铜绿假单胞菌黏液型转变阻遏因子MucA-MucB在受控膜内蛋白水解通路(RIP)下降解的结构基础与分子机制

Pseudomonas aeruginosa is one of the major pathogen causing chronic respiratory infection. Alginate that overproduced from its mucoid strains is the main component of the extracellular polysaccharide in Pseudomonas aeruginosa biofilm, which is the main reason of its high resistance to drug and difficulty in its treatment. Alginate biosynthesis is primarily regulated by regulated intramembrane proteolysis (RIP) pathway, it includes cascade degradation of anti-mucoid conversion factors MucA-MucB via protease such as AlgW and MucP, releasing extra-cytoplasmic function sigma factor AlgU to express genes which associated with alginate producing, to complete the mucoid conversion and biofilm formation. Due to the importance of RIP, enzymes involved in this pathway are thought to be feasible targets for the treatment of Pseudomonas aeruginosa infection. However, the structural basis of the specific recognition among the proteins and extracellular stimulus sensing mechanism in this system remain elusive. Our study will focus on MucA-MucB and the critical enzymes involved in its degradation. Based on protein crystal structure study and enzymatic assay, we will design corresponding mutants and test the effects on Pseudomonas aeruginosa phenotye and its infection efficiency, to further investigate MucA-associated RIP mechanism and provide basic theory for clinical treatment against Pseudomonas aeruginosa-caused persistent infection.

铜绿假单胞菌是慢性呼吸道感染的重要病原体之一，其黏液型菌株分泌的藻酸盐是它生物被膜的主要胞外多糖，是导致其耐药和难以彻底清除的重要原因。藻酸盐合成主要由受控膜内蛋白水解通路调节，即通过蛋白酶如AlgW、MucP对黏液型转变阻遏因子MucA-MucB的有序降解，释放调控因子AlgU以激活藻酸盐合成相关基因的表达，完成黏液性转变和菌膜的形成。由于该调控通路的重要性，其相关蛋白酶被视作抗铜绿假单胞菌感染的新靶点。目前对于该通路各关键因子间特异性识别的结构机制以及其对胞外诱导信号感知的分子机制尚不明确。本课题以阻遏因子MucA-MucB及其降解过程中关键蛋白酶为研究对象，基于蛋白质晶体结构和酶学实验，结合基因敲除和突变设计并通过细菌表型检测及细胞侵染模型印证，进一步阐明MucA降解相关的受控膜内蛋白水解通路的机制，为临床治疗铜绿假单胞菌引发的持续性感染提供基础。

铜绿假单胞菌是肺囊肿性纤维化感染中最普遍存在又极难根除的病原体。铜绿假单胞菌分泌的藻酸盐作为生物膜的主要胞外多糖赋予其很强的环境适应性和耐药性，而受控膜内蛋白级联酶解（RIP）通路中的阻遏因子与膜锚定蛋白酶是藻酸盐形成过程中的关键信号分子。本课题的研究对象是铜绿假单胞菌中RIP通路中关键阻遏因MucA/MucB蛋白及关键蛋白酶AlgW的结构基础及功能。在本课题中，通过分子克隆，表达纯化，结晶筛选，并且基于X-射线衍射，得到了MucAperi 和MucB复合物结构以及AlgW蛋白的结构。分析结构，构建了可能对其功能具有重要影响的突变体，并通过体外生化实验和体内功能实验证实了这些突变体的作用，探明LPS脱落的Lipid-A对于激活MucA-RIP通路的重要性。我们的研究为为治疗铜绿假单胞菌感染性疾病和新型药物小分子研发提供了理论基础和科学依据。