Burkholderia Pseudomallei Induces Host Mitophagy To Promote Its Intracellular Survival

Burkholderia pseudomallei(B.pseudomallei),a kind of Gram-negative opportunistic bacterium,is the causative agent of melioidosis.Melioidosis is a tropical medical zoonosis,which is mainly prevalent in northern Australia,southeast of Asia and other regions including Guangdong,Hainan,Hong Kong,Taiwan.The disease has protean manifestations ranging from limited infection to diffuse visceral abscess,acute pneumonia,of which the mortality rate can be as high as 40% companying with sepsis.However,there are no effective antibacterial medicines and vaccines for melioidosis.In addition,the course of patients can be either quickly or developing to repeated infection,which caused ill-treatment for the disease.Therefore,exploring the underlying pathogenic mechanism will provide new approaches for the prevention and controlling of melioidosis.In order to maintain cell homeostasis to control the organelle quality and function,mitophagy,a selective form of autophagy,can eliminate damaged mitochondria and reactive oxygen species which are generated by damaged mitochondria.In recent years,growing evidence has found that pathogens could actively induce mitophagy to manipulate host’s anti-infection response to achieve the goal of long-term infection.As a facultative intracellular bacterium,B.pseudomallei infection can induce mitophagy in host cells,and mitophagy plays an important role in facilitating its infection and colonization,but the detailed mechanisms are still unclear.This research focuses on the molecular mechanism of mitophagy induced by B.pseudomallei to promote its intracellular survival in host cells,and aims to highlight that B.pseudomallei infection manipulates host mitophagy to eliminate mtROS(mitochondrial reactive oxygen species)for their own benefits.These insights provide a more in-depth understanding of the host-pathogen relationship and suggest potential therapeutic strategies to treat melioidosis caused by B.pseudomallei.【Objectives】(1)To explore the biological significance of mitophagy induced by B.pseudomallei in host cells.(2)To reveal the molecular mechanisms of mitophagy induced by B.pseudomallei from the pathogen-host interaction interface.【Methods】(1)In B.pseudomallei infected RAW264.7 and THP-1 cell models,transmission electron microscope and laser confocal microscope were used to observe the ultrastructure of mitochondria.Flow cytometry and full-wavelength microplate reader were used to detect the function of mitochondria.Q-PCR,Western-blot,electron microscope,and laser confocal microscope were used to observe mitophagy induced by B.pseudomallei.Intracellular survival assay and flow cytometry were used to detect the intracellular proliferation and mitochondrial reactive oxygen production of cells,respectively.(2)SiRNA,q-PCR,Western-blot,and flow cytometry were used to detect and analyze the relationship between classic autophagy pathways and mitophagy induced by B.pseudomallei and its impact on intracellular survival.The changes of endoplasmic reticulum stress signaling pathways before and after B.pseudomallei infection were analyzed by deep sequencing expression.Bioinformatic analysis were used to predict the key molecules of mitophagy and knockout strains of the key molecule by homologous recombination technology were constructed.QRT-PCR and laser confocal microscope were used to observe the changes of mitophagy after infection.【Results】(1)The mitochondria of the host cell underwent obvious structural and functional damage after infection by B.pseudomallei.The numbers of mitochondria enclosed with double-membrane vesicles and autophagosomes significantly increased after the infection of macrophages by B.pseudomallei,whereas mtDNA,HSP60 and TIM23 were down-regulated.(2)B.pseudomallei promotes the clearance of mitochondrial reactive oxygen species in host cells by inducing mitophagy.Mitophagy promotes B.pseudomallei intracellular loads.Combined with these two results,B.pseudomallei infection promotes its intracellular loads by inducing mitophagy to reduce host intracellular mt ROS.(3)The host cell mitophagy induced by B.pseudomallei relies on the classical autophagy pathway.Inhibition of host cell autophagy key proteins ATG7 and Beclin 1 expression,respectively,can interfere with the process of mitophagy induced by B.pseudomallei infection,which results in the accumulation of damaged mitochondria in the host cell.Meanwhile,bacterial CFU assay indicated that stimulation of mitophagy resulted in an increase in the bacterial load of B.pseudomallei,but this effect was also abolished when ATG7 and Beclin 1 were knockdown.(4)The screening of host differentially expressed genes indicate the expression of endoplasmic reticulum stress signaling pathways increased after B.pseudomallei infection,and the endoplasmic reticulum stress of host cells was activated.(5)Bioinformatics screening reveals that the type Ⅲ secretion system tip protein BipD is a key candidate target for B.pseudomallei to induce mitophagy.The mtDNA clearance ability is inhibited and the ability to induce mitophagy is obvious suppressed after the ΔbipD knockout strain after infected with RAW264.7 cells.【Main conclusions】(1)B.pseudomallei infection induces host cell mitophagy,causes mitochondrial dysfunction,and promotes intracellular proliferation by inducing mitophagy to eliminate mitochondrial reactive oxygen species in the host cell;(2)The endoplasmic reticulum stress activation of the host cells infected by B.pseudomallei may be an upstream regulatory factor of mitophagy,and its Ⅲ type secretion system tip protein BipD may mediate the host cell mitophagy induced by B.pseudomallei.【Significance】In this study,we report that host mitophagy induced by B.pseudomallei and its biological significance in intracellular proliferation through the elimination of mtROS.From the pathogen-host interaction interface,we discuss that the molecule mechanisms of B.pseudomallei inducing mitophagy.These results reveal the regulatory role of classical autophagy and endoplasmic reticulum stress involved in the mitophagy induced by B.pseudomallei.BipD,as a type Ⅲ secretion system tip protein,was identified as a potential factor for regulating mitophagy induced by B.pseudomallei infection.Taken together,this study provides a theoretical and experimental basis for the molecular mechanisms of B.pseudomallei and formulates new strategies for the prevention and treatment of melioidosis.