StkP/PhpP-CiaH/CiaR信号网络及相关microRNA调控肺炎链球菌耐药分子机制

Penicillin binding proteins (PBPs)-independent resistant phenomenon of Streptococcus pneumoniae caused by CiaH kinase inactivation and StkP kinase mutation was found recently, but molecular mechanism for the resistance is unknown. We found that ciaH or stkP gene-deleted mutant of the bacterium displayed the high resistance to both penicillin and cefotaxim and PBPs expression levels of the two mutants was significantly down-regulated, while the CiaH-downstream transcription factor CiaR was confirmed only to bind to the promotors of partial pbps genes and stkP gene. It has been shown that the CiaR regulates five microRNA genes and the StkP possesses a PBP domain. Thus, in the present project, we plan to determine the binding ability between the PBP domain in StkP and penicillin or cefotaxim by using some certain techniques such as site mutation and SPR spectrum. Subsequently, the correlations of function and regulation among the CiaH/CiaR and StkP/PhpP, the CiaR and PBPs or microRNAs expression, the microRNAs and PBPs expression by using some certain methods such as target gene deletion, ESMA and Northern Blot. The aim of this project is to illuminate the molecular mechanism of StkP/PhpP-CiaH/CiaR signaling network down-regulating PBPs expression levels of Streptococcus pneumoniae directly by the CiaR or indirectly by microRNAs to cause the resistance to β-lactam antibiotics.

近年发现肺炎链球菌激酶CiaH失活和StkP突变可产生与青霉素结合蛋白PBPs突变无关的耐药现象，但机制不明。我们发现ciaH与stkP基因敲除突变株对青霉素和头孢噻肟高度耐药且PBPs表达显著下调，但CiaH下游转录因子CiaR仅能结合部分pbps及stkP基因启动子。有证据显示CiaR可调控5个microRNA基因且StkP有PBP结构域。本项目拟采用定位突变及等SPR光谱技术确定StkP中PBP结构域与青霉素和头孢噻肟结合能力，采用基因敲除、ESMA、Northern Blot等方法确定CiaH/CiaR与StkP/PhpP、CiaR与PBPs及microRNAs、microRNAs与PBPs表达之间功能及调控关系，以期阐明StkP/PhpP-CiaH/CiaR信号网络经CiaR直接下调或经microRNAs间接下调肺炎链球菌PBPs表达水平而导致对β-内酰胺类抗生素耐药的分子机制。

肺炎链球菌是人细菌性肺炎、脑膜炎、中耳炎主要病原体，临床上常用β-内酰胺类抗生素进行治疗肺炎链球菌感染性疾病。肺炎链球菌不产β-内酰胺酶，但近年该菌对β-内酰胺类抗生素耐药性及耐药率不断升高，其耐药机制不明。本研究采用基因克隆和重组表达、基因敲除、ESMA、激光共聚焦显微镜、分子杂交、表面等离子共振技术等方法确定CiaH/CiaR与StkP/PhpP、CiaR与PBPs及csRNAs之间功能及调控关系，以期阐明StkP/PhpP-CiaH/CiaR信号网络下调肺炎链球菌青霉素结合蛋白（PBPs）表达水平而导致对β-内酰胺类抗生素耐药的分子机制。本项目主要研究结果如下：①构建了肺炎链球菌ciaH、ciaR、stkP、phpP基因原核表达系统并制备了表达产物抗血清及其IgG，为后继实验研究提供了有效手段；②证明StkP胞外区通过其PBP-Ser/Thr激酶联合结构域（PBP and serine/threonine kinase associated domain，PASTA-domain）中两个SXXK基序与β-内酰胺类抗生素结合；③发现β-内酰胺类抗生素可诱导CiaH和StkP活化、CiaH和StkP磷酸化激活CiaR、CiaR下调PBPs编码基因表达使肺炎链球菌产生PBPs突变非依赖β-内酰胺类抗生素耐药性；④证明CiaR通过结合靶基因启动子下调PBP1a、PBP1b和PBP2a表达或上调csRNAs表达；⑤证明肺炎链球菌PhpP具有PP2c型磷酸酶活性，β-内酰胺类抗生素可诱导phpP-mRNA水平升高；⑥构建了肺炎链球菌stkP、ciaH和ciaR基因敲除突变株，通过靶基因敲除和野生株在β-内酰胺类抗生素诱导后磷酸化水平及其mRNAs水平变化，了解了各基因之间的调控关系。本项目已在国家核心期刊发表论文6篇、录用1篇、SCI杂志和核心期刊修回论文各1篇，2篇SCI论文将完成撰写拟投SCI杂志，2016年浙江省微生物学会年会大会报告论文1篇，上述论文均标注本项目资助。本项目主要科学意义：从细菌信号传导网络角度了解耐药机制是细菌耐药研究新领域。通过本项目研究，培养硕士生7名，本项目主持人入选浙江省卫生高层次创新人才、浙江省微生物学会理事和医学微生物学专委会副主任委员，获得细菌耐药相关国家发明专利1项和浙江省科技成果三等奖1项。综上所述，我们全面完成了本项目研究任务。