MurA酶突变体在屎肠球菌对磷霉素耐药中作用及机制研究

Enterococcus faecium is a clinically important conditional pathogen. The old drug, fosfomycin, has been paid more and more attention in the treatment of infectioncaused by drug-resistant Enterococcus faecium. However, the number of fosfomycin-resistant Enterococcus faecium has increased year by year, and the resistance mechanism is not completely clear. In the previous study, the codons at nucleotide position 355-357 were found in four strains of fosfomycin-resistant Enterococcus faecium from TGT Cys119 to GAT Asp119, which means the MurAenzyme C119D mutation. It is highly resistant (MIC >1024 mg/L) and the mutant in MurA enzyme is hard to inactivation by fosfomycin binding. We will continue to collect fosfomycin-resistant Enterococcus faecium from multicenter. Through in-depth study of the MurA enzyme mutation site, the main mutant pattern of MurA enzyme in the domestic fosfomycin-resistant Enterococcus faecium is clarified. Through the difference of protein activity, function expression and the fitness cost, to explore the mechanism and action of MurA enzyme mutant in fosfomycin-resistant Enterococcus faecium. It provides the laboratory basis for formulating the strategy of prevention and treatment for fosfomycin resistance and finding new targets for anti-fosfomycin resistance.

屎肠球菌是临床重要条件致病菌，磷霉素在当前耐药屎肠球菌感染治疗中逐渐受到关注。但磷霉素耐药的屎肠球菌逐年增多，耐药机制不完全明确。课题组前期在4株磷霉素耐药的屎肠球菌中发现在核苷酸位置355-357处的密码子由TGT Cys119 变化为GAT Asp119即MurA酶C119D突变，该酶突变菌株对磷霉素呈现高度耐药性（MIC>1024 mg/L），而且该突变MurA酶更不易被磷霉素结合失活。课题组将继续系统收集多中心磷霉素耐药屎肠球菌，通过对MurA酶突变位点深入研究，明确国内磷霉素耐药屎肠球菌中MurA酶主要突变型别，并针对MurA酶突变体的蛋白活性和功能表达以及适应性代价差异，探讨MurA酶突变体在磷霉素耐药中的作用机制。为制定磷霉素耐药的防治策略和寻找抗磷霉素耐药的新靶位提供实验室依据。

磷霉素耐药屎肠球菌检出逐年上升，是临床阳性菌抗感染治疗一大挑战。MurA酶是磷霉素的靶酶，MurA酶突变体的蛋白质结构改变，可能引起对磷霉素的高水平耐药。本项目围绕国内MurA酶突变体在屎肠球菌对磷霉素耐药中作用开展研究，得到以下结论：（1）磷霉素耐药屎肠球菌中主要存在fosX基因（86.3%）、fosB基因（12.8%）与MurA酶的点突变（0.9%）。（2）MurA酶突变体可使细菌对磷霉素的敏感性下降，介导屎肠球菌对磷霉素的耐药。对两种底物UNAG和PEP的亲和力均明显高于野生型MurA酶，需要更高浓度的磷霉素方可抑制活性。（3）携带fosX基因的屎肠球菌在细菌对磷霉素耐药过程中起作用，介导细菌对磷霉素的MIC上升（32 mg/L上升至128 mg/L）。fosX基因的相对表达水平与肠球菌对磷霉素的耐药水平存在相关性。.本项目的研究成果为首次报道Asp（天冬氨酸）50Glu（谷氨酸）突变型MurA酶，并证明该突变体介导屎肠球菌对磷霉素产生耐药。发现屎肠球菌中磷霉素主要耐药基因fosX基因的表达水平与屎肠球菌对磷霉素的耐药水平相关。.在项目执行期间，发表了研究相关学术论文4篇，其中SCI收录3篇，中文核心期刊收录1篇；培养了硕士研究生1名。.