假定蛋白PA3692的结构特点及其通过维持细胞膜完整性在铜绿假单胞菌致病中的作用研究

The integrity of cell membrane of Pseudomonas aeruginosa (PA) is the basis of its bacterial characteristics such as wide distribution, diversity of transmission pathways, and multiple drug resistance. However, there is a lack of information about the mechanism for PA to maintain the integrity its cell membrane. Therefore, it is important to clarify the mechanism of PA to maintain the integrity of cell membrane, which will lead to better understanding of the pathogenesis of PA and to discover new targets for anti-PA drugs. In our previous studies, we found that hypothetical protein PA3692 from PA was able to bind to peptidoglycan to maintain the integrity of cell membrane. Compared with the reported peptidoglycan binding proteins, the way of the binding of PA3692 to peptidoglycans has distinct characteristics, suggesting that there is a special active site in PA3692, which could be a new target for anti-PA drugs. In this project, we plan to solve the crystal structure of the PA3692 and PA3692 complexed with DAP (diaminopimelic acid), a component of peptidoglycan. And we will confirm the binding characteristics by in vitro biochemical experiments. Then, the PA mutant will be constructed and the change of membrane integrity and pathogenic effects will be evaluated at cell and animal levels. Thus, the contribution of PA3692-peptidoglycan interaction in pathogenicity of PA could be clarified, which help elucidate the pathogenesis of PA and discover new targets for anti-PA drugs.

铜绿假单胞菌（PA）细胞膜的完整性是其具有分布广泛、传染途径多样、多重耐药等生物学特征的基础，但目前对于PA维持自身细胞膜完整性的机制还缺乏明确的认识。因此阐明PA维持细胞膜完整性的机制对全面揭示PA的致病过程和发现新的抗PA药物靶标具有重要的意义。课题组研究新发现PA基因组未知功能蛋白PA3692通过与细胞膜周浆间隙中肽聚糖特异结合，在维持PA细胞膜完整性中发挥重要作用。与已知的肽聚糖结合蛋白相比，PA3692与肽聚糖的结合具有鲜明特点，提示其结构上具有特殊的活性位点，可作为抗PA药物新的作用靶点。本课题拟在此基础上通过解析PA3692、PA3692-肽聚糖成分DAP的晶体结构和体外生化实验等手段，揭示PA3692与肽聚糖相互作用的独特方式，并在细胞和动物水平明确PA3692-肽聚糖相互作用在PA致病中所发挥的效应，为进一步阐明PA的致病机制和发现抗PA药物靶点提供理论和实验依据。

铜绿假单胞菌（PA）细胞膜的完整性是其具有分布广泛、传染途径多样、多重耐药等生物学特征的基础，但目前对于PA维持自身细胞膜完整性的机制还缺乏明确的认识。课题组研究新发现PA基因组未知功能蛋白PA3692通过与细胞膜周浆间隙中肽聚糖特异结合，在维持PA细胞膜完整性中发挥重要作用。本课题拟采用晶体结构解析、定点突变分析、生化特性分析等手段，解析PA3692与肽聚糖复合物的晶体结构，揭示PA3692与肽聚糖独特的作用方式，并在细胞和动物水平进一步明确PA3692通过维持细胞膜完整性在PA抵抗环境压力、耐药、粘附细胞和抗宿主杀伤过程中的作用，为阐明PA的致病机制和发现抗PA药物作用靶点提供新的理论和实验依据。. 本研究成功制备高质量PA3692重组蛋白和晶体，并解析了PA3692的晶体结构，在结构中观察到其与肽聚糖结合的特异方式；构建了PA3692互作的关键氨基酸突变体，通过体外生化实验验证了PA3692的特殊作用方式。构建了PA3692缺失突变PAO1菌株和回复突变株，通过耐酸、耐盐、细胞粘附等试验发现PA3692与肽聚糖相互作用在细菌生存和耐药过程发挥关键作用。本研究还发现滴鼻免疫PA3692可诱导高保护性Th17免疫应答，并鉴定其诱导Th17的特异性表位，顺利完成预期研究内容。本项目研究结果发表SCI论文4篇，培养研究生2名，达到预期研究目标。