Study On The Mechanism Of Prophage Gene Rv2650c In Mycobacterium Tuberculosis Promoting The Survival Of Mycobacterium In Macrophages Andthe Environment

As the pathogen causing tuberculosis（TB）,Mycobacterium tuberculosis is one of the most dangerous pathogens in the world.M.tuberculosis can invade many organs in the body and cause TB.With the spread of drug-resistant M.tuberculosis and the emergence of HIV-associated TB,the incidence of drug-resistant TB has been on the rise in recent years.As one of the 3 countries with a high burden of TB in the world,the current TB epidemic of China is relatively severe.Finding the pathogenic genes of M.tuberculosis and clarifying its treatment mechanism are of great significance for developing new therapeutic drugs and advancing the treatment of drug-resistant TB in the world.M.tuberculosis was identified by Robert Koch,a German scientist,in 1882.Special cell wall structure and complex pathogenic mechanism make the pathogen resistant to different harsh environments.In order to study the pathogenic mechanism of M.tuberculosis,to explore the unique virulence factors existing in M.tuberculosis,and to find the target of new anti-tuberculosis drugs,we turned our attention to the phages and prophages.Phages have been regarded as natural enemies of bacteria.However,according to the latest developments in phage molecular biology,especially the data from omics analysis,the interaction between phages and their hosts is far more complicated than originally thought.Bacteriophages can be divided into lytic phages and temperate phages.Some temperate phages called prophages can be integrated into their host bacterial genome during the evolution process.Prophages can replicate with their host genome replication.Most bacterial genomes in nature carry one or more prophages.Besides,many studies have shown that prophages have an important influence on the evolution and adaptability of host bacteria,especially playing an important role in the pathogenicity of pathogenic bacteria.The current study found that there are two prophages,phi Rv1 and phi Rv2 in the M.tuberculosis H37Rv genome.Their functions are still unknown.Our previous study found that the prophage phi Rv1 and phi Rv2 are functional units.At the transcriptome level,the date showed that some genes in the prophage phi Rv1 and phi Rv2 are in an active transcriptional state and may encode some functional proteins to help bacteria adapt to the environment.In this study,we focus on prophage phi Rv2 gene Rv2650c.The size of Rv2650c gene is 1449 bp and it is predicted to encode capsid protein.Transcriptomic data found that Rv2650c gene is up-regulated in the macrophage infection model and persister model.After analyzing the Rv2650c gene using the method of Louis-Marie Bobaya et al.,we found that its Ka/Ks value was 1.33.This data indicates that Rv2650c gene is strongly positively selected in M.tuberculosis.In summary,the Rv2650c gene may play an important role in the response of M.tuberculosis to stress and pathogenicity.In order to study the important role of prophage in the pathogenicity of M.tuberculosis,we used Mycobacterium smegmatis as a model organism to study the function of the prophage gene Rv2650c.In this study,the gene Rv2650c of the prophage phi Rv2 in the M.tuberculosis H37Rv genome was synthesized in Beijing Qingke Xinye Biotechnology Co.,Ltd.and ligated into the plasmid p NIT-Myc vector.Then the recombinant plasmid p NIT-Myc-Rv2650c was successfully constructed.The recombinant plasmid p NIT-Myc-Rv2650c and plasmid p NIT-Myc were transferred into M.smegmatis by electrotransformation,and the recombinant M.smegmatis MS＿Rv2650c and the control bacteria MS＿Vec were successfully constructed.In the study,Western Blot experiment were performed on recombinant bacteria.Using the recombinant plasmid p NIT-Myc-Rv2650c as a template,the gene Rv2650c was amplified and subcloned into the plasmid p ET28a（+）and transformed into E.coli.The expression of the recombinant protein was detected by SDS-PAGE.In order to study the effect of prophage protein Rv2650c on recombinant M.smegmatis,the experiment performed morphological aggregation observation and fatty acid analysis on recombinant M.smegmatis and then plotted the growth curve of recombinant M.smegmatis.In order to simulate some adverse environmental factors of the human lungs faced by M.tuberculosis,we studied the survival ability of recombinant bacteria under 3%H₂O₂,acidic environment（p H=4.5）and 2%SDS.In addition,this experiment studied the intracellular survival rate of recombinant bacteria and cytokine changes of macrophages,and conducted transcriptome analysis of macrophages infected by recombinant M.smegmatis.The results showed that the gene Rv2650c was successfully heterologously expressed in M.smegmatis.The results of Western Blot experiment showed that the protein Rv2650c was localized on the cell wall of M.smegmatis.The gene Rv2650c did not significantly affect the surface morphology and cell structure of the recombinant bacteria.The colony diameter statistics result showed that the prophage protein Rv2650c restricted the proliferation of the recombinant bacteria in the presence of Tween80.Fatty acid analysis results showed that there was no significant difference between the composition of recombinant bacteria MS＿Rv2650c and MS＿Vec.The growth curve results showed that the growth of the control group and the experimental group was basically the same,indicating that the overexpression of the prophage protein Rv2650c had no significant effect on the growth rate of recombinant M.smegmatis.Compared with the recombinant bacteria MS＿Vec,the survival rate of recombinant M.smegmatis MS＿Rv2650c increased significantly,and the intracellular survival rate was significantly higher than that of the control under the condition of 3%H₂O₂,acidic environment（p H=4.5）and 2%SDS.Combined with the growth curve results,the prophage protein Rv2650c does not enhance the resistance of M.smegmatis to adverse environments by enhancing cell growth.Transcriptome analysis revealed that the recombinant bacteria MS＿Rv2650c can upregulate the expression of Nrf2 and its downstream factor NQO1 and NEAT1 in macrophages.Protein Nrf2 is a key regulator of cell protection genes in the anti-oxidative stress response,and its activation has anti-inflammatory effects.Further ELISA results showed that the inflammatory cytokines TNF-α,IL-10,IL-1βand IL-6 were indeed inhibited by the prophage protein Rv2650c.The inflammatory factors of macrophages play an important role in killing of M.tuberculosis.Finally,this experiment purified the prophage protein Rv2650c and used it as a bait to interact with the infected macrophage lysate.And some proteins were screened out by interaction which may be the target of the Rv2650c protein.We speculate that the prophage protein Rv2650c can act as a protein to induce the Nrf2 pathway by interacting with some proteins in macrophages.Nrf2 pathway can indirectly or directly inhibit the expression of macrophage inflammatory factors TNF-α,IL-1βand IL-6,which can reduce the killing of M.tuberculosis by macrophages and promote the survival of Mycobacteria.This study provides important new information for learning the role of prophage in the pathogenic mechanism of M.tuberculosis and provides new insights for the discovery of the pathogenic and virulence factors of M.tuberculosis.It also lays the foundation for the development of new drugs to control M.tuberculosis.