| Mycobacterium tuberculosis(MTB)is a bacteria that causes zoonotic diseases through aerosol transmission.1.4 million people died of diseases caused by this bacteria in 2019,and it is one of the top ten causes of death in the world.Macrophages are the main effector cells in the immune response of MTB.Macrophages can swallow MTB and inhibit its survival.However,MTB has evolved a range of immune evasion mechanisms that allow it to evade host defense mechanisms by blocking phagosome maturation,mediating inflammatory responses and manipulating host cell death programs through proteins encoded by its own array of virulence genes.In this study,it was found that Rv3435 c is highly conserved in pathogenic mycobacteria,but does not exist in non-pathogenic mycobacteria,so it may be a potential virulence gene.Recombinant mycobacteria expressing Rv3435 c heterologously were constructed by Mycobacterium smegmatis(MS).The function of MTB Rv3435 c gene and its effect on host macrophages were studied.1.Construction and protein expression characterization of Rv3435 c recombinant Mycobacterium smegmatisRv3435c was found to be highly conserved in pathogenic mycobacterium with four transmembrane domains,and the integrated shuttle plasmid p MV361 was used to heterologously express MTB Rv3435 c gene in MS.Identification by The results of PCR and acid-fast staining showed that the recombinant plasmid was successfully electrotransformed into MS.The expression of MTB Rv3435 c protein was verified by the tag antibody,and the protein size was consistent with the prediction.The results showed that Rv3435 c recombinant MS was successfully constructed.2.Phenotype analysis of MTB Rv3435 c geneThe subcellular location indicated that Rv3435 c was a cell wall-related protein.By measuring the growth curve of the recombinant strain,it was found that Rv3435 c significantly inhibited the growth of the recombinant strain.Scanning electron microscopy and 7H10 solid culture revealed that both the individual and colonies of the recombinant strain were changed,and the expression of Rv3435 c was enhanced.The tolerance of the recombinant strain to the unfavorable external environment was analyzed.The analysis of fatty acid differences revealed significant changes in long-chain fatty acid levels in the strains,including a significant increase in linoleic acid content,so we hypothesized that the slower growth of Ms-Rv3435 c was related to the upregulation of linoleic acid levels.3.The effect of MTB Rv3435 c on host macrophagesTHP-1 and RAW264.7 two macrophage cell lines was used to explore the effect of Rv3435 c on host macrophages.By counting the strains in the macrophages,Rv3435 c was found to significantly enhance the survival of recombinant strains in macrophages,although it did not change the invasive ability of recombinant strains.The results of ELISA and RT-q PCR,showed that Rv3435 c significantly inhibited the secretion of inflammatory factors.Further analysis of macrophage apoptosis by flow cytometry revealed that Rv3435 c inhibited the apoptosis of host macrophages.In summary,this study found that Rv3435 c is a cell wall-related protein that can alters the morphology of bacteria and colonies,inhibits the growth rate of recombinant mycobacteria,and enhances its resistance to various stresses.Meanwhile,it was evident that Rv3435 c inhibited inflammatory factors and host cell apoptosis,and enhanced the survival of recombinant bacteria in macrophages.Experimental data showed that Rv3435 c may play an important role in MTB infection. |
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