Mycobacterium Tuberculosis PE＿PGRS1 Promotes Intracellular Survival Of Mycobacteria Via Inhibiting Calcium-mediated PERK-eIF2α-ATF4 Pathway

Tuberculosis（TB）is a type of infectious disease that respiratory droplets as the primary transmission route,and its pathogenic pathogen is Mycobacterium tuberculosis（M.tuberculosis）.There are around 10 million new latent infections in 2019,and around1.4 million deaths.The incidence of TB has dropped off,but the rate of decline is not obvious,and it is still one of the main reasons in the death of global infectious diseases.Therefore,it has become imperative to in-depth exploration of the infection mechanism of M.tuberculosis,to screen new drug targets and improve the efficacy of vaccines.The PE/PPE family is an important effector in M.tuberculosis,accounting for about10%coding capacity of the entire genome.According to the conservative sequence of Pro-Glu and Pro-Pro-Glu at the N-terminal,the family can be subset into PE subfamily and PPE subfamily.Among them,the PE＿PGRS belongs to the PE subfamily,which is characterized by the presence of a repetitive sequence formed by the concatenation of Gly-Gly-Ala/Gly-Gly-Asn domains at the C-terminal region.The unique PE domain and PGRS domain serves multiple functions,such as antigens presentation,T cells recruitment,interaction with macrophages to trigger a series of immune responses including apoptosis,necrosis,and autophagy.Endoplasmic reticulum（ER）is a membrane vesicle network structure connecting the nuclear membrane and cytoplasm.Its main function is to synthesize proteins and carbohydrates,and process the synthesized proteins.In addition,the other function of ER is that as a central intracellular Ca²⁺reservoir to maintain the Ca²⁺homeostasis.As a pivotal intracellular signaling molecule,Ca²⁺can impact almost every aspect of cellular processes,including the activation of macrophages.When cells are stimulated,the ER will initiate stress response to reduce protein misfolding and balance Ca²⁺levels inside and outside the lumen.Endoplasmic reticulum stress（ERS）in mammals is mainly activated via three ER transmembrane proteins:PERK,IRE1αand ATF6.Moreover,pathogens can regulate apoptosis,autophagy,and other physiological processes by activating the effector in ERS.In this study,we used the Mycolicibacterium smegmatis（M.smegmatis）as a model bacterium to perform functional characterization of PE＿PGRS family member PE＿PGRS1.Previous studies predicted that PE＿PGRS1 was a virulence-related factor,but the function is unknown.After analyzing the amino acid sequence,it is found that PE＿PGRS1 contained 7 Ca²⁺binding domains of GGx Gx D/Nx Ux（x is any amino acid）.Subsequently,we heterologously expressed PE＿PGRS1 in M.smegmatis,and infected macrophages with recombinant strain Ms＿PE＿PGRS1.The experimental results showed that PE＿PGRS1 reduced intracellular Ca²⁺levels,accompanied by the down-regulation of calcium-related proteins Calnexin and Calpain1.The ERS-associated proteins CHOP,Bip,p-PERK,p-e IF2αand ATF4 all down-regulated expressed to varying degree after the Ms＿PE＿PGRS1 infected for 24 h and 48 h.Meanwhile,PE＿PGRS1 could increase the level of anti-apoptotic Bcl-2,decrease the level of pro-apoptotic Bax,and inhibit the Caspase-3/Caspase-9 to reduce macrophage apoptosis.It is remarkably that two splicing variation of Bax/Bcl-2,Baxβand Bcl-2α,were differentially expressed after infection with Ms＿PE＿PGRS1,and may be involved in the regulation of apoptosis.Further,the transcription levels of inflammatory factors IL-1β,IL-6 and IL-12p40 were also decrease.In summary,we have identified PE＿PGRS1 as a calcium-associated protein,which can inhibit the PERK-e IF2α-ATF4 axis signaling by affecting intracellular Ca²⁺levels,and reduce cell apoptosis and promote the survival of M.smegmatis within macrophage.