LsrK体外磷酸化AI-2抑制肺炎克雷伯菌碳青霉烯酶耐药基因水平转移机制研究

Carbapenem resistant Klebsiella pneumoniae is one of the extensively drug resistant bacteria, which is difficult to treat, and has the highest mortality rate. Our preliminary experimental results show that quorum sensing has effect on plasmid conjugation (Preliminary experimental results); Combined with previous studies: at present, only AI-2 signal system has found in Klebsiella pneumoniae, LsrK is an activated phosphorylase for AI-2, Activated AI-2 has changed on polarity and unable to enter the bacteria, Thus we proposed that whether AI-2 participated in horizontal transfer of carbapenem resistance gene? whether phosphorylated AI-2 in vitro can inhibit the horizontal transfer of carbapenem resistance gene (scientific problems)? Accordingly we propose the hypothesis: AI-2 was phosphorylated by LsrK in vitro, and unable to transfer through ABC system, Intracellular, LsrR operon transcription was inhibited under the action of repressor protein LsrR, regulatory effects of AI-2 on resistance gene horizontal transfer was affected (scientific hypothesis). AI-2 report strain real-time monitoring, homologous mutation, transcriptome and differential proteomics analysis was proposed in this study, to clarify regulation mechanism of AI-2 on horizontal transfer of carbapenemases, which activated by LsrK in vitro(Innovation), to provide scientific basis for inhibiting the spread of extensively drug resistant bacteria.

碳青霉烯类耐药肺炎克雷伯菌是目前临床治疗最困难，致死率最高的一类广泛耐药菌。本课题组前期研究发现群体感应对质粒接合有影响（预实验结果）；结合已有研究：目前发现肺炎克雷伯菌只存在AI-2信号系统，LsrK是AI-2的磷酸化酶，活化的AI-2极性发生改变无法进入细菌内，由此我们提出AI-2是否参与了碳青霉烯酶耐药基因水平转移？体外磷酸化AI-2是否能抑制碳青霉烯酶耐药基因水平转移（科学问题）？据此我们提出假设：体外AI-2在LsrK的作用下被磷酸化，极性发生改变，无法通过ABC转运系统，菌体内lsr操纵子在阻遏蛋白LsrR作用下转录处于抑制状态，AI-2对耐药基因水平转移的调控受到影响（科学假说）。本项目拟采用AI-2报告菌株实时监测，同源突变，转录组及差异蛋白组分析等方法，阐明LsrK体外磷酸化AI-2对碳青霉烯酶耐药基因水平转移的调控机制（创新点），为抑制泛耐药基因水平传播提供科学依据。

碳青霉烯类耐药肺炎克雷伯菌是目前临床治疗最困难，致死率最高的一类广泛耐药菌。本课题组前期研究发现群体感应对质粒接合有影响（预实验结果）；结合已有研究：目前发现肺炎克雷伯菌只存在AI-2信号系统，LsrK是AI-2的磷酸化酶，活化的AI-2极性发生改变无法进入细菌内，由此我们提出AI-2是否参与了碳青霉烯酶耐药基因水平转移？体外磷酸化AI-2是否能抑制碳青霉烯酶耐药基因水平转移（科学问题）？据此我们提出假设：体外AI-2在LsrK的作用下被磷酸化，极性发生改变，无法通过ABC转运系统，菌体内lsr操纵子在阻遏蛋白LsrR作用下转录处于抑制状态，AI-2对耐药基因水平转移的调控受到影响（科学假说）。本项目通过体外合成AI-2类群体感应信号分子，并重组LsrK蛋白对其进行体外磷酸化，干扰AI-2进入细菌内启动lsr操纵子。实时监测干扰组、突变组细菌接合实验，并评价其接合效率。发现亚抑菌浓度组，以1/16MIC组接合效率较高。ST11与ST15肺炎克雷伯菌，以ST11型接合效率较高。AI干扰+突变6h接合组较对照接合抑制率为31%，AI干扰+突变12h接合组较对照接合抑制率为33%。明确了LsrK体外磷酸化AI-2能干扰AI-2类群体感应系统。阐述了体外磷酸化AI-2能够一定程度上抑制碳青霉烯酶耐药基因水平转移。