Rv3091,An Extracellular Patatin-like Phospholipase In Mycobacterium Tuberculosis,prolongs Intracellular Survival By Mediating Phagosomal Escape

Tuberculosis is a severe consumptive disease that is caused by infection with Mycobacterium tuberculosis(Mtb).So far it has become the second leading cause of death after AIDS.Due to the emergence of drug-resistant Mtb,its co-infection with HIV,and the lack of effective vaccines to prevent tuberculosis,the prevention and control of the disease is in a great dilemma.The main reason is that Mtb is an intracellular pathogen with a complex structure,unique secretory system,and various immune evasion mechanisms.It can escape the killing of host immune defense and cause long-term existence in host cells,thereby forming latent infection of chronic tuberculosis.Therefore,it is urgent to clarify the Immune escape and persistent infection mechanism of tuberculosis and find effective methods to combat Mtb.Mtb has a special lipid structure cell envelope that is different from Gram-positive bacteria and Gram-negative bacteria.These lipids are essential for the viability and pathogenicity of Mtb.Therefore,enzymes(esterase,lipase,phospholipase,etc.)related to lipid anabolism are considered to be the key virulence factors of Mtb.The free fatty acids hydrolyzed by phospholipases provide an energy source for Mtb to grow and replicate in cells.Free fatty acids provide an energy source for Mtb to grow and replicate in host cells.Therefore,Mtb phospholipase may play an important role in destroying the membrane structure of host cells(eg,phagosome membrane structure),especially in the adaptation and survival of Mtb macrophages.However,the current research on Mtb phospholipase is only limited to activity identification and cytotoxicity analysis,and its mechanism has not been studied in depth,and whether it contributes to the immune escape and persistent infection of Mtb has been found few reports.In this study,Mtb genome was predicted and analyzed by bioinformatics method of searching phospholipase conserved amino acid residues.The Mtb genome contains Rv3091 has been described based on the occurrence of four conserved sequence blocks.Here,the rv3091 gene of Mtb H37Rv,which has been previously described as a putative Patatin-like protein superfamily gene in NCBI(National Center for Biotechnology Information).We cloned and expressed the rv3091 gene from Mtb H37Rv genome in M.smegmatis.We named the recombinant M.smegmatis overexpressing Rv3091 protein as Ms3091.And subsequently conducted protein purification and characterization.Using nitrophenyl ester,dioleoylphosphatidylcholine(DOPC)dioleoylphosphatidylglycerol(DOPC)as a substrate,with or without inhibitors and eukaryotic cofactors in different temperatures the spectrophotometric method was used to verify that it possesses phospholipase A1,phospholipase A2 and lipase activity.We confirm the putative active site residues,namely,Ser214 and Asp407,using site directed mutagenesis,protein expression,purification and activity analysis.The Rv3091 is an extracellular protein that mainly found on the mycobacterial cell wall by polyclonal antibodies combined with Western blotting and mass spectrometry detection.And alters the colony morphology of M.smegmatis.The presence of Rv3091 enhances the intracellular survival capability of M.smegmatis in murine peritoneal macrophages.Furthermore,through transmission electron microscope observation,Rv3091 promoted the escape of M.smegmatis from phagosome and increased the damage of macrophages.Additionally,Rv3091 polyclonal antibody can block Rv3091 activity,thereby reducing the co-localization of recombinant M.smegmatis and lysosomal-associated membrane protein 1(LAMP-1)by antibody blocking,immunofluorescence and extracellular PLA activity analysis.It is clear that the enzymatic activity of Rv3091 plays an important role in promoting the escape of M.smegmatis from macrophage phagosomes.Moreover,Rv3091 significantly increased the survival of M.smegmatis and aggravated lesions in C57BL/6J murine lungs in vivo.Taken together,our results indicate that Rv3091 as an extracellular Patatin-like phospholipase that is critical to the pathogenicity of mycobacterium as it allows mycobacterium to utilize phospholipids for its growth and provides resistance to phagosome killing,resulting in its enhanced intracellular survival.To lay a foundation for the prevention and control of tuberculosis and the development of new anti-tuberculosis agents.