Mycobacterium Tuberculosis Mce2E Suppresses Innate Immune Responses And Promotes Tumor Cell Proliferation

Mycobacterium tuberculosis is a facultative intracellular pathogens.Mycobacterium tuberculosis can survive and grow in macrophages.Mce(mammalian cell entry)proteins are a group of secreted and membrane surface adhesion proteins coded by mce operons.It has already been shown that Mycobacterium tuberculosis mce operons could promote the internalization of mycobacteria into host cells,the mce2 operon mu-tant's attenuation in mice was associated with decreased bacterial burden and delayed granuloma formation.But,the specific function of the Mce2E protein is not clear yet.The infection of pathogens and LPS can activate NF-?B(nuclear factor kappa-light-chain-enchancer of activated B cell)and MAPK(mitogen-activated protein kinase)signaling pathway,so as to promote the release of proinflammatory factor,such as TNF?,IL-1?,and to activate the innate immune re-sponses.The LAM of Mycobacterium tuberculosis has similar function.We thus tran-siently transfected Mce2E into HEK293T cells and examined the activation of NF-?B or MAPK pathways using dual-luciferase reporter system.The experimental results showed that Mce2E blocks ERK1/2 pathway activation induced by either RasV12,v-Raf(constitutive active Raf)or MEK1-ED(constitutive active MEK1).Mce2E might block the JNK/p38 signaling induced by RacL61,but not NF-?B induced by TNFa.Next,we examined the effects of M.tuberculosis Mce2E on the activation of ERK pathway,and found that Mce2E might block the ERK1/2 signaling downstream or at the level of MEK1 along the canonical Ras-Raf-MEK-ERK cascade.Using co-immunoprecipitation assay,pull-down assay,we demonstrated that Mce2E directly inter-acted with ERK2,but not MEK1.We futher demonstrated that Mce2E directly interacted with ERK2 in a D motif(module and sequence of eukaryotic cells)-dependent manner,since the AAA mutation of D motif efficiently abolished Mce2E-mediated inhi-bition of ERK1/2 activation.QRT-PCR,ELISA and CFU analysis data indicated that expression of Mce2E in either Mycobacterium smegmatis or M.bovis BCG greatly down-regulated the expression of Tnf and 116 and enhanced bacterial survival in macrophage cells during mycobacterial infection.While conducting the above experiments,we surprisingly noticed that Mce2E can enhance epithelial cell proliferation.In addition,we also found that there were a few proteins involved in cell proliferation and migration among the host-interacting proteins we identified for Mce2E through yeast two-hybrid analysis.Furthermore,there have been some studies suggesting that Mce proteins promotes cell proliferation and migration.Taken together,we hypothesized that Mce2E might promote lung caner development through regulating cell proliferation and migration.We thus infected A549 cells with the Mce2E-overexpressing and Mce2E-knockout strains,and we found that Mce2E was able to promote cell proliferation as indicated by the CCK-8 analysis data.At the same time,we found that Mce2E was able to promote cell migration as shown by the transwell experiment.We further demonstrated that the volum of transplanted tumor infected with the Mce2E-overexpressing strain was smaller than that of the Mce2E.We then confirmed that eEF1A1 plays an important role in the regulation of cell proliferation.Mce2E could stablize eEFlA1 expression as demonstrated in the CHX(inhibiting protein synthesis)experiment.Mce2E can inhibit the K48-linked polyubiquitin chain(contain substitution of arginine for all lysine residues except the lysine at position 48)formation of eEF1A1 as shown in a vivo ubiquitination assay.In conclusion,our study not only characterizes the specific mechanisms by which Mycobacterium tuberculosis Mce2E impedes host innate immune responses,but also demonstrates that Mce2E promotes cell proliferation and migration,wich provides a new perspective for the research of chronic infection and inflammation-associated cancers.