Mycobacterium Tuberculosis PPE10(Rv0442c) Alters Host Cell Apoptosis And Cytokines Profile Via Linear Ubiquitin Chain Assembly Complex HOIP-NF-?B Signaling Axis

Tuberculosis caused by Mycobacterium tuberculosis infection remains one of the top ten causes of deaths worldwide.The success of mycobacteria as an intracellular pathogen within macrophages is in part due to its ability to actively manipulate host signaling.The genome of M.tuberculosis contains many virulence factors.During to infection and residing in the host cell,M.tuberculosis applies many virulence strategies to subvert host cell immune responses,pathogen virulence determinants play significant roles in host-pathogen interaction and determination of infection outcome.PE/PPE protein family has been identified as a source of virulence and antigenic variation.Many PE/PPE proteins were interacting with host cell receptors and interfere in cell signaling transduction to modulate the immune response.M.tuberculosis PPE10(Rv0442c)is one of the highly repeated PPE proteins family,has been shown to interfere in capsule integrity,increase the virulence of M.marinum and reduce the recruitment of ubiquitin.Moreover,transpose insertion in PPE10 loci of BCG reduces its ability to inhibits phagosome acidification,also,PPE10 can allow the observation of T-cell responses in mice when immunized with BCG contain PPE10 antigen.To explore the role of PPE10 in pathogens-host interaction,PPE10 encoding gene Rv0442 c was heterologously expressed in the nonpathogenic M.smegmatis strain.We found that overexpression of Rv0442c(PPE10)altered the bacterial cell surface properties,colony morphology,and biofilm formation.PPE10 increases the resistance of M.smegmatis to stress conditions such as low pH and oxidative stresses(diamide)in comparison to M.smegmatis harboring vector only,this resistance to stress conditions accompanied by higher survival rate within the host macrophages.Furthermore,PPE10 increased uptake of Ethidium bromide and Nile red,which indicate the increase of cell wall permeability in Ms_PPE10 compared to Ms_vec.PPE10 significantly decreases the transcription levels of host's cell proinflammatory cytokine such as IL-1?,IL-6,IL-12p40 and TNF-?,as well as highly increasing transcription levels of anti-inflammatory cytokines IL-10.Also,Ms_PPE10 modulates host cell apoptosis that via down-regulation the expression of caspase-3,7,8,and cleaved caspase-3.Moreover,host cell death analysis via fluorescent microscope and flow cytometry indicates a significant reduction in earlier apoptosis in Ms_PPE10 infected cell compared to Ms_vec infected cell.Cell signaling analysis indicates that,Ms_PPE10 down-regulates transcription levels of pro-inflammatory cytokines and modulates host cell apoptosis through Linear Ubiquitin Chain Assembly Complex(LUBAC)HOIP-NF-?B signaling axis as Ms_PPE10 significantly decreased the transcription levels of LUBAC subunits,furthermore,pre-infection treatments of NF-?B signaling axis through its general TPCK inhibitor led to relieve the cytokines transcription inhibitory effects of Ms_PPE10 in host cells.We also found That Ms_PPE10 reduces the expression and phosphorylation of p65/RelA NF-?B.In summary,this study revealed novel insights into the mechanism of action of the PPE family.