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钩端螺旋体OmpR家族相关TCS鉴定及功能初探OA北大核心CSTPCD

Identification and functional exploration of OmpR family TCS in Leptospira

中文摘要英文摘要

目的 建立钩端螺旋体(钩体)OmpR家族双组分系统(TCS)的鉴定方法,初步探讨OmpR家族TCS在钩体感染人巨噬细胞过程中对钩体主要外膜蛋白(OMPs)表达水平的调控作用.方法 采用生物信息学技术预测致病性钩体赖株TCS的组氨酸激酶(HK)和应答调节蛋白(RR)OmpR功能结构域.采用激酶活性检测试剂盒和细菌双杂交技术进一步验证各HK激酶活性以及各HK与RR的相互作用.采用qRT-PCR法检测HK抗血清封闭前后钩体主要OMPs基因mRNA水平的变化.结果 钩体56601株中LA2828、LA1710含有HATPase_c激酶催化结构域,LA2827、LA1709含有Response_reg与OmpR功能结构域,提示其分别可构成HK/OmpR-TCS.体外重组HK-2828和HK-1710蛋白均具有激酶活性,且分别与OmpR-2827和OmpR-1709存在直接的相互作用.HK-2828抗体封闭处理可使感染过程中显著下降的外膜蛋白编码基因li-pL32、lipL41和ompL1的mRNA水平明显回升.结论 成功构建钩体OmpR家族-TCS鉴定及功能探究体系,为阐明感染过程中问号钩体OMPs表达改变调控机制,以及制定基因工程疫苗OMPs抗原筛选新策略提供依据.

This study was aimed at establishing a method for identifying the OmpR family two-component system(TCS)in Leptospira interrogans,and to explore the effects of these proteins in regulating changes in the expression of leptospiral major outer membrane protein(OMP)during infection of human macrophages.Bioinformatics methods were used to analyze the do-mains of HK/OmpR-TCS proteins in Leptospira.A kinase activity detection kit and bacterial two-hybrid analysis were used to detect HK kinase activity,and the interactions between HKs and RRs,respectively.Real-time fluorescence quantitative RT-PCR(qRT-PCR)was used to detect changes in the expression of leptospiral OMP genes during infection of macrophages with or without HK-antibody block assay.LA1710 and LA2828 contained the HATPase_c domain,and LA1709 and LA2827 con-tained the Response reg and OmpR functional domain.The recombinant HK-2828 and HK-1710 proteins showed kinase activity and direct interaction with OmpR-2827 and OmpR-1709,respectively.The treatment with HK-2828 antibody partly reversed the infection-based down-regulated mRNA levels of lipL32,lipL41 and ompL1 genes(P<0.05).The system for identifica-tion and functional investigation of OmpR family TCS was successfully constructed.This study provides a basis for elucidating the regulatory mechanisms of OMP expression changes in leptospires during infection,and developing a new OMP antigen screening strategy for genetically engineered vaccines.

戴欣珏;夏文浩;任雨轩;车名花;胡玮琳

江苏省宜兴市中医医院,宜兴 214200||浙江省生物标志物与体外诊断转化重点实验室,杭州 310053浙江大学医学院微生物学系,杭州 310058江苏省宜兴市中医医院,宜兴 214200江苏省宜兴市中医医院,宜兴 214200||浙江大学医学院微生物学系,杭州 310058

基础医学

钩端螺旋体双组分系统外膜蛋白

Leptospiratwo component systemouter membrane proteins

《中国人兽共患病学报》 2024 (003)

197-202 / 6

国家自然科学基金(No.81971951),浙江省生物标志物与体外诊断转化重点实验室开放基金(No.KFJJ2023002) Supported by the National Natural Science Foundation of Chi-na(No.81971951)and the Opening Fund of Key Laboratory of Biomarkers and In Vitro Diagnosis Translation of Zhejiang Province(No.KFJJ2023002)

10.3969/j.issn.1002-2694.2024.00.041

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