) in Helicobacter pylori., Data Sources: The data used in this review were identified by searching of PUBMED (1995 - 2007) online resources using the key terms 'Type IV secretion system' and 'Helicobacter pylori'., Study Selection: Mainly original articles and critical reviews written by major pioneer investigators of this field were selected., Results: The research progress on T4SS in Helicobacter pylori was summarized. The structure and function was discussed., Conclusions: T4SS is not only involved in toxin secretion and injection of virulence factors into eukaryotic host target cells, but also involved in horizontal DNA transfer to other bacteria and eukaryotic cells, through DNA uptake from or release into the extracellular milieu. It provides a new insight into the pathogenicity of Helicobacter pylori and a novel target for antimicrobials development. However, many challenges remain for us in understanding the biological role of T4SS in Helicobacter pylori.

Helicobacter pylori is a human-specific gastric pathogen that colonizes over half the world's population. Infection with this bacterium is associated with a spectrum of gastric pathologies ranging from mild gastritis to peptic ulcers and gastric cancer. A strong predictor of severe disease outcome is infection with a bacterial strain harbouring the cag (cytotoxin associated gene) pathogenicity island (PAI), a 40 kb stretch of DNA that encodes homologues of several components of a type IV secretion system (TFSS). One gene within the cag PAI, cagA, has been shown to encode a substrate for the TFSS which is translocated into host cells, inducing the dephosphrylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. It appeears to play a key role in H. pylori pathogenesis. Very little is known about the H. pylori cag PAI-encoded TFSS, the expression of Cag proteins in H. pylori, and the functions of individual proteins encoded by the cag PAI. Only by exploring the mechanistic details of the interplay between H. pylori and eukaryotic cells can we endeavour to understand how these cellular interactions play out at the tissue and organismal level during the lifelong coexistence of bacterium and host.

Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density, the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.

Aim: To construct ltB-ureB fusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein., Methods: The ureB gene from a clinical Helicobacter pylori (H pylori) strain Y06 and the ltB gene from Escherichia coli (E. coli) strain 44851 were linked into ltB-ureB fusion gene by PCR. The fusion gene sequence was analyzed after T-A cloning. A prokaryotic recombinant expression vector pET32a inserted with ltB-ureB fusion gene (pET32a-ltB-ureB) was constructed. Expression of the recombinant LTB-UreB protein (rLTB-UreB) in E. coli BL21DE3 induced by isopropylthio-beta-D-galactoside (IPTG) at different concentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of H pylori and a self-prepared rabbit anti-rUreB serum, respectively, and determine the antigenicity of the recombinant protein on inducing specific antibody in rabbits. GM1-ELISA was used to demonstrate the adjuvanticity of rLTB-UreB. Immunoreaction of rLTB-UreB to the UreB antibody positive sera from 125 gastric patients was determined by using ELISA., Results: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ltB-ureB fusion gene were 100%. IPTG with different dosages of 0.1-1.0 mmol/L could efficiently induce pET32a-ltB-ureB-E.coli BL21DE3 to express the rLTB-UreB. The output of the target recombinant protein expressed by pET32a-ureB-E.coli BL21DE3 was approximately 35% of the total bacterial proteins. rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori and anti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB binding bovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from the patients (125/125) were positive for rLTB-UreB., Conclusion: A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established. The expressed rLTB-UreB showed qualified immunogenicity, antigenicity and adjuvanticity. All the results mentioned above laid a firm foundation for further development of H pylori genetically engineered vaccine.

Aim: To construct a prokaryotic expression system of a Helicobacter pylori (H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA(+) H pylori) isolates cause diseases., Methods: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed, and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively. Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients' sera, and the correlations between infection with CagA(+) H pylori and gastritis as well as peptic ulcer were analyzed., Results: Of all the clinical specimens obtained, 80.8% (126/156) were found to have H pylori isolates and 97.2% of the isolates (106/109) were positive for cagA gene. In comparison with the reported data, the cloned cagA1 fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences, respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein. rCagA1 was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109) expressed CagA and 88.1% of the patients' serum samples (96/109) were CagA antibody-positive. The percentage of CagA(+) H pylori strains (97.9%) isolated from the biopsy specimens of peptic ulcer appeared to be higher than that from gastritis (88.5%), but the difference was not statistically significant (chi (2)=3.48, P>0.05)., Conclusion: rCagA1 produced by the prokaryotic expression system constructed in this study possesses good immunoreactivity and antigenicity, and the established ELISAs can be used to detect CagA of H pylori and its antibody. H pylori isolates show high frequencies of cagA gene and CagA expression, but the infections by CagA(+) H pylori strains are not the most decisive factors to cause gastric diseases.