结核枝杆菌中毒素-抗毒素系统的鉴定

Characterization of Toxin-antitoxin systems in Mycobacterium tuberculosis

【目的】鉴定结核分枝杆菌基因组上MazF同源蛋白基因与其上游基因是否组成毒素-抗毒素系统,阐明毒素蛋白的作用机理,并初步探讨毒素-抗毒素系统在营养缺乏时的表达调控。【方法】在大肠杆菌和耻垢分枝杆菌中将MazF同源蛋白单独表达或与其对应的抗毒素蛋白共同表达,鉴定MazF同源蛋白对细菌生长的抑制作用以及其对应的抗毒素蛋白能否消除这种生长抑制;通过体外RNA切割实验,检测MazF同源蛋白是否具有RNA切割活性;检测正常生长条件下和饥饿条件下毒素-抗毒素系统的启动子活性,探讨其在应激条件下的表达调控。【结果】结核分枝杆菌MazF同源蛋白中,Rv0659c、Rv1495和Rv1942c不具有抑制细菌生长的毒素蛋白活性,Rv1991c、Rv2801c、Rv1102c和mtPemK能够抑制细菌生长,而且它们的抑制作用可以分别被其对应的抗毒素Rv1991a、Rv2801a、Rv1103c和mtPemI解除。Rv1991c、Rv2801c和Rv1102c具有RNA切割活性,mtPemK则不能切割RNA。Rv1991a-1991c和Rv2801a-2801c系统的启动子在饥饿条件下活性显著升高。【结论】结核分枝杆菌基因组上Rv1991a-1991c、Rv2801a-2801c、Rv1103c-1102c和mtPemI-mtPemK是毒素-抗毒素系统。毒素蛋白Rv1991c、Rv2801c和Rv1102c通过切割RNA发挥抑菌或杀菌活性,mtPemK具体作用机理目前还不清楚。Rv1991a-1991c和Rv2801a-2801c系统可能参与结核分枝杆菌在营养匮乏条件下的生长调控。

[Objective] To characterize the toxin-antitoxin system（TA system） in Mycobacterium tuberculosis,which consist of MazF homologue gene and its upstream gene.[Methods]Seven M.tuberculosis MazF homologues were induced alone or co-expressed with their upstream genes respectively in Escherichia coli and Mycobacterium smegmatis,to test the toxic effects of the MazF homologues on bacteria growth,and the antitoxic effects of protein encoded by their upstream genes.The RNA cleavage activity of MazF homologous was identified in vitro with Rv0707 mRNA as the substrate.The promoter region of the identified toxin-antitoxin loci in M.tuberculosis was cloned in front of the lacZ reporter gene in pSD5B vector.The promoter activity was measured under the normal or starvation condition.[Results]The growth of either E.coli or M.smegmatis was inhibited by four MazF homologous proteins,among which Rv1102c,Rv1991c and Rv2801c,but not mtPemK,had the RNA cleavage activities.The toxic effects and RNA cleavage activities of Rv1102c,Rv1991c and Rv2801c were inhibited by their corresponding antitoxin Rv1103c,Rv1991a and Rv2801a,respectively.The other three MazF homologues,Rv1942c、Rv0659c and Rv1495,were not toxic to E.coli and M.smegmatis and also could not cleave RNA.It was found that the promoter activities of Rv2801a-2801c and Rv1991a-1991c systems were significantly increased under the complete starvation condition.[Conclusion]Our results demonstrated that Rv1103c-1102c,Rv1991a-1991c,Rv2801a-2801c and mtPemI-mtPemK were typical toxin-antitoxin systems in M.tuberculosis.Rv1102c,Rv1991c and Rv2801c were toxin proteins which inhibited cell growth through their RNA cleavage activities,while the mechanism of mtPemK toxin is still unknown.It is possible that Rv2801a-2801c and Rv1991a-1991c systems are involved in the starvation stress response.