耻垢分枝杆菌精氨酸代谢变化对细菌毒力及肺上皮细胞线粒体功能的影响

Influence of arginine metabolism of Mycolicibacterium smegmatis on the virulence of bacteria and mitochondrial function of alveolar epithelial cells

目的探讨精氨酸代谢变化对耻垢分枝杆菌(M.smeg)毒力、致病性及宿主生理的影响。方法通过构建M.smeg argR敲除株,以野生型M.smeg为对照,测定细菌体外生长能力变化;采用实时荧光定量聚合酶链反应(RT-qPCR)检测细菌脂类代谢相关基因fadD2、fadA5、kstR等的表达。用M.smeg侵染人非小细胞肺癌上皮细胞系A549,检测CCK8细胞活力,采用RT-qPCR检测细胞线粒体功能相关基因UCP1和DNM1L,观察细菌对宿主细胞生理的影响。结果敲除argR可提高细菌脂质储存能力,在以葡萄糖为单一碳源的培养基中更具生长优势(P<0.001);同时,M.smeg分枝菌酸合成上调;侵染A549细胞2 h后,入胞率显著增加(P<0.001),细菌毒力增强,对A549细胞的侵染能力提高,并通过影响线粒体功能影响宿主细胞的活力。结论M.smeg精氨酸代谢负转录调控因子argR的敲除提升了细菌毒力,并可抑制宿主细胞线粒体功能。

Objective To investigate the influence of arginine metabolism on the virulence,pathogenicity and host physiology of Mycolicibacterium smegmatis(M.smeg).Methods A M.smeg knockout isolate of argR was constructed.Wild-type M.smeg was used as control,and the growth ability of bacteria in vitro was determined.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to determine the expressions of fadD2,fadA5 and kstR.Epithelial cells A549 were infected with M.smeg.Cell activity was determined as well.UCP1 and DNM1L were determined by RT-qPCR.The physiological effect on host cells was observed.Results It was found that argR knockout increased the lipid storage capacity of bacteria and had a growth advantage in the medium with glucose as a single carbon source(P<0.001).The synthesis of M.smeg mycoacid was up-regulated,and the virulence of epithelial cells A549 was increased.After the infection of epithelial cells A5492 h,the cell entry rate was increased(P<0.001),and the infection ability of epithelial cells A549 was improved.The deletion of argR affected cell viability by affecting its mitochondrial function.Conclusions The knockout of argR,a negative transcriptional regulator of arginine metabolism in M.smeg,enhances bacterial virulence and inhibits mitochondrial function in alveolar epithelial cells.