Screening And Pathogenesis Investigation Of PE/PPE Proteins Associated With The Intracellular Survival Of Mycobacterium Tuberculosis

Mycobacterium tuberculosis(M.tuberculosis,Mtb)is the causative agent of human and animal tuberculosis(TB).It has always accompanied the evolutionary history of animals and humans and has never been eliminated,representing one of the most successful infectious agents.It is now known to have accompanied mankind since we emerged from Africa approximately50,000 years ago and remained one of the top 10 causes of death worldwide.The most important reason why M.tuberculosis is difficult to scavenge lies in its complex structure and composition,special secretion system and multiple mechanisms of escaping host immune responses.Type?secretion system of M.tuberculosis is the latest type of secretion system discovered,and is still one of the most unknown secretion systems to the world.Though little is known about the details of the mechanism involved in the processing and substrate transportation of type?secretion system,some researches have predicted that PE/PPE family proteins in Mtb are most likely to be secreted by the special secretion system.There are many members in Mycobacterium genus,whereas PE/PPE family has been merely found in pathogenic species such as M.tuberculosis,Mycobacterium africanum,Mycobacterium bovis,indicating that PE/PPE proteins might be closely related to the pathogenicity of Mtb.PE/PPE family,which builds up 10%of the genome encoding capacity,consists of PE and PPE subfamilies,exclusively existing in Mtb.The characteristic of PE subfamily is the conserved P(Pro)-E(Glu)sequence in N terminal,sequence length varies,with no specificity in C terminal.PPE subfamily contains conserved P(Pro)-P(Pro)-E(Glu)sequence in N terminal,and most members of PPE contain typical motifs in C terminal,while some of it have none.The conservative characteristic sequences of PE/PPE family proteins might be associated with the subcellular localization and secretion of the PE/PPE proteins in the virulence mycobacteria,and the diverse variable range of PE/PPE family proteins might be related to the mycobacteria adapt to the host environment and infection host causes disease.Macrophage is an important defending barrier of native immune system,distributed throughout various parts of the body,and capable of capturing and removing invasive pathogens in various ways.M.tuberculosis is intracellular parasitic pathogens,which is mainly living in macrophages and capable of survive and replicate within macrophages without being killed.During infection of macrophage,expression level of PE/PPE proteins displays significant difference compared to in vitro cultivation.Several studies proved that PE/PPE family members are involved in the structure of cell wall of mycobacteria,and as bacterial surface proteins,they usually play a key role in the interaction with host cells.Based on the above analysis,the author concludes that PE/PPE proteins might be correlated with the survival in macrophages and the immune escape of mycobacteria.This study confirmed that PE17,a member of PE/PPE family,can enhance the intracellular survival capacity of M.smegmatis in macrophage,and increase the pathogenicity of M.smegmatis to macrophage and mice.First of all,the 156 of M.tuberculosis H37Rv PE/PPE family overexpressing plasmids were constructed by using the shuttle vector pMVTC-C.These plasmids were electroporated into M.smegmatis mc~2155 strains(MS)one by one,establishing the library contains 144 strains of recombinant M.smegmatis.Based on mice peritoneal macrophage infection model,recombinant M.smegmatis library was screened,and 6recombinant M.smegmatis overexpressing RS23(Rv0124),RS48(Rv2853),PE17(Rv1646),PE30(Rv1651c),PE35(Rv3872)and PPE8(Rv0355c)were identified to remarkably enhance intracellular survival.Then PE17 was chosen for more investigation.To investigate the subcellular location of PE17 protein in mycobacteria,pAIC-PE17-MS strain of overexpressing PE17 protein was constructed.Differential centrifugation was used to separate pAIC-PE17-MS strain subcellular fractions of culture filtrate(CF),whole cell lysates(WCL),cell wall(CW),cell membrane(CM)and cytosol(Cy).The Western Blotting results showed that the PE17protein bands that exhibited strong intensity were recognized in the cell wall(CW)and the cytosol(Cy)fractions.The pMV262-PE17-MS strain of the expressed PE17 protein was also constructed for studying the effects of PE17 protein on the morphology and vivo/vitro infection of mycobacteria.The results showed that the expression of PE17 protein did not affect the colonial morphology and the cell wall structure of mycobacteria.Results from mice peritoneal macrophage infection model showed that PE17 can inhibit secretion of inflammatory factor of macrophage,and cause macrophage necrosis.Intravenous infection confirmed that PE17 can prolong the survival period of M.smegmatis in mice,and enhance the damage to organs of infected mice.Meanwhile,the continuing high level of IFN-?and IL-1?indicated that the bacteria are not easy to eliminate,partly revealing the reason for more damage to organs by recombinant M.smegmatis overexpressing PE17.From a total of 144 M.smegmatis transformants harbouring PE/PPE family genes,6 strains that can significantly promote survival in macrophage were screened.The contribution of M.tuberculosis PE17 protein to the pathogenicity of M.tuberculosis was first verified by cellular infection model and in vivo infection model in mice.This study identified the new virulence factor of M.tuberculosis,which provides a new candidate target for the development of drugs to treat tuberculosis,and adds a new theoretical basis for the prevention and control of tuberculosis.