类鼻疽菌通过诱导线粒体自噬促进其胞内存活的研究

Burkholderia pseudomallei promotes its intracellular survival by inducing host mitophagy

目的探讨类鼻疽菌感染人单核细胞THP-1后诱导线粒体自噬对其胞内存活的影响。方法类鼻疽菌BPC006感染THP-1细胞后2 h,以未感染组作为对照,通过透射电镜和激光共聚焦显微镜观察线粒体结构变化,流式细胞仪检测类鼻疽菌感染后不同时间点线粒体膜电位的变化,全波长酶标仪检测感染后各时间点ATP含量变化;同时,通过Western blot和实时定量聚合酶链反应(quantitative real time polymerase chain reaction,Q-PCR)分别检测感染后各时间点线粒体蛋白HSP60、TIM23和线粒体DNA mtCO1的变化,激光共聚焦显微镜观察线粒体与自噬小体共定位的情况;最后,分别通过线粒体自噬诱导剂CCCP和抑制剂Mdivi-1预处理后,检测各感染时间点胞内细菌载量的变化。结果类鼻疽菌感染THP-1细胞后2 h观察到宿主细胞线粒体超微结构受损,线粒体膜电位和ATP含量在各感染时间点均降低(P<0.01),且呈时间递减趋势,提示类鼻疽菌感染后线粒体结构和功能受损;Western blot检测显示HSP60和TIM23蛋白水平随感染时间延长而减少,同时Q-PCR检测也显示mtCO1基因水平随感染时间延长而减少,免疫荧光分析显示线粒体蛋白TIM23与自噬小体GFP-LC3共定位明显增多(P<0.01),提示类鼻疽菌感染后诱导了线粒体自噬的发生;使用线粒体自噬诱导剂CCCP以及抑制剂Mdivi-1处理细胞后,与对照组比较,激活线粒体自噬类鼻疽菌胞内细菌载量增多(P<0.05),而抑制线粒体自噬其胞内细菌载量降低(P<0.05)。结论类鼻疽菌感染THP-1细胞后引起宿主细胞线粒体结构和功能受损进而诱导线粒体自噬的发生,从而有利于类鼻疽菌的胞内存活。

Objective To investigate the effect of mitophagy induced by Burkholderia pseudomallei(B.pseudomallei)infection on the intracellular survival in human monocyte THP-1 cells.Methods THP-1 cells were infected with B.pseudomallei BPC006 for 2 h,and the cells without infection were taken as the control group.The structural changes of mitochondria were observed by transmission electron microscopy and confocal laser microscopy,mitochondria membrane potential(MMP)were detected by flow cytometry,and amount of ATP was detected by full-wave length microplate reader at different time points.The expression levels of mitochondria proteins HSP60 and TIM23 were detected with Western blotting,and that of mitochondria DNA(mtCO1)was detected with quantitative real time polymerase chain reaction(Q-PCR).Confocal laser microscopy was used to observe the co-localization of mitochondria and autophagosome after infection.After the pretreatment with mitophagy activator CCCP or inhibitor Mdivi-1,its intracellular survival was examined by counting the viable bacteria in the cells.Results B.pseudomallei infection resulted in changes in mitochondria ultrastructure and decreases in MMP level and ATP content in time-dependent manners(P<0.01),suggesting that mitochondria were dysfunctional after the infection in THP-1 cells.The results of Western blotting and Q-PCR assay respectively showed the protein levels of HSP60 and TIM23 and the mRNA level of mtCO1 were decreased after the infection.Immunofluorescence assay showed increased colocalization of mitochondrial protein TIM23 and autophagosome GFP-LC3 after infection(P<0.01).These results suggest B.pseudomallei infection induced mitophagy in THP-1 cells.After pretreatment with CCCP or Mdivi-1,the results of bacterial colony forming units showed that activation of mitophagy by CCCP significantly increased intracellular bacterial load(P<0.05),while inhibition of mitophagy by Mdivi-1 decreased the intracellular replication(P<0.05).Conclusion B.pseudomallei infection can lead to mitochondria structural and functional damage of host cells and then induce mitophagy,and thus help the intracellular survival of the bacteriuum.