耻垢分枝杆菌的蚯蚓血红蛋白样蛋白MSMEG_3312影响其大环内酯类药物的敏感性

A hemerythrin-like protein MSMEG_ 3312 influences erythromycin resistance in mycobacteria

【目的】活性氧类分子是机体有氧代谢的自然产物,可以引起氧化损伤,导致细胞DNA突变、蛋白质失活,是细菌耐药产生的原因之一。蚯蚓血红蛋白家族是能携带氧、可逆地结合氧的一类蛋白,在氧代谢过程中发挥重要作用,与活性氧类分子介导的细菌耐药相关。预测耻垢分枝杆菌(Mycobacterium smegmatis)MSMEG_3312是蚯蚓血红蛋白样蛋白,其功能可能与细菌抗药性有关。【方法】通过生物信息学预测耻垢分枝杆菌MSMEG_3312的结构特征。通过基因敲除、遗传互补和抗药性分析以及启动子表达测定等方法研究MSMEG_3312与耻垢分枝杆菌抗药的相关性。【结果】与野生菌株mc2155相比,msmeg_3312敲除菌株表现为抗大环内酯类抗生素,而且回补msmeg_3312部分丧失了这种耐药表型。此外,大环内酯类抗生素的胁迫在统计上显著下调msmeg_3312启动子的表达。另外,对作用于核糖体的其他药物,敲除菌株和野生菌株没有抗药性差异。【结论】生物信息学分析显示MSMEG_3312的氨基酸序列具有典型的蚯蚓血红蛋白保守的HHE结构域,预测其二级结构含有4个α-螺旋组成的典型蚯蚓血红蛋白结构。MSMEG_3312与耻垢分枝杆菌对大环内酯类抗生素的药物敏感性相关,其可能通过影响药物与核糖体50S亚基的作用来发挥功能。

[ Objective] Reactive oxygen species are natural products of metabolism in aerobic organisms, which lead to oxidative damage, such as DNA mutation, protein inactivation and drug resistance. MSMEG_3312 was predicted as a hemerythrin-like protein, which can carry oxygen and reversibly bind to oxygen, thus it might play important roles in the process of oxygen metabolism. In this study, we explored the role of MSMEG_3312 in drug resistance. [ Methods] On the basis of bioinformatics, we identified the conserved sequence of HHE domain in MSMEG_3312 and it was predicted to have typical a-helix at secondary structure. To explore potential functions of MSMEG_3312, we constructed the msmeg_ 3312 knockout strain and compare the susceptibility to various drugs to its parent strain, mc2155. In addition, we also measured the promoter response when treatment of erythromycin. [ Results] Genetic results showed that MSMEG 3312 is not necessary for M. smegmatis growth at 7H9 rich medium. The msmeg_3312 knockout strain showed increased erythromycin resistance. Moreover, the drug resistance is only limited to erythromycin which its mechanism of action is by binding to the 50S subunit of the bacteria ribosomal complex and then inhibit protein synthesis. However, there were no different MICs of other antibiotics, targets for protein synthesis inhibition, but not 50S subunit, such as tetracyclines, aminoglycosides and chloramphenicol. Moreover, we also showed that the promoter of msmeg_3312 responses to erythromycin. [ Conclusions] Hemerythin-like protein MSMEG_3312 is involved in erythromycin resistance.