淋球菌肽聚糖乙酰化对大肠杆菌溶解性转糖酶A酶切作用的影响  

Effects of peptidoglycan acetylation in Neisseria gonorrhoeae on catalysis activity of murein lytic transglycosylase A from E.coli

作　　者：吴腾飞[1] 张巧灵[1] 邓旭明[1] 刘波[1] 

机构地区：[1]吉林大学畜牧兽医学院基础兽医学教研室,吉林省长春市130062

出　　处：《中国组织工程研究与临床康复》2010年第46期8643-8646,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30972170);项目名称:2型猪链球菌消化道感染机制及其诱导的自噬作用研究;科学前沿与交叉学科创新项目(200903337);项目名称:2型猪链球菌(SS2)经消化道粘膜感染的研究资助~~

摘　　要：背景:有研究表明大肠杆菌(E.coli)肽聚糖乙酰化抑制溶解性转糖酶的酶切活性,但对淋球菌胞壁质乙酰化对该类酶的影响未见报道。目的:观察淋球菌的肽聚糖乙酰化对革兰阴性菌溶解性转糖酶家族二的酶切效果的影响。方法:运用同源重组方法敲除淋球菌的肽聚糖乙酰化基因A和B(△pacAB),制备3H标记的野生株和突变株的肽聚糖。应用分子克隆和蛋白表达纯化技术获得溶解性转糖酶A,体外测定溶解性转糖酶A对淋球菌和突变株(△pacAB)的肽聚糖酶切效果和最适酶切温度。结果与结论:溶解性转糖酶A经克隆、表达和纯化得到浓度为26.12g/mL蛋白;氚代葡糖胺标记并纯化得到氚代淋球菌肽聚糖多聚体;溶解性转糖酶A对40%乙酰化的淋球菌FA19肽聚糖酶切效果仅为9.71%,但对去乙酰化的突变株肽聚肽的酶切效果达93.45%;溶解性转糖酶对淋球菌肽聚糖酶的最佳酶反应温度为30℃。结果提示,淋球菌的肽聚糖乙酰化抑制溶解性转糖酶蛋白的切割活性,淋球菌可能通过胞壁酸乙酰化抑制溶解性转糖酶A引起的自溶的发生。BACKGROUND：Studies have demonstrated that E.coli peptidoglycan（PG） acetylation suppresses catalysis activity of lytic transglycosylase.However,whether PG acetylation in Neisseria gonorrhoeae（N.gonorrhoeae） has the same effect remains poorly understood.OBJECTIVE：To identify effect of PG acetylation in N.gonorrhoeae on catalysis activity of lytic transglycosylase family 2 in gram-negative bacteria.METHODS：PG acetylation genes（pacAB） in N.gonorrhoeae FA19 were knocked out though transformation by homologous recombination.3H-glucosamine labeled PG from either wild type N.gonorrhoeae FA19（PG1） or pacAB mutant strain（PG2） were isolated respectively.We amplified,cloned,expressed and purified lytic transglycosylase（MltA） from E.coli.The catalysis activity of MltA on PG1 and PG2 was detected,and the optimum reaction temperature was measured.RESULTS AND CONCLUSION：The recombinant MltA was obtained at the concentration of 26.12 g/mL.Only 9.71% PG1 was cleaved,but around 93.45% of no-acetylation PG2 was cleaved.The optimum reaction temperature was 30 ℃.PG with O-acetylation decreased catalysis capability of MltA.It suggests acetylation of PG in N gonorrhoeae inhibited the autolysis caused by MltA.

关 键 词：淋球菌 肽聚糖 肽聚糖乙酰化基因 溶解性转糖酶A 组织构建 

分 类 号：R318[医药卫生—生物医学工程]