铜绿假单胞菌DsbM调控抗氧化系统导致细菌耐药机制的研究

Pseudomonas aeruginosa is an important opportunistic pathogen which can cause severe hospital acquired infection. The antibiotic resistance problem is increasingly serious, that makes great trouble to anti-infection treatment. In the previous study, our group has used Mu transpositional recombination technology to find out an aminoglycoside antibiotic resistance suppressor gene dsbM, and proved that the expression of the gene dsbM could increase sensitivity of P. aeruginosa against antibiotic drugs when the experiments including genetic complementation, gene knockout, gene over-expression and DNA microarray were carried out. It is also shown that DsbM is a novel disulfide oxidoreductase. We suppose that antioxidant system regulation by DsbM may cause the change of aminoglycoside antibiotic resistance in P. aeruginosa. The yeast two-hybrid screening has proved that the interaction between DsbM and OxyR, a regulation factor in antioxidant system, was existed. We will use in vitro Mu mutations generate system to mutate the gene dsbM shortly and randomly, and to find out the enzymic activity center and regulation dots of the DsbM. We will also estimate what affection of the DsbM against a series of antioxidant factors such as OxyR, KatB, AhpB and AhpCF by using the yeast two-hybrid system, genetic recombination and gene expression, detection of enzymic activities and proteome analysis. Our final aim is to elucidate the inhibitory mechanism of the DsbM against antibiotic resistance in P. aeruginosa.

铜绿假单胞菌是条件性致病菌，其耐药性问题日趋严重。项目组前期应用Mu转座重组技术发现了一个氨基糖苷类抗生素的耐药性抑制基因dsbM，并通过遗传互补、基因敲除、过表达以及微阵列表达谱检测等手段，证明dsbM的表达能够增加宿主菌对抗生素药物的敏感性。实验证实DsbM是一种新型的二硫键氧化还原酶,推测铜绿假单胞菌可能通过细菌抗氧化系统来调节其对氨基糖苷类抗生素的耐药性。酵母双杂交实验证实DsbM蛋白与抗氧化调节系统OxyR蛋白存在相互作用。项目组拟采用Mu体外突变产生系统对dsbM进行随机插入突变以期发现其活性中心和调控位点；通过酵母双杂交技术、基因重组与表达、酶活性测定和转录组学分析，评估DsbM对细菌抗氧化系统诸因子如katB, ahpB和ahpCF的影响，以及dsbM与其上游基因的相互关系，最终阐明DsbM对耐药性的抑制机制，探讨通过激活或增强抑制基因的表达降低细菌耐药的新途径。

项目组利用基因敲除、过表达、报告基因融合、实时荧光定量PCR技术、凝胶阻滞和RNAseq等分析手段，对铜绿假单胞菌dsbM基因及其所在耐药性相关的基因簇以及DsbM潜在的调控对象进行了全面的研究分析，进一步证明dsbM基因产物的催化功能，发现了DsbM包括PA0056在内的潜在底物蛋白，DsbM对细菌抗氧化系统的影响是通过OxyR和PA0056等调控蛋白来实现的。项目组证明了PA0056是一个耐药性相关的调控因子并且DsbM蛋白与PA0056蛋白的相互作用方式与DsbM蛋白-OxyR蛋白作用方式相似，PA0057是一种金属β-内酰胺酶，而PA0055蛋白能够与DNA结合从而调控基因表达。研究发现，过表达PA0055基因对PA0057基因、dsbM基因表达产生负调控，并导致细菌对β-内酰胺类抗生素亚胺培南的耐药性下降。PA0055、PA0056和DsbM之间相互影响，并在基因簇之外的因子参与下，以一种极其复杂的方式协同调控铜绿假单胞菌的抗生素耐药性。由于细菌对不同抗生素产生耐药性的机制不同，项目组应用不同种类的抗生素处理病原菌，研究DsbM与PA0056缺陷对不同耐药机制的影响，并利用真菌疏水蛋白复配抑菌肽进行抗菌增效和抑制生物被膜的产生。总之，阐明DsbM及相关调控因子对细菌耐药性的影响机制，可以找到降低细菌耐药性的新途径。目前发表SCI收录论文2篇，培养博士研究生4名（毕业1名），硕士研究生3名（毕业1名）。