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Characterization and use of Helicobacter pylori SecAN68 ATPase for probing SecA inhibitors as antibacterial agents

Author(s): Han, Qiong, Tiantian Jian, Yan Liu, Han Li, Junda Zhou, Hyuk-kyu Seoh, Sen-Fang Sui, Xu Jia, Phang-Cheng Tai, Xinhe Huang

SecA, the only known energy conversion ATPase in the bacterial protein transport pathway, is a unique protein not found in mammalian cells. H. pylori SecA is closely related to the secretion of the virulence factor vacuolating toxin (VacA) in mediating H. pylori infection and diseases. We previously identified several potential compounds through virtual screening that may inhibit SecA function. In this study for testing inhibitory activity, the HpSecAN68 fragments were constructed and cloned into pET-28a (+) to obtain recombinant plasmid for transforming into E. coli BL21 (DE3). NI-Nitrilotriacetic acid resins were employed to purify the HpSecAN68 protein that has high intrinsic ATPase activity. The recombinant HpSecAN68 protein was successfully expressed, optimized and purified in an active soluble form. The ATPase activity assay system using NADH colorimetry for the purified HpSecAN68 protein was established. The determined Km and Vmax of HpSecAN68 are 2.997±0.697 mM and 0.231±0.038 μmol·mg-1·min-1 respectively in the reaction system. The purified HpSecAN68 ATPase system was used to probe the inhibitory effects of three small molecule inhibitors identified in our study, two of these have IC50 values about 10 μM. In addition, using the micro-broth method, we also observed the significant antibacterial growth effects of these molecules for three Gram-positive and Gram-negative bacteria. This study provides a solid foundation for further research on H. pylori SecA and the development of SecA inhibitors as antimicrobial targets.

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    Editor In Chief

    Masashi Emoto

  • Professor of Laboratory of Immunology
    Department of Laboratory Sciences
    Gunma University Graduate School of Health Sciences
    Gunma, Japan

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