群体感应调控基因abaR在鲍曼不动杆菌生物膜形成中的作用机制

Acinetobacter baumannii (A. baumannii) is a major pathogen that causes nosocomial infection. Owing to its ability to frequently form biofilm and develop drug resistance, an infection of Candida albicans is difficult to be eradicated in clinic. An increasing number of evidence has revealed that the bacterial quorum sensing (QS) is tightly correlated with the biofilm formation and drug resistance in bacteria including the A. baumannii. The QS system of A. baumannii is regulated by abaI gene product, N-acylhomoserine lactomes (AHLs). Sequentially, AHL is able to regulate the expression of QS-controlled genes by binding to and forming a complex with its receptor or regulator AbaR, a protein encoded by abaR gene. To date, the structure and function of the abaI gene in the biofilm formation and drug resistance of A. baumannii have been extensively interrogated. However, the role of the abaR gene in the biofilm formation and drug resistance of AB remains elusive. The proposal is therefore aimed to explore the potential functions and underlying of abaR gene in the biofilm formation and drug resistance in this bug, on a basis of the isolation of clinical multiple drug-resistant A. baumannii strains, with following 4 specific aims: 1) establishment of an in vitro biofilm model and a method of QS detection method for clinical A. baumannii isolates; 2) a RNA-seq analysis will be employed to identify differentially expressing genes of A. baumannii biofilms in response to QS inhibitors; 3) interrogating functions of QS in the biofilm formation and multidrug resistance in clinical MDR AB isolates; and 4) mutating the abaR gene using a CRISPR-CAS9 gene editing strategy to generate A. baumannii mutants harboring a mutated abaR gene for functional validation. As a consequence, these studies will allow us to explore the potential functions and mechanisms of abaR gene in the biofilm formation and drug resistance of A. baumannii, which will lay a foundation for further study of the mechanism of biofilm formation and drug resistance of A. baumannii, as well as provide novel targets for development of effective antimicrobial agents.

多重耐药鲍曼不动杆菌(Acinetobacter baumannii, AB)是医院感染的主要病原菌之一， 研究表明群体感应 (quorum sensing, QS) 和生物被膜的形成与其临床耐药密切相关。AB的QS系统是由AbaI指导合成的自诱导因子N-酰基高丝氨酸内酯分子 (AHL)与其受体(调控子)分子abaR基因产物AbaR结合后调控QS相关效应基因的表达，但对于abaR基因在AB的生物膜形成和耐药中的作用和机制还未阐明。本项目拟在临床分离多重耐药AB的基础上，1) 建立AB生物被膜模型和QS检测方法; 2)分析RNA-Seq转录组变化；3) 探讨QS在临床AB生物膜形成和耐药中的作用；4)通过CRISPR-CAS9基因编辑手段构建abaR基因突变鲍曼不动杆菌突变株，验证abaR基因在AB生物膜形成及耐药中的作用与机制，为寻找抗多重耐AB感染临床治疗药物的研究提供新的靶点。

本项目分析了临床多重耐药鲍曼不动杆菌群体 感应abaR 基因表达，以及群体感应和生物膜形成的相关性基因的表达与群体感应和生物膜形成的相关性。，通过研究abaR 基因对临床多重耐药鲍曼不动杆菌的生物膜形成及耐药基因表达差异的影响，分析临床多重耐药鲍曼不动杆菌的生物膜形成及耐药基因表达差异性，进一步探讨了群体密度感应信号小分子对生物被膜的影响。同时构建 CRISPR-Cas9载体系统质粒 pet41a-cas9-gRNA 并表达重组Cas9蛋白，为细菌基因的编辑研究奠定了实验基础。采用生物信息学方法分析鲍曼不动杆菌ATCC17978中第二信使环鸟苷磷酸（c-di-GMP）相关代谢蛋白，预测其性质和编码能，获得了如下4个方面的研究结果：1）发现菌毛集合系统基因与生物被膜形成密切相关，群体密度感应系统小分子 3-oxo-C8-AHL对菌毛集合系统等相关基因的表达具有下调作用，从而抑制鲍曼不动杆菌的运动，致使生物被膜结构形成缓慢；2）转录组学分析两种生长状态下的鲍曼不动杆菌共获得504个差异表达基因，其中264个下调，240个上调。群体感应系统相关基因A1S_0109、A1S_0112~ A1S_0114及乙醇脱氢酶基因A1S_2098上调。QRT-PCR验证差异表达基因的表达情况，结果与测序数据一致。群体感应分子C12-HSL的加入使鲍曼不动杆菌生物膜的形成能力增强，且细菌运动性亦增强。双硫仑（200M）处理后，鲍曼不动杆菌生物膜内细菌乙醇脱氢酶的活性减弱，群体感应相关基因的表达下调，生物膜形成能力下降。双硫仑对鲍曼不动杆菌生物膜形成的抑制作用在临床分离株中同样有效。鲍曼不动杆菌生物膜的形成与群体感应系统相关。群体感应增强，鲍曼不动杆菌的生物膜形成能力和细菌运动性增强。乙醇脱氢酶在鲍曼不动杆菌生物膜形成中发挥重要作用。乙醇脱氢酶活性减弱，群体感应基因表达下调，生物膜形成能力下降；3）通过双酶切法成功构建CRISPR- Cas9载体系统质粒pet41a- cas9-gRNA并表达重组Cas9 蛋白并原核细菌中表达，为细菌基因的编辑研究奠定了实验基础；4）采用生物信息学分析了鲍曼不动杆菌ATCC17978含有12个与c-di-GMP代谢相关GGDEF/EAL结构域蛋白，为探索c-di-GMP代谢网络奠定了基础。这些工作为进一步研究鲍曼不动杆菌的耐药机制提供了理论依据和实验基础。