Down-regulation Of FABP5 By The Infection Of Mycobacterium Tuberculosis Inhibits Cardiolipin Synthesis By Which Affects Mitochondrial Functions In Macrophages

Tuberculosis(TB)is a chronic immune disease caused by Mycobacterium tuberculosis(Mtb)infection,which seriously endangers human life quality and health.As a highly successful pathogen,Mtb has evolved unique immune escape mechanisms,which are widely attributed to its ability to adapt to and manipulate in the intracellular environment of macrophage(MΦ).Mitochondria play a crucial role in various physiological processes that are essential for cellular homeostasis,including ATP synthesis and bioenergetic metabolism.Therefore,mitochondria are major cell organelles which are affected by Mtb infection thus introducing various physiological responses of the host to Mtb infection.Mtb has evolved a series of strategies to alter mitochondrial functions to promote its immune escape,support its survival,replication,and dissemination.mitochondrial metabolism closely corresponds to the activation and functionality of immune cells,mitochondrial fatty acid metabolism is critical for regulating cellular fatty acid storage,inflammatory responses,and lipid homeostasis,and it plays an important role in resisting pathogen infection.Mitochondria regulate their own lipid metabolism based on the signals introduced by Mtb infection or their environment changes to introduce the activation and functionality of MΦ.However,it is currently unclear whether Mtb infection affects mitochondrial function by regulating mitochondrial fatty acid metabolism.Therefore,this study explores whether Mtb infection regulates mitochondrial fatty acid metabolism to orient the differentiation of MΦ and affect their functions,which will further expand our understanding of the pathophysiological mechanisms of TB and provide potential targets for the development of effective anti-TB drug.Mtb infection changes mitochondrial morphology and physiological functionIn order to investigate the effect of Mtb infection on mitochondria,we used THP-1monocyte line and differentiated it into MΦ by PMA treatment,then MΦ was infected with Mtb,and the morphology of mitochondria inside Mtb infected MΦ was observed under transmission electron microscopy.The results showed that the morphology of mitochondria changed from filament to puncta compared to that in MTB uninfected MΦ.Furthermore,we observed the changes of quantity of intracellular ATP in MΦ after Mtb infection,and it was found that ATP decreased significantly after Mtb infection.In addition,we extracted mitochondria from Mtb infected MΦ,and Western blot was applied to detect if the expression of mitochondrial respiratory chain complexes was affected by Mtb infection,ROS-specific probe DCFH-DA was used to detect the changes in the quantity of reactive oxygen species(ROS)in the Mtb infected cells.We also used membrane-permeable fluorescent probe Calcein AM to detect the permeability transition pore(MPTP)of the mitochondria in MΦ.The results of above experiments showed that after Mtb infection,the expression of mitochondrial respiratory chain complexes was downregulated,ROS increased,MPTP increased,and mitochondrial respiration decreased.Effect of Mtb infection on mitochondrial lipid metabolism in macrophagesWe observed that Mtb infection significantly affected mitochondrial morphology and functions,but it is not yet known whether the changes were introduced by Mtb infection which might regulate lipid metabolism in mitochondria.Cardiolipin(CL)is a unique phospholipid found only in the inner membrane of mitochondria and is essential for mitochondrial function,bioenergetics,and the formation of respiratory super-complexes.We detected the CL quantity in Mtb infected MΦs,and found that the CL significantly decreased in Mtb infected MΦ in comparison with that in Mtb uninfected MΦ.We then examined the expression of genes involved in CL synthesis(CPT2,HADHA,HADHB,ACAT1,ACOT1,ACOT2,ACAD8,ELOVE6,SCD1,SCD6,FADS1,FADS2)and found that Mtb infection led to a significant decrease in the expression of these genes at m RNA level.Effect of Mtb infection on lipid metabolism of macrophage mitochondria by downregulating FABP5 The fatty acid binding protein(FABP)family is an important group of proteins that control intracellular lipid metabolism.Fatty acid binding protein 5(FABP5)is a member of the FABP family and plays an important role in the transport of fatty acids to organelles such as mitochondria.However,it is unclear whether FABP5 is involved in the regulation of mitochondrial fatty acid metabolism in MΦ during the Mtb infection.In this study,we investigated the changes in FABP5 expression in Mtb(H37Ra)infected MΦ.We found that the expression level of FABP5 was significantly downregulated after H37 Ra infection.To verify whether the downregulation of FABP5 affects mitochondrial lipid metabolism,we established a THP-1 cell line with stable knock-down of FABP5 using a lentiviral system expressing FABP5 specific sh RNA.We measured the expression of fatty acid metabolismrelated genes,cardiolipin synthesis-related genes,cardiolipin,mitochondrial respiratory efficiency,ROS,and ATP in the FABP5 knockdown cells.The results showed that Mtb infection inhibited fatty acid metabolism-related genes and cardiolipin synthesis in mitochondria partially due to the downregulation of FABP5 which caused by Mtb infection.However,there may be other regulatory mechanisms involved,which is worthy of further experimental investigation.In conclusion,our study results indicate that Mtb infection downregulates FABP5 expression which inhibits mitochondrial cardiolipin synthesis,thereby affecting mitochondrial morphology and function.