Burkholderia pseudomallei promotes its intracellular survival by inducing host mitophagy

Abstract

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.

Keywords

• burkholderia pseudomallei
• thp-1 cells
• mitochondria
• mitophagy