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Outer inflammatory protein of Helicobacter pylori impacts IL-8 expression, adherence, cell apoptosis and cell cycle of gastric cells independent of its copy number

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Abstract

Outer inflammatory protein (OipA) is an important virulence factor of Helicobacter pylori (H. pylori), but the correlation between oipA copy number and its virulence remains unknown. The study was designed to investigate whether the duplicate oipA gene loci showed more virulent than one oipA gene in vitro. H. pylori strain CCS9803 (China Chongqing Strain 9803) that carries duplicate oipA loci was used to construct one or two oipA knockout mutant strain, which was further verified by qPCR and western blot. Gastric epithelial cells AGS and GES-1 were infected with wild-type (WT) or oipA mutants for 6 or 24 h. The expression levels of IL-8, bacterial adhesion, cell apoptosis and cell cycle were performed to analyze the function of oipA. The WT and oipA mutant strains induce significantly higher mRNA and protein levels of IL-8 than the uninfected group (P < 0.05), but only oipA2 mutants induced significantly decreased expression levels than the WT-infected group (P < 0.05). Adherence to gastric cells was significantly decreased by inactivated two oipA loci (P < 0.05). The WT strain caused a significant rising proportion of early apoptosis cell, which had dropped after duplicate oipA genes were both knockout (P < 0.05). WT and oipA1 mutants failed to affect cell cycle; however, the oipA2 mutants increased M phase and reduced S phase when compared to the uninfected group. In conclusion, our study demonstrated that oipA impacts IL-8 expression, adherence, cell apoptosis and cell cycle of gastric cells independent of its gene copy number.

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Acknowledgements

We thank Professor QM Zou (the third military medical university, China) for kindly providing H. pylori CCS9803 WT strain for our experiment. We thank Professor HK BI (Department of pathogenic biology, Nanjing Medical University, China) for kindly gifting us H. pylori G27 for our experiment. We thank Shanghai YH biotechnology co., LTD for cooperation with the oipA gene knockout work. We thank ABclonal Biotechnology co., Ltd. for preparation of anti-recombinant OipA antisera E12446.

Funding

The work was supported by the National Key Research and Development Program of China (No. 2016YFC1302201), National Natural Science Foundation of China (No. 81970502, No. 81860107, No. 81260076) and Leading Talent Training Plan of the Gan-Po Outstanding Talents 555 Project of Jiangxi Province (2010-3-61).

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Authors and Affiliations

  1. Department of Gastroenterology, First Affiliated Hospital of Nanchang University, No.17 Yongwaizheng Street, Donghu District, Nanchang, 330006, Jiangxi, China

    Qiaoyun Zhao, Rulin Zhao, Youhua Wang, Conghua Song, Huan Wang, Jianfang Rong, Fangfei Wang & Yong Xie

  2. Microbiology Laboratory, Gastroenterology Institute of Jiangxi Province, Nanchang, 330006, Jiangxi, China

    Qiaoyun Zhao, Rulin Zhao, Conghua Song, Huan Wang, Jianfang Rong, Fangfei Wang & Yong Xie

  3. Gastroenterology Research Center, Key Laboratory of Digestive Diseases of Jiangxi Province, Nanchang, 330006, Jiangxi, China

    Qiaoyun Zhao, Rulin Zhao, Youhua Wang, Conghua Song, Huan Wang, Jianfang Rong, Fangfei Wang & Yong Xie

  4. Department of Gastroenterology, Affiliated PuRen Hospital of Wuhan University of Science and Technology, Wuhan, 430080, Hubei, China

    Wenzhu Yin

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  1. Qiaoyun Zhao
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Contributions

XY and ZQY designed the research; ZQY wrote the manuscripts; ZQY and ZRL carried out verification experiments of knockout oipA gene and bacterial and cell culture; YWZ and WFF contributed to HCS experiments of cell apoptosis and cell cycle. WH and RJF contributed to bacterial protein extraction and western blot analysis. WYH and SCH contributed to data statistics and paper review. All the authors read and agreed to the final manuscript.

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Correspondence to Yong Xie.

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Zhao, Q., Yin, W., Zhao, R. et al. Outer inflammatory protein of Helicobacter pylori impacts IL-8 expression, adherence, cell apoptosis and cell cycle of gastric cells independent of its copy number. Med Microbiol Immunol 209, 621–630 (2020). https://doi.org/10.1007/s00430-020-00688-w

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