Burkholderia Pseudomallei Inhibits Lipid Metabolism To Benefit Its Persistent Intracellular Infection In Lung Epithelial Cells Through Lipophagy Suppression

Burkholderia pseudomallei,a gram-negative pathogen,is the causative agent of melioidosis,which is endemic in Southeast Asia,Northern Australia and other tropical regions,such as Hainan,Guangdong,Hong Kong.The clinical manifestations of melioidosis are diverse,which from subclinical infections,local abscesses to pneumonia,acute sepsis,and the mortality can be up to 40%.Because melioidosis carries a high fatality rate and strong drug resistance,Burkholderia pseudomallei is classified as category I potential bioterrorism agent by the Center for Disease Control and NIAID in 2006.With the development of research,a lot of evidence shows that melioidosis may be a zoonotic disease.In particular,in recent years,some non-epidemic sources have also reported cases of melioidosis,such as South Africa,the Middle East and other places.It has now been well accepted that it is a key component of the pathogenesis of Burkholderia pseudomallei in cooperation between the two sides of the strait,the construction of the “Belt and Road” and South China Sea Strategy,therefore,it is urgent to study the pathogenic mechanism of Burkholderia pseudomallei and find new strategies for clinical treatment.As a kind of intracellular parasitic and harmful bacteria,the persistent infection caused by Burkholderia pseudomallei escaping from host immunity is the key to its prevention and control.Burkholderia pseudomallei are able to rapidly invade various cells in the host and fuse them to create conditions for their own proliferation and dissemination.And these infected cells not only serve as a habitat for Burkholderia pseudomallei,but also provide energy and nutrients.The interaction between host lipid metabolism and pathogenic infection play an important role in the immune process of pathogenic infection.In addition,we foundthat lipid metabolism of the lung epithelial cells A549 is abnormal after the infection of Burkholderia pseudomallei,and the lipids accumulated heavily.What the relationship between the abnormal lipid metabolism and the infection of Burkholderia pseudomallei,what role it plays in escape immunity,and what biological significance it has for the pathogenesis of Burkholderia pseudomallei are still unclear.Lipid metabolism is involved in many important activities of cells,providing various energy changes such as dynamic changes in organelle formation,expansion and contraction,and cell endoplasmic reticulum protein degradation.The intracellular lipids can be transported to lysosomes by autophagosomes to decompose which a process is called lipophagy.As a subcellular process,lipophagy has been reported to be closely related to the pathogenesis and infection of a variety of bacteria,viruses and other microorganisms.Previously,Dr.Li qian found that Burkholderia pseudomallei infected A549 cells could induce the up-regulation of MIR4458,MIR4667-5p and MIR4668-5p,and inhibited the expression of target gene ATG10 in real time and continuously and negatively regulated autophagy,thus affected the immune clearance of host cells and promoted their intracellular survival.However,it remains to be clarified how Burkholderia pseudomallei regulate lipid metabolism through autophagy,thereby affecting the proliferation and replication of bacteria in cells and leading to persistent infection.To explore how autophagy regulates lipid metabolism during the infection of Burkholderia pseudomallei,we first established a model of A549 cells infected with Burkholderia pseudomallei.The changes of lipid metabolism and autophagy levels of A549 cells infected with Burkholderia pseudomallei were detected by transmission electron microscopy,Western Blot and laser confocal microscopy,and we found that lipids accumulated in lung epithelial cells A549,and the key enzyme activities of lipid metabolism decreased.At the same time,the intracellular AKT-mTOR autophagy pathway was significantly inhibited.Second,we further validated the relationship between autophagy and lipid metabolism.Under the conditions of drug activation and inhibition of autophagy or siRNA treatment,the lipid metabolism of A549 cells was observed by Western Blot and laser confocal microscopy.It was found that activation of autophagy significantly promoted intracellular lipid metabolism and the ability of A549 cells to eliminate intracellular bacteria was enhanced by bacterial intracellular counting experiments.Next,to identify how Burkholderia pseudomallei regulate lipid metabolismthrough autophagy,we screened the expression profiles of A549 cells infected by Burkholderia pseudomallei and analysis and prediction of lipid metabolism related genes by Cluster 3.0 and TreeView,and found that NR1D2 mRNA expression was significantly up-regulated.NR1D2(Nuclear receptor subfamily 1 group D member2)is a member of the orphan nuclear hormone receptor superfamily,which belongs to one of the ligand-activated transcription factors.It has been reported that NR1D2 can not only regulate genes involved in metabolic function,such as lipid metabolism and inflammatory response related genes,but also act as an important signal to induce autophagy that it can interact with the key proteins LC3 and Beclin-1 of autophagy signaling pathway to achieve the regulation of autophagy.Therefore,to confirm that NR1D2 mediated autophagy regulates lipid metabolism during Burkholderia pseudomallei infection of A549 cells,we first used RT-PCR and Western Blot to confirm the up-expression of NR1D2 in A549 cells.At the same time,we constructed NR1D2 interference plasmid to block the expression of NR1D2,and we found that the accumulation of lipid droplets in lung epithelial cells A549 was reduced and promoted the metabolism of cellular lipids and the rate of?-oxidation.In addition,we also observed that the key molecules LC3 and Beclin-1of autophagic signaling pathway were enhanced by the down-regulated expression of NR1D2 after Burkholderia pseudomallei infection.Further,under the level of autophagy was inhibited with the interference in NR1D2,we observed that the lipid level of host cells were no significant change compared with the control group(shRNA+DMSO),indicating that NR1D2 mediates autophagy to regulate lipid metabolism in A549 cells infected with Burkholderia pseudomallei.Host lipid metabolism plays an important role in pathogen infection,so we found that activating the lipid metabolism rate of host cells can enhance the ability of host cells clear intracellular clearance of Burkholderia pseudomallei by supplement triacsin C exogenously.Briefly,this study explores the significance of lipid metabolism in the host-pathogen relationship and proposes the mode of “Burkholderia pseudomallei,lipophagy and persistent infection”.Specifically,we found that the underlying mechanism for lipid accumulation after exposure to Burkholderia pseudomallei in A549 cells is through increased NR1D2 expression mediated autophagy inhibition,causing persistent infection.These insights increase our understanding of the lipophagy-pathogen relationship,lay the groundwork for strategies aimed at combating infectious diseases,and may provide useful information for developing potential therapeutic interventions against pathogens.