Mycobacterium Tuberculosis H37Rv Lipolytic Enzyme Rv1076(LipU) Promotes Bacteria Survival And Host Cell Cytokines Expression

Tuberculosis(TB),a serious threat to human health,is a zoonosis caused by Mycobacterium tuberculosis(Mtb)complexand a global public health problem.According to the World Health Organization(WHO)report 2018,the resurgence of tuberculosis in recent years,together with AIDS and malaria,is serious concern.About one-third of the world's population is latently infected with M.tuberculosis.There are millions of new infections each year and the death toll is as high as millions.China is the third most burdened tuberculosis countries.The occurrence and widespread of multi-drug resistant(MDR),extensively drug resistant(XDR),and HIV co-infection haveexacerbated the difficulty in TB control.Different from other bacteria,mycobacterial cell wall contains a large number of lipids,accounting for about 30 ~ 40% of the dry weight of cells.However,their biological functions,synthetic pathways,and intricate structures are still mysteries.Studies have shown that M.tuberculosis can accumulate large amounts of liposomes(including lipids from host cells)during infection and store them in the form of triacylglycerols(TAGs)to maintain thesurvival.M.tuberculosis in the non-replicating stage can persist in host cells for several decades and is particularly difficult to eliminate.During reactivation,TAG is used as energy to stimulate the cells replication,resulting in active infection.There are also a large number of genes in the M.tuberculosis genome that encode enzymes(about 250)related to lipid metabolism.Among them,lipolytic enzymes,which hydrolyze lipids stored by bacteria,provide energy for M.tuberculosis survival.These enzymes are mainly divided into five major families: Lip family,phospholipase family,cutinase family,?-lactamase family and PE/PPE family.They play a key role in the life cycle,retention and virulence of mycobacteria.An in-depth study of the properties and functions of these enzymes will help develop new anti-tuberculosis drugs or diagnostic reagents.M.tuberculosis H37 Rv Rv1076(Lip U),a member of lipase family,is homologous to the human Hormone Sensitive Lipase(HSL)and has been annotated as a putative lipase/esterasebased on the presence of conserved motif‘GXSXG'.To define the enzymatic characteristics of Rv1076,the gene was cloned,and expressed in Escherichia coli.The protein was purified for enzymatic characterization.Lip U showed high specific activity for the hydrolysis of short carbon chain substrates with optimal activity at 40 ? /p H 8.0 and stability at low temperature and near-neutral p H.The specific activity,Km and Vmax of Lip U was calculated to 176.7U/mg,1.73?M and62.24?M/min respectively.Ionic detergents can inhibitits activity.The active-site residues of Lip U were determined to be Ser140,Asp244 and His269 by site-directed mutagenesis.The upregulation of M.tuberculosis Rv1076 under nutritive stress implicates a role in starvation.The knockout of Lip U was detrimental to the growth of Mycobacterium smegmatis under nutrient pressure conditions,and resulted in a decrease in the ability of M.smegmatis to use short-chain fatty acids.In addition,we also found that Lip U facilitated the secretion of cytokines TNF-? and IL-12,indicating that it is involved in the host immune response and has a good immunogenicity.These studies detailed the characteristics and functions of M.tuberculosis Lip U,and can improve our understanding of the Lip family lipases.New anti-tuberculosis drugs and diagnostic reagents can be inspired by the study.