Objective Secreted proteins of Helicobacter pylori （H. pylori） interact with gastric epithelium cells and may contribute to cell damage. Considering the fact that HPO17S and hypothetical conserved protein HP1286 are...Objective Secreted proteins of Helicobacter pylori （H. pylori） interact with gastric epithelium cells and may contribute to cell damage. Considering the fact that HPO17S and hypothetical conserved protein HP1286 are included in the group and that HP0175 is a well-known apoptosis-induced factor, the present study aims to clarify whether HP1286 plays a role in bacterial pathogenicity or even functions as an apoptosis-induced factor in human stomach. Methods Two genes encoding HP1286 and HPO175 were cloned into pET32a vector and expressed as recombinant proteins in Escherichia coil （E. coil） BL21. Signal peptide sequences were not included. The recombinant proteins were purified with His SpinTrap and desalted by using HiTrap Desalting. Immunoreactivity of the proteins was determined by Western blot. Human gastric epithelial cell AGS was challenged with these endotoxin-free proteins; and apoptosis of cells was assayed with the Cell Death ELISA kit. Results Recombinant proteins and their respective products whose N-terminal his-tag were removed with thrombin were recognized by serum from the patient infected with H. pylori. Apoptotic AGS cells challenged by HP1286 of H. pylori 26695 were four times more than untreated cells. In addition, apoptosis-induced ability of HP1286 of SS1 was not as strong as that of H. pylori 26695 strain. Conclusion HP1286 of H. pylori 26695 induces apoptosis of AGS cells in a time-dependent manner, however the apoptosis-induced ability of HP1286 may differ due to variations between different strains.展开更多
BACKGROUND The high prevalence and persistence of Helicobacter pylori(H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are vari...BACKGROUND The high prevalence and persistence of Helicobacter pylori(H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor.AIM To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric(AGS) cells.METHODS Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0,3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via, I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling.RESULTS HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells.Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells.Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori. Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found;modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage.CONCLUSION The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.展开更多
Helicobacter pylori (HP) infection is a global problem that affects about half of the world’s population and requires sufficient attention in clinical and scientific work. Due to differences in economic and medical c...Helicobacter pylori (HP) infection is a global problem that affects about half of the world’s population and requires sufficient attention in clinical and scientific work. Due to differences in economic and medical conditions among countries around the world, there is currently no unified treatment plan for anti-HP. In China, empirical quadruple therapy is mainly used. With the abuse of antibiotics, many patients face the problem of secondary eradication after failure, and the resistance rate of HP is gradually increasing. After eradication failure, drug sensitivity cultivation is carried out to choose sensitive antibiotics for treatment. A new strategy is currently needed to address how to improve the eradication rate of HP during the first eradication. This article aims to discuss the first-line treatment plans and research progress for eradicating HP based on drug sensitivity testing before eradication. Compared with traditional empirical therapies, treatment based on drug sensitivity results can effectively improve the eradication rate of HP, and reduce drug resistance rates, and adverse reactions, among other benefits. .展开更多
基金supported by the Major Technology Project as part of "Prevention and Control of Major Infectious Diseases including AIDS and Viral Hepatitis" (No. 2008ZX10004-002)
文摘Objective Secreted proteins of Helicobacter pylori （H. pylori） interact with gastric epithelium cells and may contribute to cell damage. Considering the fact that HPO17S and hypothetical conserved protein HP1286 are included in the group and that HP0175 is a well-known apoptosis-induced factor, the present study aims to clarify whether HP1286 plays a role in bacterial pathogenicity or even functions as an apoptosis-induced factor in human stomach. Methods Two genes encoding HP1286 and HPO175 were cloned into pET32a vector and expressed as recombinant proteins in Escherichia coil （E. coil） BL21. Signal peptide sequences were not included. The recombinant proteins were purified with His SpinTrap and desalted by using HiTrap Desalting. Immunoreactivity of the proteins was determined by Western blot. Human gastric epithelial cell AGS was challenged with these endotoxin-free proteins; and apoptosis of cells was assayed with the Cell Death ELISA kit. Results Recombinant proteins and their respective products whose N-terminal his-tag were removed with thrombin were recognized by serum from the patient infected with H. pylori. Apoptotic AGS cells challenged by HP1286 of H. pylori 26695 were four times more than untreated cells. In addition, apoptosis-induced ability of HP1286 of SS1 was not as strong as that of H. pylori 26695 strain. Conclusion HP1286 of H. pylori 26695 induces apoptosis of AGS cells in a time-dependent manner, however the apoptosis-induced ability of HP1286 may differ due to variations between different strains.
基金Supported by the Federal Funds,HIM/2009/037.SSA851 and HIM/2014/012.SSA 1098the grant from Secretaría de Investigación y Posgrado,SIP 20161878the Instituto Politécnico Nacional by Consejo Nacional de Ciencia y Tecnología,CB-222180.
文摘BACKGROUND The high prevalence and persistence of Helicobacter pylori(H. pylori) infection, as well as the diversity of pathologies related to it, suggest that the virulence factors used by this microorganism are varied. Moreover, as its proteome contains 340hypothetical proteins, it is important to investigate them to completely understand the mechanisms of its virulence and survival. We have previously reported that the hypothetical protein HP0953 is overexpressed during the first hours of adhesion to inert surfaces, under stress conditions, suggesting its role in the environmental survival of this bacterium and perhaps as a virulence factor.AIM To investigate the expression and localization of HP0953 during adhesion to an inert surface and against gastric(AGS) cells.METHODS Expression analysis was performed for HP0953 during H. pylori adhesion. HP0953 expression at 0,3, 12, 24, and 48 h was evaluated and compared using the Kruskal-Wallis equality-of-populations rank test. Recombinant protein was produced and used to obtain polyclonal antibodies for immunolocalization. Immunogold technique was performed on bacterial sections during adherence to inert surfaces and AGS cells, which was analyzed by transmission electron microscopy. HP0953 protein sequence was analyzed to predict the presence of a signal peptide and transmembrane helices, both provided by the ExPASy platform, and using the GLYCOPP platform for glycosylation sites. Different programs, via, I-TASSER, RaptorX, and HHalign-Kbest, were used to perform three-dimensional modeling.RESULTS HP0953 exhibited its maximum expression at 12 h of infection in gastric epithelium cells.Immunogold technique revealed HP0953 localization in the cytoplasm and accumulation in some peripheral areas of the bacterial body, with greater expression when it is close to AGS cells.Bioinformatics analysis revealed the presence of a signal peptide that interacts with the transmembrane region and then allows the release of the protein to the external environment. The programs also showed a similarity with the Tip-alpha protein of H. pylori. Tip-alpha is an exotoxin that penetrates cells and induces tumor necrosis factor alpha production, and HP0953 could have a similar function as posttranslational modification sites were found;modifications in turn require enzymes located in eukaryotic cells. Thus, to be functional, HP0953 may necessarily need to be translocated inside the cell where it can trigger different mechanisms producing cellular damage.CONCLUSION The location of HP0953 around infected cells, the probable posttranslational modifications, and its similarity to an exotoxin suggest that this protein is a virulence factor.
文摘Helicobacter pylori (HP) infection is a global problem that affects about half of the world’s population and requires sufficient attention in clinical and scientific work. Due to differences in economic and medical conditions among countries around the world, there is currently no unified treatment plan for anti-HP. In China, empirical quadruple therapy is mainly used. With the abuse of antibiotics, many patients face the problem of secondary eradication after failure, and the resistance rate of HP is gradually increasing. After eradication failure, drug sensitivity cultivation is carried out to choose sensitive antibiotics for treatment. A new strategy is currently needed to address how to improve the eradication rate of HP during the first eradication. This article aims to discuss the first-line treatment plans and research progress for eradicating HP based on drug sensitivity testing before eradication. Compared with traditional empirical therapies, treatment based on drug sensitivity results can effectively improve the eradication rate of HP, and reduce drug resistance rates, and adverse reactions, among other benefits. .
