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Sain S, Solanki B, Kumar N. Helicobacter pylori CagA and CagT antibodies arrest the translocation of CagA into gastric epithelial cells. 3 Biotech 2025; 15:179. [PMID: 40406400 PMCID: PMC12092867 DOI: 10.1007/s13205-025-04343-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2024] [Accepted: 05/04/2025] [Indexed: 05/26/2025] Open
Abstract
Cytotoxic-associated gene A (CagA) is a key virulence factor of Helicobacter pylori, associated with gastric ulcers and stomach cancer. The bacterium employs a Cag-type IV secretion system for translocation of CagA into the host cells. This study investigates the impact of CagA antibodies on CagA translocation into gastric epithelial cells in vitro. Our findings reveal that CagA synthesis and translocation across bacterial membranes is a continuous process initiated upon host-cell contact. Notably, the treatment of Helicobacter pylori with CagA-specific antibodies significantly inhibited the translocation of CagA into host cells during infection. These results suggest that the CagA antibody may serve as a potential therapeutic strategy to combat Helicobacter pylori pathogenesis. A similar result was obtained when CagT antibody was used under the same conditions. Notably, the CagT antibody exhibited a more pronounced pathoblocking effect, likely due to its accessibility on the bacterial surface as a structural component of Cag-T4SS. Taken together, this study provides insights into the therapeutic potential of CagA and CagT antibodies to mitigate Helicobacter pylori infection, while also advancing our understanding of the mechanisms involved in CagA translocation across the bacterial membranes to the host cell. Supplementary Information The online version contains supplementary material available at 10.1007/s13205-025-04343-0.
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Affiliation(s)
- Swagata Sain
- School of Biotechnology, Gautam Buddha University, Greater Noida, India
| | - Bhawna Solanki
- School of Biotechnology, Gautam Buddha University, Greater Noida, India
| | - Navin Kumar
- School of Biotechnology, Gautam Buddha University, Greater Noida, India
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Bryant KN, Frick-Cheng AE, Solecki LE, Kroh HK, McDonald WH, Lacy DB, McClain MS, Ohi MD, Cover TL. Species-specific components of the Helicobacter pylori Cag type IV secretion system. Infect Immun 2025; 93:e0049324. [PMID: 40208031 PMCID: PMC12070742 DOI: 10.1128/iai.00493-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2024] [Accepted: 03/08/2025] [Indexed: 04/11/2025] Open
Abstract
Helicobacter pylori strains containing the cag pathogenicity island (PAI) deliver an effector protein (CagA) and non-protein substrates into gastric cells through a process that requires the Cag type IV secretion system (T4SS). The Cag T4SS outer membrane core complex (OMCC) contains multiple copies of five proteins, two of which are species-specific proteins. By using modifications of a previously described OMCC immunopurification method and optimized mass spectrometric methods, we have now isolated additional cag PAI-encoded proteins that are present in lower relative abundance. Four of these proteins (CagW, CagL, CagI, and CagH) do not exhibit sequence relatedness to T4SS components in other bacterial species. Size exclusion chromatography analysis of immunopurified samples revealed that CagW, CagL, CagI, and CagH co-elute with OMCC components. These four Cag proteins are copurified with the OMCC in immunopurifications from a Δcag3 mutant strain (lacking peripheral OMCC components), but not from a ΔcagX mutant strain (defective in OMCC assembly). Negative stain electron microscopy analysis indicated that OMCC preparations isolated from ΔcagW, cagL::kan, ΔcagI, and ΔcagH mutant strains are indistinguishable from wild-type OMCCs. In summary, by using several complementary methods, we have identified multiple species-specific Cag proteins that are associated with the Cag T4SS OMCC and are required for T4SS activity.
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Affiliation(s)
- Kaeli N. Bryant
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | | | - Lauren E. Solecki
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Heather K. Kroh
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - W. Hayes McDonald
- Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
- Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA
| | - D. Borden Lacy
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
| | - Mark S. McClain
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Melanie D. Ohi
- Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA
| | - Timothy L. Cover
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
- Vanderbilt Institute for Infection, Immunology, and Inflammation, Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
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Xue ZJ, Gong YN, He LH, Sun L, You YH, Fan DJ, Zhang MJ, Yan XM, Zhang JZ. Amino acid deletions at positions 893 and 894 of cytotoxin-associated gene A protein affect Helicobacter pylori gastric epithelial cell interactions. World J Gastroenterol 2024; 30:4449-4460. [PMID: 39534413 PMCID: PMC11551673 DOI: 10.3748/wjg.v30.i41.4449] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2024] [Revised: 09/29/2024] [Accepted: 10/12/2024] [Indexed: 10/23/2024] Open
Abstract
BACKGROUND Helicobacter pylori (H. pylori) persistently colonizes the human gastric mucosa in more than 50% of the global population, leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma. Cytotoxin-associated gene A (CagA) protein, an important oncoprotein, has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus, which play crucial roles in pathogenesis. Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer. AIM To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function. METHODS We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894. The cagA gene was amplified and mutated into cagA-NT and cagA-NE (sequence characteristics of strains from nongastric cancer patients), cloned and inserted into pAdtrack-CMV, and then transfected into AGS cells. The expression of cagA and its mutants was examined using real-time polymerase chain reaction and Western blotting, cell elongation via cell counting, F-actin cytoskeleton visualization using fluorescence staining, and interleukin-8 (IL-8) secretion via enzyme-linked immunosorbent assay. RESULTS The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA-NT and pAdtrack/cagA-NE (40.88 ± 3.10 vs 32.50 ± 3.17, P < 0.001 and 40.88 ± 3.10 vs 32.17 ± 3.00, P < 0.001) at 12 hours after transfection. At 24 hours, pAdtrack/cagA-NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA-NT (46.02 ± 2.12 vs 53.90 ± 2.10, P < 0.001 and 46.02 ± 2.12 vs 51.15 ± 3.74, P < 0.001). The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA-NT and pAdtrack/cagA-NE (27.54 ± 17.37 vs 41.51 ± 11.90, P < 0.001 and 27.54 ± 17.37 vs 41.39 ± 14.22, P < 0.001) at 12 hours after transfection. Additionally, pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA-NT and pAdtrack/cagA-NE at different times after transfection. CONCLUSION Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity, which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H. pylori.
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Affiliation(s)
- Zhi-Jing Xue
- Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan 250013, Shandong Province, China
| | - Ya-Nan Gong
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
| | - Li-Hua He
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
| | - Lu Sun
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
| | - Yuan-Hai You
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
| | - Dong-Jie Fan
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
| | - Mao-Jun Zhang
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
| | - Xiao-Mei Yan
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
| | - Jian-Zhong Zhang
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
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Mok CY, Chu HY, Lam WWL, Au SWN. Structural insights into the assembly pathway of the Helicobacter pylori CagT4SS outer membrane core complex. Structure 2024; 32:1725-1736.e4. [PMID: 39032488 DOI: 10.1016/j.str.2024.06.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Revised: 05/16/2024] [Accepted: 06/25/2024] [Indexed: 07/23/2024]
Abstract
Cag type IV secretion system (CagT4SS) translocates oncoprotein cytotoxin-associated gene A (CagA) into host cells and plays a key role in the pathogenesis of Helicobacter pylori. The structure of the outer membrane core complex (OMCC) in CagT4SS consists of CagX, CagY, CagM, CagT, and Cag3 in a stoichiometric ratio of 1:1:2:2:5 with 14-fold symmetry. However, the assembly pathway of OMCC remains elusive. Here, we report the crystal structures of CagT and Cag3-CagT complex, and the structural dynamics of Cag3 and CagT using hydrogen deuterium exchange-mass spectrometry (HDX-MS). The interwoven interaction of Cag3 and CagT involves conformational changes of CagT and β strand swapping. In conjunction with biochemical and biophysical assays, we further demonstrate the different oligomerization states of Cag3 and Cag3-CagT complex. Additionally, the association with CagM requires the pre-formation of Cag3-CagT complex. These results demonstrate the generation of different intermediate sub-assemblies and their structural flexibility, potentially representing different building blocks for OMCC assembly.
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Affiliation(s)
- Chin Yu Mok
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Hoi Yee Chu
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Wendy Wai Ling Lam
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Shannon Wing Ngor Au
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
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Xue Z, Li W, Ding H, Pei F, Zhang J, Gong Y, Fan R, Wang F, Wang Y, Chen Q, Li Y, Yang X, Zheng Y, Su G. Virulence gene polymorphisms in Shandong Helicobacter pylori strains and their relevance to gastric cancer. PLoS One 2024; 19:e0309844. [PMID: 39250512 PMCID: PMC11383249 DOI: 10.1371/journal.pone.0309844] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2024] [Accepted: 08/20/2024] [Indexed: 09/11/2024] Open
Abstract
BACKGROUND Helicobacter pylori (H. pylori) virulence factors, particularly the cagA and vacA genotypes, play important roles in the pathogenic process of gastrointestinal disease. METHODS The cagA and vacA genotypes of 87 H. pylori strains were determined by PCR and sequencing. The EPIYA and CM motif patterns were analyzed and related to clinical outcomes. We examined the associations between the virulence genes of H. pylori and gastrointestinal diseases in Shandong, and the results were analyzed via the chi-square test and logistic regression model. RESULTS Overall, 76 (87.36%) of the strains carried the East Asian-type CagA, with the ABD types being the most prevalent (90.79%). However, no significant differences were observed among the different clinical outcomes. The analysis of CagA sequence types revealed 8 distinct types, encompassing 250 EPIYA motifs, including 4 types of EPIYA or EPIYA-like sequences. Additionally, 28 CM motifs were identified, with the most prevalent patterns being E (66.67%), D (16.09%), and W-W (5.75%). Notably, a significant association was discovered between strains with GC and the CM motif pattern D (P < 0.01). With respect to the vacA genotypes, the strains were identified as s1, s2, m1, m2, i1, i2, d1, d2, c1, and c2 in 87 (100%), 0 (0), 26 (29.89%), 61 (70.11%), 73 (83.91%), 14 (16.09%), 76 (87.36%), 11 (12.64%), 18 (20.69%), and 69 (79.31%), respectively. Specifically, the vacA m1 and c1 genotypes presented a significantly greater prevalence in strains from GC compared to CG (P < 0.05). Following adjustment for age and sex, the vacA c1 genotype demonstrated a notable association with GC (OR = 5.174; 95% CI, 1.402-20.810; P = 0.012). This association was both independent of and more pronounced than the correlations between vacA m1 and GC. CONCLUSIONS CagA proteins possessing CM motif pattern D were more frequently observed in patients with GC (P < 0.01), implying a potentially higher virulence of CM motif pattern D than the other CM motif patterns. Moreover, a strong positive association was identified between the vacA c1 genotype and GC, indicating that the vacA c1 genotype is a robust risk indicator for GC among male patients aged ≥55 years in Shandong.
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Affiliation(s)
- Zhijing Xue
- Department of Gastroenterology, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
- Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Weijia Li
- Department of Gastroenterology, Qilu Hospital of Shandong University, Jinan, Shandong, China
| | - Hailing Ding
- The Faculty of Medicine, Qilu Institute of Technology, Jinan, Shandong, China
| | - Fengyan Pei
- Medical Research & Laboratory Diagnostic Center, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
| | - Jianzhong Zhang
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Yanan Gong
- National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Ruyue Fan
- Shandong Center for Disease Control and Prevention, Jinan, Shandong, China
| | - Fang Wang
- Department of Gastroenterology, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
| | - Youjun Wang
- Department of Gastroenterology, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
| | - Qing Chen
- Department of Gastroenterology, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
| | - Yanran Li
- Department of Gastroenterology, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
| | - Xinyu Yang
- Department of Gastroenterology, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
- Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
| | - Yan Zheng
- Department of Gastroenterology, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
- Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
| | - Guohai Su
- Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
- Department of Cardiovascular Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
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Moosavian M, Kushki E, Navidifar T, Hajiani E, Mandegari M. Is There a Real Relationship between the Presence of Helicobacter pylori in Dental Plaque and Gastric Infection? A Genotyping and Restriction Fragment Length Polymorphism Study on Patient Specimens with Dyspepsia in Southwest Iran. Int J Microbiol 2023; 2023:1212009. [PMID: 38021088 PMCID: PMC10645488 DOI: 10.1155/2023/1212009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2022] [Revised: 07/26/2023] [Accepted: 10/20/2023] [Indexed: 12/01/2023] Open
Abstract
Background The oral cavity can act as an extra gastric reservoir for H. pylori, and the presence of the bacteria in the oral cavity is associated with a higher risk of dental caries development. This study aimed to determine the genotype and evaluate the association between the presence of H. pylori in dental plaque and gastric biopsy specimens in dyspeptic patients in Ahvaz, Southwest Iran. Methods In this study, 106 patients with recruited dyspeptic complaints were selected, and from each patient, two gastric antral biopsy specimens and two dental plagues were examined. The presence of H. pylori was identified by the rapid urease test (RUT) and the amplification of ureAB and 16S rRNA genes. Also, to verify a hypothetical mouth-to-stomach infection route, the enzymatic digestions of three genes of cagA, vacA, and ureAB in H. pylori strains isolated from dental plaques and stomach samples were compared for each same case. Results H. pylori was found in the stomach of 52.8% (56/106) and the dental plaques of 17.9% (19/106) of the studied cases. On the other hand, H. pylori was recognized in the stomach of all 19 cases with oral colonization. Following a combination of restriction fragment lengths 21 polymorphism (RFLP) patterns of these three known genes on stomach and dental plague samples, 14 and 11 unique patterns were seen, respectively. However, for all H. pylori-positive cases (19), the comparison of RLFP patterns of these genes in dental plaque and gastric biopsy specimens was different for the same case. Conclusions In this study, it seems that there is no significant association between the presence of H. pylori in dental plaque and the stomach of the same case.
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Affiliation(s)
- Mojtaba Moosavian
- Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Elyas Kushki
- Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Tahereh Navidifar
- Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Eskandar Hajiani
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mahdi Mandegari
- School of Dentistry, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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Li B, Rong Q, Du Y, Zhang R, Li J, Tong X, Geng L, Zhang Y. Regulation of β1-integrin in autophagy and apoptosis of gastric epithelial cells infected with Helicobacter pylori. World J Microbiol Biotechnol 2021; 38:12. [PMID: 34873651 DOI: 10.1007/s11274-021-03199-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2021] [Accepted: 11/30/2021] [Indexed: 01/22/2023]
Abstract
Helicobacter pylori infection is an essential factor in the development of human gastric diseases, but its pathogenic mechanism is still unclear. In this work we have showed that, the LC3II levels were increased and β1-integrin levels were decreased in H. pylori-positive human gastric tissue samples and H. pylori co-cultured GES-1 cells. There was significant upregulation of LC3II levels and downregulation of P62 levels in GES-1 cells after β1-integrin knockdown co-cultured with H. pylori. This indicated that β1-integrin downregulation promoted autophagy in GES-1 cells after H. pylori infection. The cell apoptosis rate and poly ADP-ribose polymerase (PARP) and caspase-3 activities were increased in GES-1 cells pretreated with 3-methyladenine (3-MA ) after H. pylori infection. Furthermore, there was a significant decrease in apoptosis of β1-integrin knockdown GES-1 cells co-cultured with H. pylori; apoptosis was also downregulated in β1-integrin knockdown- and 3-MA-treated GES-1 cells co-cultured with H. pylori. Correspondingly, PARP and caspase-3 activities were decreased in β1-integrin knockdown cells co-cultured with H. pylori and β1-integrin knockdown-3-MA-treated-1 cells with H. pylori infection. Thus, β1-integrin is a novel autophagy and apoptosis regulator during H. pylori infection. However, inhibition of autophagy did not reverse the decrease in apoptosis caused by downregulation of β1-integrin.
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Affiliation(s)
- Boqing Li
- School of Basic Medical Sciences, Binzhou Medical University, 346# Guanhai Road, Yantai, 264003, China
| | - Qianyu Rong
- Yantai Affiliated Hospital of Binzhou Medical University, Yantai, 264100, China
| | - Yunqiu Du
- School of Basic Medical Sciences, Binzhou Medical University, 346# Guanhai Road, Yantai, 264003, China
| | - Ruiqing Zhang
- School of Basic Medical Sciences, Binzhou Medical University, 346# Guanhai Road, Yantai, 264003, China
| | - Jing Li
- School of Basic Medical Sciences, Binzhou Medical University, 346# Guanhai Road, Yantai, 264003, China
| | - Xiaohan Tong
- School of Basic Medical Sciences, Binzhou Medical University, 346# Guanhai Road, Yantai, 264003, China
| | - Li Geng
- School of Basic Medical Sciences, Binzhou Medical University, 346# Guanhai Road, Yantai, 264003, China
| | - Ying Zhang
- School of Basic Medical Sciences, Binzhou Medical University, 346# Guanhai Road, Yantai, 264003, China.
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Kim HW, Woo HJ, Yang JY, Kim JB, Kim SH. Hesperetin Inhibits Expression of Virulence Factors and Growth of Helicobacter pylori. Int J Mol Sci 2021; 22:ijms221810035. [PMID: 34576198 PMCID: PMC8472136 DOI: 10.3390/ijms221810035] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2021] [Revised: 09/09/2021] [Accepted: 09/13/2021] [Indexed: 12/22/2022] Open
Abstract
Helicobacter pylori (H. pylori) is a bacterium known to infect the human stomach. It can cause various gastrointestinal diseases including gastritis and gastric cancer. Hesperetin is a major flavanone component contained in citrus fruits. It has been reported to possess antibacterial, antioxidant, and anticancer effects. However, the antibacterial mechanism of hesperetin against H. pylori has not been reported yet. Therefore, the objective of this study was to determine the inhibitory effects of hesperetin on H. pylori growth and its inhibitory mechanisms. The results of this study showed that hesperetin inhibits the growth of H. pylori reference strains and clinical isolates. Hesperetin inhibits the expression of genes in replication (dnaE, dnaN, dnaQ, and holB) and transcription (rpoA, rpoB, rpoD, and rpoN) machineries of H. pylori. Hesperetin also inhibits the expression of genes related to H. pylori motility (flhA, flaA, and flgE) and adhesion (sabA, alpA, alpB, hpaA, and hopZ). It also inhibits the expression of urease. Hespereti n downregulates major virulence factors such as cytotoxin-associated antigen A (CagA) and vacuolating cytotoxin A (VacA) and decreases the translocation of CagA and VacA proteins into gastric adenocarcinoma (AGS) cells. These results might be due to decreased expression of the type IV secretion system (T4SS) and type V secretion system (T5SS) involved in translocation of CagA and VacA, respectively. The results of this study indicate that hesperetin has antibacterial effects against H. pylori. Thus, hesperetin might be an effective natural product for the eradication of H. pylori.
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Affiliation(s)
- Hyun Woo Kim
- Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Korea; (H.W.K.); (J.-B.K.)
| | - Hyun Jun Woo
- Department of Clinical Laboratory Science, Semyung University, Jecheon 27136, Korea;
| | - Ji Yeong Yang
- Division of Crop Foundation, National Institute of Crop Science (NICS), Rural Development Administration (RDA), Wanju 55365, Korea;
| | - Jong-Bae Kim
- Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Korea; (H.W.K.); (J.-B.K.)
| | - Sa-Hyun Kim
- Department of Clinical Laboratory Science, Semyung University, Jecheon 27136, Korea;
- Correspondence:
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9
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Evodiamine Inhibits Helicobacter pylori Growth and Helicobacter pylori-Induced Inflammation. Int J Mol Sci 2021; 22:ijms22073385. [PMID: 33806161 PMCID: PMC8036659 DOI: 10.3390/ijms22073385] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Revised: 03/22/2021] [Accepted: 03/23/2021] [Indexed: 12/13/2022] Open
Abstract
Helicobacter pylori (H. pylori) classified as a class I carcinogen by the World Health Organization (WHO) plays an important role in the progression of chronic gastritis and the development of gastric cancer. A major bioactive component of Evodia rutaecarpa, evodiamine, has been known for its anti-bacterial effect and anti-cancer effects. However, the inhibitory effect of evodiamine against H. pylori is not yet known and the inhibitory mechanisms of evodiamine against gastric cancer cells are yet to be elucidated concretely. In this study, therefore, anti-bacterial effect of evodiamine on H. pylori growth and its inhibitory mechanisms as well as anti-inflammatory effects and its mechanisms of evodiamine on H. pylori-induced inflammation were investigated in vitr. Results of this study showed the growth of the H. pylori reference strains and clinical isolates were inhibited by evodiamine. It was considered one of the inhibitory mechanisms that evodiamine downregulated both gene expressions of replication and transcription machineries of H. pylori. Treatment of evodiamine also induced downregulation of urease and diminished translocation of cytotoxin-associated antigen A (CagA) and vacuolating cytotoxin A (VacA) proteins into gastric adenocarcinoma (AGS) cells. This may be resulted from the reduction of CagA and VacA expressions as well as the type IV secretion system (T4SS) components and secretion system subunit protein A (SecA) protein which are involved in translocation of CagA and VacA into host cells, respectively. In particular, evodiamine inhibited the activation of signaling proteins such as the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and the mitogen-activated protein kinase (MAPK) pathway induced by H. pylori infection. It consequently might contribute to reduction of interleukin (IL)-8 production in AGS cells. Collectively, these results suggest anti-bacterial and anti-inflammatory effects of evodiamine against H. pylori.
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Dos Santos Pereira E, Magalhães Albuquerque L, de Queiroz Balbino V, da Silva Junior WJ, Rodriguez Burbano RM, Pordeus Gomes JP, Barem Rabenhorst SH. Helicobacter pylori cagE, cagG, and cagM can be a prognostic marker for intestinal and diffuse gastric cancer. INFECTION GENETICS AND EVOLUTION 2020; 84:104477. [PMID: 32736040 DOI: 10.1016/j.meegid.2020.104477] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Revised: 07/21/2020] [Accepted: 07/23/2020] [Indexed: 01/15/2023]
Abstract
It is known that Helicobacter pylori is the main cause of peptic ulceration and gastric cancer. However, there is a lack of information on whether H. pylori strains may differ in gastric cancer histological subtypes. This study aimed to investigate different H. pylori strains considering six cag Pathogenicity Island - cagPAI genes (cagA, cagE, cagG, cagM, cagT, and virb11), and vacuolating cytotoxin - vacA alleles, and their relation to gastric cancer histologic subtypes. For this purpose, tumor samples from 285 patients with gastric carcinoma were used. H. pylori infection and genotypes were determined by polymerase chain reaction (PCR). H. pylori was detected in 93.9% of gastric tumors. For comparative analyzes between histopathological subtypes considering H. pylori cagPAI genes the strains were grouped according to the vacA s1/s2 alleles. In the vacAs1 group, the strains cagA(-)cagE(+), cagA(+)cagE(+)cagG(+), cagA(+)cagM(+), or only cagE(+) strains were more frequent in the intestinal subtype (P = .009; P = .024; P = .046, respectively). In contrast, cagM(+)cagG(+)cagA(-) and cagE(-) were associated with diffuse tumors (P = .036), highlighting the presence of cagE in the development of intestinal tumors, and the presence of cagG and absence of cagE in diffuse tumors. Furthermore, WEKA software and Decision Tree (CART) analyses confirmed these findings, in which cagE presence was associated with intestinal tumors, and cagE absence and cagG(+) with diffuse tumors. In conclusion our results showed that vacAs1 (cagG + cagM) strains, mainly cagG positive with cagE absence, were relevant in the studied population for the diffuse outcome, while the presence of cagE was relevant for the intestinal outcome. These findings suggest the relevance of these H. pylori genes as potential markers for gastric cancer histological outcomes.
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Affiliation(s)
- Eliane Dos Santos Pereira
- Department of Pathology and Forensic Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil
| | | | - Valdir de Queiroz Balbino
- Department of Genetics, Biomedical Center, Federal University of Pernambuco, Recife, Pernambuco, Brazil
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11
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Genetic variation in the cag pathogenicity island of Helicobacter pylori strains detected from gastroduodenal patients in Thailand. Braz J Microbiol 2020; 51:1093-1101. [PMID: 32410092 DOI: 10.1007/s42770-020-00292-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2020] [Accepted: 04/30/2020] [Indexed: 12/26/2022] Open
Abstract
There is a lack of evidence of genetic variation in the Helicobacter pylori cag-PAI in Thailand, a region with the low incidence of gastric cancer. To clarify this issue, variation in the H. pylori cag-PAI in strains detected in Thailand was characterized and simultaneously compared with strains isolated from a high-risk population in Korea. The presence of ten gene clusters within cag-PAI (cagA, cagE, cagG, cagH, cagL, cagM, cagT, orf13, virB11, and orf10) and IS605 was characterized in H. pylori strains detected from these two countries. The cagA genotypes and EPIYA motifs were analyzed by DNA sequencing. The overall proportion of the ten cag-PAI genes that were detected ranged between 66 and 79%; additionally, approximately 48% of the strains from Thai patients contained an intact cag-PAI structure, while a significantly higher proportion (80%) of the strains from Korean patients had an intact cag-PAI. A significantly higher proportion of IS605 was detected in strains from Thai patients (55%). Analysis of cagA genotypes and EPIYA motifs revealed a higher frequency of Western-type cagA in Thai patients (87%) relative to Korean patients (8%) who were predominately associated with the East Asian-type cagA (92%). Variations in the Western-type cagA in the Thai population, such as EPIYA-BC patterns and EPIYA-like sequences (EPIYT), were mainly detected as compared with the Korean population (p < 0.05). In summary, H. pylori strains that colonize the Thai population tend to be associated with low virulence due to distinctive cag-PAI variation, which may partially explain the Asian paradox phenomenon in Thailand.
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Chehelgerdi M, Doosti A. Effect of the cagW-based gene vaccine on the immunologic properties of BALB/c mouse: an efficient candidate for Helicobacter pylori DNA vaccine. J Nanobiotechnology 2020; 18:63. [PMID: 32316990 PMCID: PMC7175550 DOI: 10.1186/s12951-020-00618-1] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2020] [Accepted: 04/13/2020] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Helicobacter pylori (H. pylori) infect more than half of the world population, and they cause different serious diseases such as gastric carcinomas. This study aims to design a vaccine on the basis of cagW against H. pylori infection. After pcDNA3.1 (+)-cagW-CS-NPs complex is produced, it will be administered into the muscles of healthy BALB/c mice in order to study the effect of this DNA vaccine on the interleukin status of mice, representing its effect on the immune system. After that, the results will be compared with the control groups comprising the administration of cagW-pCDNA3.1 (+) vaccine, the administration of chitosan and the administration of PBS in the muscles of mice. METHODS The cagW gene of H. pylori was amplified by employing PCR, whose product was then cloned into the pcDNA3.1 (+) vector, and this cloning was confirmed by PCR and BamHI/EcoRV restriction enzyme digestion. CagW gene DNA vaccine was encapsulated in chitosan nanoparticles (pcDNA3.1 (+)-cagW-CS-NPs) using a complex coacervation method. The stability and in vitro expression of chitosan nanoparticles were studied by DNase I digestion and transfection, and the immune responses elicited in specific pathogen-free (SPF) mice by the pcDNA3.1 (+)-cagW-CS-NPs were evaluated. Apart from that, the protective potential pcDNA3.1 (+)-cagW-CS-NPs was evaluated by challenging with H. pylori. RESULTS The pcDNA3.1 (+)-cagW-CS-NPs comprises cagW gene of H. pylori that is encapsulated in chitosan nanoparticles, produced with good morphology, high stability, a mean diameter of 117.7 nm, and a zeta potential of + 5.64 mV. Moreover, it was confirmed that chitosan encapsulation protects the DNA plasmid from DNase I digestion, and the immunofluorescence assay showed that the cagW gene could express in HDF cells and maintain good bioactivity at the same time. In comparison to the mice immunized with the control plasmid, in vivo immunization revealed that mice immunized with pcDNA3.1 (+)-cagW-NPs showed better immune responses and prolonged release of the plasmid DNA. CONCLUSIONS This research proves chitosan-DNA nanoparticles as potent immunization candidates against H. pylori infection and paves the way for further developments in novel vaccines encapsulated in chitosan nanoparticles.
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Affiliation(s)
- Mohammad Chehelgerdi
- Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
| | - Abbas Doosti
- Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
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13
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Woo HJ, Yang JY, Lee MH, Kim HW, Kwon HJ, Park M, Kim SK, Park SY, Kim SH, Kim JB. Inhibitory Effects of β-Caryophyllene on Helicobacter pylori Infection In Vitro and In Vivo. Int J Mol Sci 2020; 21:ijms21031008. [PMID: 32028744 PMCID: PMC7037973 DOI: 10.3390/ijms21031008] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Revised: 01/29/2020] [Accepted: 01/30/2020] [Indexed: 01/01/2023] Open
Abstract
The human specific bacterial pathogen Helicobacter pylori (H. pylori) is associated with severe gastric diseases, including gastric cancer. Recently, the increasing resistance makes the usage of antibiotics less effectively. Therefore, development of a new antimicrobial agent is required to control H. pylori infection. In the current study, the inhibitory effect of β-caryophyllene on H. pylori growth, as well as the antibacterial therapeutic effect, has been demonstrated. β-caryophyllene inhibited H. pylori growth via the downregulation of dnaE, dnaN, holB, and gyrA and also downregulated virulence factors such as CagA, VacA, and SecA proteins. β-caryophyllene inhibited expression of several T4SS components, so that CagA translocation into H. pylori-infected AGS gastric cancer cells was decreased by β-caryophyllene treatment. β-caryophyllene also inhibited VacA entry through the downregulation of T5aSS. After β-caryophyllene administration on Mongolian gerbils, the immunohistochemistry (IHC) and Hematoxylin&Eosin stains showed therapeutic effects in the treated groups. Hematological data, which was consistent with histological data, support the therapeutic effect of β-caryophyllene administration. Such a positive effect of β-caryophyllene on H. pylori infection potently substantiates the natural compound as being capable of being used as a new antimicrobial agent or functional health food to help patients who are suffering from gastroduodenal diseases due to H. pylori infection.
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Affiliation(s)
- Hyun Jun Woo
- Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Korea; (H.J.W.); (J.Y.Y.); (H.W.K.); (H.J.K.)
- Department of Clinical Laboratory Science, College of Medical Sciences, Daegu Haany University, Gyeongsan 38610, Korea
| | - Ji Yeong Yang
- Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Korea; (H.J.W.); (J.Y.Y.); (H.W.K.); (H.J.K.)
| | - Min Ho Lee
- Forensic DNA Division, National Forensic Service, Wonju 26460, Korea;
| | - Hyun Woo Kim
- Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Korea; (H.J.W.); (J.Y.Y.); (H.W.K.); (H.J.K.)
| | - Hye Jin Kwon
- Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Korea; (H.J.W.); (J.Y.Y.); (H.W.K.); (H.J.K.)
| | - Min Park
- Department of Biomedical Laboratory Science, Daekyeung University, Gyeongsan 38547, Korea;
| | - Sung-kyu Kim
- SFC BIO Co., Ltd. 1505-1ho, Daerung-town, 25, Gasan digital 1 ro, Geumcheon-gu 08594, Seoul, Korea;
| | - So-Young Park
- College of Pharmacy, Dankook University, 119 Dandae-ro, Cheonan-si, Chungnam 31116, Korea;
| | - Sa-Hyun Kim
- Department of Clinical Laboratory Science, Semyung University, Jaecheon 27136, Korea
- Correspondence: (S.-H.K.); (J.-B.K.)
| | - Jong-Bae Kim
- Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 26493, Korea; (H.J.W.); (J.Y.Y.); (H.W.K.); (H.J.K.)
- Correspondence: (S.-H.K.); (J.-B.K.)
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Khan M, Khan S, Ali A, Akbar H, Sayaf AM, Khan A, Wei DQ. Immunoinformatics approaches to explore Helicobacter Pylori proteome (Virulence Factors) to design B and T cell multi-epitope subunit vaccine. Sci Rep 2019; 9:13321. [PMID: 31527719 PMCID: PMC6746805 DOI: 10.1038/s41598-019-49354-z] [Citation(s) in RCA: 105] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2019] [Accepted: 08/23/2019] [Indexed: 12/30/2022] Open
Abstract
Helicobacter Pylori is a known causal agent of gastric malignancies and peptic ulcers. The extremophile nature of this bacterium is protecting it from designing a potent drug against it. Therefore, the use of computational approaches to design antigenic, stable and safe vaccine against this pathogen could help to control the infections associated with it. Therefore, in this study, we used multiple immunoinformatics approaches along with other computational approaches to design a multi-epitopes subunit vaccine against H. Pylori. A total of 7 CTL and 12 HTL antigenic epitopes based on c-terminal cleavage and MHC binding scores were predicted from the four selected proteins (CagA, OipA, GroEL and cagA). The predicted epitopes were joined by AYY and GPGPG linkers. Β-defensins adjuvant was added to the N-terminus of the vaccine. For validation, immunogenicity, allergenicity and physiochemical analysis were conducted. The designed vaccine is likely antigenic in nature and produced robust and substantial interactions with Toll-like receptors (TLR-2, 4, 5, and 9). The vaccine developed was also subjected to an in silico cloning and immune response prediction model, which verified its efficiency of expression and the immune system provoking response. These analyses indicate that the suggested vaccine may produce particular immune responses against H. pylori, but laboratory validation is needed to verify the safety and immunogenicity status of the suggested vaccine design.
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Affiliation(s)
- Mazhar Khan
- The CAS Key Laboratory of Innate Immunity and Chronic Diseases, Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, CAS Center for Excellence in Molecular Cell Science, University of Science and Technology of China (USTC), Collaborative Innovation Center of Genetics and Development, Hefei, 230027, Anhui, China
| | - Shahzeb Khan
- Centre for Biotechnology and Microbiology, University of Swat, Swat, Khyber Pakhtunkhwa, Pakistan
| | - Asim Ali
- The CAS Key Laboratory of Innate Immunity and Chronic Diseases, Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, CAS Center for Excellence in Molecular Cell Science, University of Science and Technology of China (USTC), Collaborative Innovation Center of Genetics and Development, Hefei, 230027, Anhui, China
| | - Hameed Akbar
- Laboratory of Cellular Dynamics, School of Life Sciences, University of Science and Technology of China (USTC), Anhui Sheng, P.R. China
| | - Abrar Mohammad Sayaf
- Centre for Biotechnology and Microbiology, University of Swat, Swat, Khyber Pakhtunkhwa, Pakistan
| | - Abbas Khan
- Centre for Biotechnology and Microbiology, University of Swat, Swat, Khyber Pakhtunkhwa, Pakistan
- Department of Bioinformatics and Biological Statistics, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, P.R. China
| | - Dong-Qing Wei
- Department of Bioinformatics and Biological Statistics, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, P.R. China.
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15
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Bakhti SZ, Latifi‐Navid S, Zahri S, Yazdanbod A. Inverse association of Helicobacter pylori cagPAI genotypes with risk of cardia and non-cardia gastric adenocarcinoma. Cancer Med 2019; 8:4928-4937. [PMID: 31273955 PMCID: PMC6712521 DOI: 10.1002/cam4.2390] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Revised: 06/19/2019] [Accepted: 06/19/2019] [Indexed: 12/17/2022] Open
Abstract
Iran is a high-risk country for cardia gastric adenocarcinoma (CGA) in Central Asia, with an incidence rate five times the average global rate, and shows a high infection rate for Helicobacter pylori (69%). The aim was to examine the associations of multiple H. pylori cagPAI genotypes (ie cagH, cagL, cagG, and orf17) with the risk of CGA, non-CGA, and different histological types of GA in Iran. A large number of H. pylori strains (N = 336) were successfully cultured and genotyped. Histopathological evaluations were performed. The analysis showed an inverse association between the cagH+ genotype and the risk of CGA and intestinal-type gastric adenocarcinoma (IGA) (adjusted ORs; 0.312 and 0.283, respectively), where the controls were nontumors. The orf17+ genotype decreased the risk of non-CGA and diffuse-type gastric adenocarcinoma (DGA)(adjusted ORs; 0.310 and 0.356, respectively). When the controls were those with nonatrophic gastritis, the cagG+ genotype was negatively associated with the risk of CGA, non-CGA, IGA, and DGA (adjusted ORs; 0.324, 0.366, 0.306, and 0.303, respectively). We did not find such a significant association for the cagL+ genotype in multiple logistic regression analysis. Combination of the vacA c2 and cagPAI genotypes further decreased the risk estimates for GAs. This study showed the reverse association of H. pylori cagPAI genotypes-cagH+ and cagG+ -with the risk of CGA in male patients aged ≥ 55 in Iran. Presence of the vacA c2 genotype in combination with cagPAI genotypes showed strong inverse associations with the risk of CGA and non-CGA. These findings may reveal a coordinated relationship between the vacA c2 and cagPAI genotypes.
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Affiliation(s)
- Seyedeh Zahra Bakhti
- Department of Biology, Faculty of SciencesUniversity of Mohaghegh ArdabiliArdabilIran
| | - Saeid Latifi‐Navid
- Department of Biology, Faculty of SciencesUniversity of Mohaghegh ArdabiliArdabilIran
| | - Saber Zahri
- Department of Biology, Faculty of SciencesUniversity of Mohaghegh ArdabiliArdabilIran
| | - Abbas Yazdanbod
- Digestive Diseases Research CenterArdabil University of Medical SciencesArdabilIran
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16
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Junaid M, Li CD, Shah M, Khan A, Guo H, Wei DQ. Extraction of molecular features for the drug discovery targeting protein-protein interaction of Helicobacter pylori CagA and tumor suppressor protein ASSP2. Proteins 2019; 87:837-849. [PMID: 31134671 DOI: 10.1002/prot.25748] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2019] [Revised: 05/04/2019] [Accepted: 05/22/2019] [Indexed: 12/13/2022]
Abstract
Half of the world population is infected by the Gram-negative bacterium Helicobacter pylori (H. pylori). It colonizes in the stomach and is associated with severe gastric pathologies including gastric cancer and peptic ulceration. The most virulent factor of H. pylori is the cytotoxin-associated gene A (CagA) that is injected into the host cell. CagA interacts with several host proteins and alters their function, thereby causing several diseases. The most well-known target of CagA is the tumor suppressor protein ASPP2. The subdomain I at the N-terminus of CagA interacts with the proline-rich motif of ASPP2. Here, in this study, we carried out alanine scanning mutagenesis and an extensive molecular dynamics simulation summing up to 3.8 μs to find out hot spot residues and discovered some new protein-protein interaction (PPI)-modulating molecules. Our findings are in line with previous biochemical studies and further suggested new residues that are crucial for binding. The alanine scanning showed that mutation of Y207 and T211 residues to alanine decreased the binding affinity. Likewise, dynamics simulation and molecular mechanics with generalized Born surface area (MMGBSA) analysis also showed the importance of these two residues at the interface. A four-feature pharmacophore model was developed based on these two residues, and top 10 molecules were filtered from ZINC, NCI, and ChEMBL databases. The good binding affinity of the CHEMBL17319 and CHEMBL1183979 molecules shows the reliability of our adopted protocol for binding hot spot residues. We believe that our study provides a new insight for using CagA as the therapeutic target for gastric cancer treatment and provides a platform for a future experimental study.
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Affiliation(s)
- Muhammad Junaid
- State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
| | - Cheng-Dong Li
- State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
| | - Masaud Shah
- Department of Molecular Science and Technology, Ajou University, Suwon, South Korea
| | - Abbas Khan
- State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
| | - Haoyue Guo
- Department of Physiology, McGill University, Montreal, Quebec, Canada
| | - Dong-Qing Wei
- State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
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17
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Abstract
Type IV secretion systems (T4SSs) are nanomachines that Gram-negative, Gram-positive bacteria, and some archaea use to transport macromolecules across their membranes into bacterial or eukaryotic host targets or into the extracellular milieu. They are the most versatile secretion systems, being able to deliver both proteins and nucleoprotein complexes into targeted cells. By mediating conjugation and/or competence, T4SSs play important roles in determining bacterial genome plasticity and diversity; they also play a pivotal role in the spread of antibiotic resistance within bacterial populations. T4SSs are also used by human pathogens such as Legionella pneumophila, Bordetella pertussis, Brucella sp., or Helicobacter pylori to sustain infection. Since they are essential virulence factors for these important pathogens, T4SSs might represent attractive targets for vaccines and therapeutics. The best-characterized conjugative T4SSs of Gram-negative bacteria are composed of twelve components that are conserved across many T4SSs. In this chapter, we will review our current structural knowledge on the T4SSs by describing the structures of the individual components and how they assemble into large macromolecular assemblies. With the combined efforts of X-ray crystallography, nuclear magnetic resonance (NMR), and more recently electron microscopy, structural biology of the T4SS has made spectacular progress during the past fifteen years and has unraveled the properties of unique proteins and complexes that assemble dynamically in a highly sophisticated manner.
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18
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Ying L, Ferrero RL. Role of NOD1 and ALPK1/TIFA Signalling in Innate Immunity Against Helicobacter pylori Infection. Curr Top Microbiol Immunol 2019; 421:159-177. [PMID: 31123889 DOI: 10.1007/978-3-030-15138-6_7] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The human pathogen Helicobacter pylori interacts intimately with gastric epithelial cells to induce inflammatory responses that are a hallmark of the infection. This inflammation is a critical precursor to the development of peptic ulcer disease and gastric cancer. A major driver of this inflammation is a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI), present in a subpopulation of more virulent H. pylori strains. The cagPAI T4SS specifically activates signalling pathways in gastric epithelial cells that converge on the transcription factor, nuclear factor-κB (NF-κB), which in turn upregulates key immune and inflammatory genes, resulting in various host responses. It is now clear that H. pylori possesses several mechanisms to activate NF-κB in gastric epithelial cells and, moreover, that multiple signalling pathways are involved in these responses. Two of the dominant signalling pathways implicated in NF-κB-dependent responses in epithelial cells are nucleotide-binding oligomerisation domain 1 (NOD1) and a newly described pathway involving alpha-kinase 1 (ALPK1) and tumour necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA). Although the relative roles of these two pathways in regulating NF-κB-dependent responses still need to be clearly defined, it is likely that they work cooperatively and non-redundantly. This chapter will give an overview of the various mechanisms and pathways involved in H. pylori induction of NF-κB-dependent responses in gastric epithelial cells, including a 'state-of-the-art' review on the respective roles of NOD1 and ALPK1/TIFA pathways in these responses.
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Affiliation(s)
- Le Ying
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia
| | - Richard L Ferrero
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Monash University, Clayton, VIC, Australia.
- Department of Molecular and Translational Medicine, Monash University, Clayton, VIC, Australia.
- Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
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19
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Junaid M, Shah M, Khan A, Li CD, Khan MT, Kaushik AC, Ali A, Mehmood A, Nangraj AS, Choi S, Wei DQ. Structural-dynamic insights into the H. pylori cytotoxin-associated gene A (CagA) and its abrogation to interact with the tumor suppressor protein ASPP2 using decoy peptides. J Biomol Struct Dyn 2018; 37:4035-4050. [PMID: 30328798 DOI: 10.1080/07391102.2018.1537895] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Helicobacter pylori (H. pylori) is one of the most extensively studied Gram-negative bacteria due to its implication in gastric cancer. The oncogenicity of H. pylori is associated with cytotoxin-associated gene A (CagA), which is injected into epithelial cells lining the stomach. Both the C- and N-termini of CagA are involved in the interaction with several host proteins, thereby disrupting vital cellular functions, such as cell adhesion, cell cycle, intracellular signal transduction, and cytoskeletal structure. The N-terminus of CagA interacts with the tumor-suppressing protein, apoptosis-stimulating protein of p53 (ASPP2), subsequently disrupting the apoptotic function of tumor suppressor gene p53. Here, we present the in-depth molecular dynamic mechanism of the CagA-ASPP2 interaction and highlight hot-spot residues through in silico mutagenesis. Our findings are in agreement with previous studies and further suggest other residues that are crucial for the CagA-ASPP2 interaction. Furthermore, the ASPP2-binding pocket possesses potential druggability and could be engaged by decoy peptides, identified through a machine-learning system and suggested in this study. The binding affinities of these peptides with CagA were monitored through extensive computational procedures and reported herein. While CagA is crucial for the oncogenicity of H. pylori, our designed peptides possess the potential to inhibit CagA and restore the tumor suppressor function of ASPP2.
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Affiliation(s)
- Muhammad Junaid
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
| | - Masaud Shah
- b Department of Molecular Science and Technology, Ajou University , Suwon , South Korea
| | - Abbas Khan
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
| | - Cheng-Dong Li
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
| | - Muhammad Tahir Khan
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
| | - Aman Chandra Kaushik
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
| | - Arif Ali
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
| | - Aamir Mehmood
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
| | - Asma Sindhoo Nangraj
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
| | - Sangdun Choi
- b Department of Molecular Science and Technology, Ajou University , Suwon , South Korea
| | - Dong-Qing Wei
- a State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai , China
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