蛋白修饰影响嗜水气单胞菌耐药功能及其作用机理研究

The protein post-translational modifications (PTMs) play important role on the bacterial antibiotics resistance. Recently, researches including our team found that many antibiotics resistance functional proteins were modified by common PTMs. However, mechanism and effects of these PTMs on bacterial antibiotics resistance are still largely unknown..In this study, to further investigate the antibiotic resistance mechanism meditated by PTMs, quantitative proteomics would be used to compare the differential expression of common PTMs, such as phosphorylation, succinylation and acetylation in antibiotics resistance Aeromonas hydrophila strains firstly; Secondly, the antibiotics resistance functions or enzymatic activities of these candidate proteins will be valued by antibiotics survival capabilities assay in related strains which genes are knocked out, rescued or site-directed mutated; Finally, to illuminate the intrinsic roles of protein-protein interaction of these PTMs in antibiotics resistance, PTMs mediated complexes would be identified by molecular biology techniques such as co-immunoprecipitation and pull down method in over-expressed and site-directed mutated strains. In this way, we try to provide novel insights into mechanisms of antibiotics resistance which mediated by common PTMs and provide novel candidate targets for new drug development.

蛋白质翻译后修饰(PTMs)在细菌耐药中起重要的作用。申请人及前人研究发现细菌中多个耐药相关蛋白具有蛋白修饰位点，但其对耐药功能的影响及作用机理尚不明确。本课题在前期研究的基础上，拟利用定量蛋白修饰组学技术，比较不同嗜水气单胞菌耐药菌株中，磷酸化、乙酰化和琥珀酰化等常见PTMs蛋白与位点的表达差异，初步了解蛋白修饰在细菌耐药中的变化；将关键差异基因缺失、补救及修饰位点突变后，进行耐药功能与相关酶活性评价，验证关键蛋白及相应修饰位点的耐药功能；利用Pull-down与免疫共沉淀等方法，比较关键修饰位点突变后对目标蛋白复合物组成的影响，明确耐药相关PTMs在蛋白相互作用中的功能。研究结果将有助于深化对蛋白质修饰介导耐药机制的认识，并为新药开发提供候选靶点。

水产养殖业重要的病原菌嗜水气单胞菌的耐药性日益严峻，蛋白质翻译后修饰(PTMs)在细菌耐药中起重要的作用，但其对耐药功能的影响及作用机理尚不明确。本项目首先利用特异抗体富集结合质谱鉴定的修饰蛋白质组学技术，构建嗜水气单胞菌乙酰化和琥珀酰化等常见PTMs蛋白修饰图谱，并以群体感应中重要的II型信号分子合成酶LuxS蛋白乙酰化和琥珀酰化修饰为例，发现LuxS蛋白K23和K30位点的琥珀酰化修饰正调控LuxS的酶活性，K156位点的乙酰化和琥珀酰化修饰分别负、正调LuxS酶活性，产生Cross-talk现象，从而调控细菌的群体感应，进而影响到细菌的耐药性。进而利用Label-free定量修饰蛋白质组学技术，比较不同嗜水气单胞菌耐药菌株中全蛋白与酰基化的修饰蛋白与位点的表达差异。生物信息学分析发现，修饰蛋白多参与蛋白核糖体翻译、TCA循环、丙酮酸代谢等生物途径；然后以LuxS、AHA_0655、LamB和铁离子吸收蛋白Fur为例，探讨其酰基化修饰对细菌耐药的影响。首先，研究发现LuxS蛋白K23与K165位点的酰基化改变了细菌对土霉素的敏感性，同时还发现Arginine ABC transporter(AHA_0655) K14和麦芽糖通道蛋白LamB K51位点影响细菌的耐药性提示酰基化修饰这些蛋白影响应对细菌耐药过程；其次，Δfur菌株导致细菌对大部分抗生素的耐药性降低，但对链霉素、硫酸新霉素和巴龙霉素的抗性增加；再次，当Fur蛋白K14突变为E时，细菌对喹诺酮类抗生素的耐药性普遍下降，特别是对恩诺沙星和巴洛沙星的最低杀菌浓度下降8倍。以上结果初步证实乙酰化/琥珀酰化修饰在细菌Fur蛋白的耐药调控功能中起重要的作用，Fur蛋白可能只需要通过改变K14位点的不同酰基化修饰来调控耐药相关基因的表达，从而应对不同类型抗生素压力。这一发现解释了有些重要转录因子的mRNA或者蛋白质水平并没有变化，却依旧可以调控目的基因表达的现象，充分体现了细菌耐药的复杂性，可能是一种新的耐药调控机制。以上研究结果将有助于深化对蛋白质修饰介导耐药机制的认识，并为新药开发提供候选靶点。