The Underlying Mechanism Of Mycobacterium Tuberculosis Rv0426c In Stress Response And Immune Evasion

Tuberculosis?TB?,caused by Mycobacterium tuberculosis?Mtb?remains one of the major threats to global public health with nearly 10 million new tuberculosis cases and 1.3 million TB deaths annually.Importantly,estimated one-third of the world's population have been latently infected with M.tuberculosis.To combat TB and its agent,the WHO launched The End TB Strategy,which aims to slash the suffering and cost of TB by 2035.However,the emergence of multidrug-resistant tuberculosis and HIV/Mtb co-infection further aggravates the prevention and control of TB.Macrophages play a central role in defense against M.tuberculosis infection.As the first line of host innate immune system,macrophages can eliminate intracellular Mtb through phagolysosomal fusion,production of reactive oxygen intermediates and the nitrogen oxides,secretion of cytokines as well as cell apoptosis and autophagy.Furthermore,macrophages are important antigen presenting cell which can present the Mtb antigen to the T cells,and active the adaptive immunity.However,as one of the most successful intracellular pathogens,Mtb can escape the host immune responses by a variety of unique mechanisms and survive for long periods inside the human macrophages.The capacity of Mtb to subvert immune responses of macrophages largely depends on the secretion of diverse effectors.These effectors are reported to use multiple strategies to impair the function of macrophages and facilitate the survival of Mtb in the host environment,including inhibition of phagolysosome fusion,blockage of phagosome acidification,modulation of host cytokine responses,and manipulation of host cell death programs.Moreover,some effectors could manipulate the inflammasome and subvert antigen presentation processes to inhibit the activation of adaptive immunity.Although more than 100 effectors have been identified up to now,it is not enough to comprehensively elucidate the virulence and pathogenic mechanism of M.tuberculosis.Therefore,it is an escalating need to identify new effectors involved in host-pathogen interaction.In this research,we concentrated on the function of a predicted M.tuberculosis effector Rv0426c.Multiple sequence alignment showed that Rv0426c is highly conserved among virulent mycobacteria and absent in avirulent strains like M.smegmatis mc²155,implying that Rv0426c might be a virulence factor.To explore the underlying function of Rv0426c,we constructed recombinant M.smegmatis strains using pNIT-Myc vector.We found that Rv0426c was a cell envelope-associated protein and overexpression of Rv0426c decreased the resistance of the recombinant strains to SDS,lysozyme,H₂O₂ and acidic stress?pH=3?,which might be the results of increased cell wall permeability as the Ms_Rv0426c accumulated more levels of ethidium bromide than Ms_vec.To further investigate the roles of Rv0426c in host-pathogen interaction,we detected the survival rates of Ms_Rv0426c and Ms_vec in PMA-differentiated THP-1 cells.The results showed that Rv0426c enhanced the early survival of mycobacteria within macrophages.Although overexpression of Rv0426c had no effect on NO production and cell necrosis,we found that Ms_Rv0426c downregulated the mRNA level of proinflammatory factors IL-1?,IL-6,IL-12p40 and TNF-?.Furthermore,fluorescence microscopy and flow cytometry showed that Ms_Rv0426c induced attenuated apoptosis.In conclusion,our research identified that Rv0426c is a novel effector involved in host-pathogen interaction.Overexpression of Rv0426c in M.smegmatis resulted in attenuated apoptosis and lower level of IL-1?,IL-6,IL-12p40,and TNF-?,which benefit the replication of mycobacteria in PMA-differentiated THP-1 cells by supplying a favorable niche.These results might contribute to further understanding the pathogenesis of M.tuberculosis.