多重耐药鲍曼不动杆菌RND家族药物外排泵的结构与调控机制研究

Acinetobacter baumannii is an important hospital-acquired pathogen causing serious pneumonia and bloodstream infections, within its clinical detection rate getting higher and multidrug resistance problem becoming more and more serious, the World Health Organization has ranked it as the most critical multidrug-resistant pathogenic bacteria all over the world...Acinetobacter baumannii multidrug resistant ability is highly correlated with its RND (Resistance-Nodulation-Division) family efflux pump anchored across the cell membrane, which can efflux antibiotics out of the cell under antimicrobial pressure. There are three RND efflux pumps, namely AdeABC, AdeIJK and AdeFGH in Acinetobacter baumannii, and they are strictly controlled by AdeRS two-component regulatory system, transcriptional regulator AdeN, and AdeL respectively...Here we will screen the gene polymorphism of the RND efflux pump together with their regulatory elements from the clinical Acinetobacter baumannii strains. Following by an integrative structural biology approach, we will reveal the atomic crystal structure of the regulatory elements AdeRS, AdeN and AdeL targeting their promoter DNA sequence to elucidate the RND efflux pump regulatory mechanism. More importantly, we will combine X-ray crystallography and cryo-Electron Microscopy to solve the structure of RND efflux pump transmembrane complex and demonstrate its efflux mechanism. Finally the knockout and mutagenesis strains will be generated and used to verify the key regulatory residues involved in the multidrug resistance from the clinical infection sample's gene polymorphisms and structural studies...Taken together, we will carry out the clinical, structural and physiological studies to reveal the mechanism insight into how RND efflux pump mediate the multidrug resistance in Acinetobacter baumannii, and our study will enlighten the antimicrobial drug discovery by targeting RND efflux pump and will pave the way for anti-infective therapy against Acinetobacter baumannii.

鲍曼不动杆菌是导致医院严重肺炎和血流感染的病原菌，其临床检出率日益增高且多重耐药问题日趋严重，已经成为临床抗感染治疗的重要挑战。鲍曼不动杆菌的多重耐药性与其跨膜RND家族药物外排泵介导的抗生素外排密切相关。本项目将筛查临床感染多重耐药鲍曼不动杆菌中三种RND药物外排泵AdeABC，AdeIJK，AdeFGH及其调控元件AdeRS，AdeN，AdeL的基因多态性；然后通过一体化结构生物学方案，解析其调控元件识别靶向DNA调控序列的晶体结构，在原子水平阐述其调控机制；进而整合X射线晶体学和冷冻电镜揭示药物外排泵跨膜复合物外排抗生素底物的结构机制；最后构建基因敲除和点突变菌株，验证在临床感染样本中的多态性和结构解析获得关键氨基酸调控位点对其多重耐药的影响，在临床，结构和生理水平三个尺度系统揭示RND药物外排泵介导鲍曼不动杆菌多重耐药的分子机制，为研发新的抗生素药物和抗感染治疗策略提供重要依据。

鲍曼不动杆菌是近些年世界卫生组织多重耐药威胁程度最高的致病菌首位，能导致严重的肺炎，血流感染和手术器械导致的严重感染等。其临床检出率日益增高且多重耐药问题日趋严重，已经成为临床抗感染治疗的重要挑战。鲍曼不动杆菌的多重耐药与其跨膜RND家族药物外排泵介导的抗生素外排密切相关。本项目以多重耐药鲍曼不动杆菌中的RND药物外排泵及其调控元件为研究对象，揭示其临床感染突变多态性和调控分子机制。.本项目的主要成果包括（1）系统阐释了AdeRS二元调控系统介导药物外排泵AdeABC的调控分子机制，在阐明AdeR通过识别AdeABC与AdeRS之间的一段顺反子调控序列而介导其表达调控的基础上，我们进一步揭示AdeR的N末端的无序序列参与了蛋白质水解降解的敏感性，对于维持其细胞质AdeR水平起重要作用，在完成AdeABC外排泵启动子的识别和激活后，AdeR二聚体可被N-末端IDR释放，并进一步进入降解和稳态循环途径。同时我们解析了AdeS激酶结构域的高分辨率晶体结构，提出了AdeS六聚体的激活工作模型。（2）解析了RND药物外排泵AdeG的高分辨率的冷冻电镜结构，提出并阐释了其外排抗生素的工作模型。（3）通过系统分析临床感染鲍曼不多杆菌的多态性和结构获取的关键调控氨基酸位点信息，我们构建了多种RND药物外排泵的基因敲除菌种，进一步在生理水平验证了药物外排泵的调控分子机制。.项目负责人在项目执行期间入选陕西省特支计划青年拔尖人才，并且获得陕西省杰出青年基金的支持。本项目将为研发靶向药物外排泵的新的抗生素药物和抗感染治疗策略提供重要依据。