| Mycobacterium tuberculosis?Mtb?is a pathogen that causes tuberculosis.With the emergence of multi-drug resistant strains and extensively drug-resistant strains,tuberculosis has re-emerged globally and is the ninth leading cause of death in the world,seriously threatening human health.The data show evidence that nearly 10.4 million people worldwide were infected with tuberculosis,1.3 million tuberculosis patients died,and 374,000 people died of TB infection in 2016.Mycobacterium tuberculosis does not have internal and external toxins,and its virulence is related to the mass reproduction and production of metabolites in the host.Iron ions play an important role in bacterial respiration and DNA synthesis,and their availability affects the ability of Mycobacterium tuberculosis to infect humans.Mycobacterium tuberculosis uses mycobactin as an iron carrier to firmly bind free iron in the host and transport it into the bacteria.This process is very important for the virulence of Mycobacterium tuberculosis.So far,14 mycobacteriophage synthetases have been found,of which MbtB,MbtD,MbtE,MbtG and MbtK are indispensable for the survival of Mycobacterium tuberculosis.In M.tuberculosis,Fe3+-carboxy mycobactin transports iron ions to the cell wall associated with mycobactin,or iron-regulated transporters A and B that deliver iron ions to the cytoplasmic membrane.At present,the regulatory mechanism of iron ion uptake and translocation in Mycobacterium tuberculosis is not fully understood.sRNAs are non-coding RNAs that play a regulatory role and are widely found in various fields of life.Bacterial sRNAs are approximately 50-500 nucleotides in length and can regulate gene expression in a variety of ways.sRNAs play an important role in the regulation of bacterial adaptive responses,metabolism,virulence and other important physiological processes.Bacterial sRNAs are also involved in the regulation of iron uptake processes.For example,sRNA ryhB found in many Gram-negative bacteria can sense iron ion concentration in the environment.Decreased iron ion concentration leads to the expression of sRNA ryhB,while ryhB promotes transferrin mRNA.The degradation of transferrin inhibits the expression of transferrin,leading to a reduction in the iron requirement for bacteria.There is no report on the involvement of sRNAs in the regulation of the iron uptake and transport of Mycobacterium tuberculosis.The aim of this study was to identify sRNAs involved in the regulation of iron uptake by Mycobacterium tuberculosis and to study their function.In this study,an iron deprivation model of Mycobacterium tuberculosis was established,and key genes related to iron metabolism under iron deprivation conditions were detected by Real-time quantitative PCR.It was found that 12 out of 14 genes were significantly upregulated,demonstrating that the model can Effectively respond to bacterial response to iron stress conditions.High-throughput sequencing and analysis of the transcriptome of the standard strain H37Rv under iron deprivation conditions revealed that there were 755 differentially expressed genes in 4008 genes of Mycobacterium tuberculosis,among which 69 genes were significantly upregulated?fold-change?2?There are 40?fold-change?2?with significantly reduced expression.GO enrichment analysis found that the biological process gene of the activity level of the transfer iron carrier,the molecular functional gene of the host iron aggregation reaction,the molecular functional gene of the nutritional symbiont acquired from the host by the siderophore,the molecular functional gene introduced by the iron ion,and the nickel and cobalt Ionic reactions have the highest degree of molecular enrichment.By analyzing the results of RNA isolation of Mycobacterium tuberculosis under the iron deprivation model,this study predicted 246 potential sRNAs,named 001-246,and used RNAplex software to predict the target genes of these candidate sRNAs.GO enrichment analysis found that the target genes of these candidate sRNAs were mainly concentrated in the glycine betaine transport process.KEGG analysis revealed that these candidate sRNA target genes were enriched in the ATP-binding cassette transporter family.In the model of iron deprivation,four differentially expressed candidate sRNAs were named ASssr,ASdapC,ncRv1354 and ASnmtR.By Northern blotting,it was found that ASssr was actually present and expressed in both the model group and the control group;Real-time quantitative PCR results confirmed that ASdapC,ncRv1354and ASnmtR also existed,but the expression level was significantly lower than that of ASssr.?more than 210 times?.By comparison with the Rfam database,it was found that ASssr is complementary to ssr,and ssr is structurally predicted to be tmRNA.The main function of tmRNA is to release the ribosomes that bind to mRNA and lose function through the rescue mechanism mediated by the SmpB protein-tmRNA complex.It enters a new translation cycle;it simultaneously releases mRNA and degrades abnormal mRNA to prevent the ribosome from extinguishing.Because it exists only in prokaryotes,it is highly likely to become a new target for the action of antimicrobial agents.The predicted target gene for ASnmtR is nmtR,a specific nickel/cobalt inhibitor of Mycobacterium tuberculosis that regulates the transcription of the cell membrane transporter that mediates nickel/cobalt efflux of cytoplasm.Therefore,ASnmtR may be involved in the extracellular nickel.We collected seven clinically isolated multidrug-resistant Mycobacterium tuberculosis strains and examined the expression levels of candidate s RNA.We found that ASssr,ASdapC,and ASnmtR were significantly lower in kanamycin-resistant Mycobacterium tuberculosis.Kanamycin-sensitive tuberculosis bacteria.We speculate that ASssr,ASdapC,and ASnmtR may be involved in the kanamycin resistance process and are potential molecular markers for kanamycin resistance,which requires more clinical samples to be validated.In order to explore the relationship between the expression of these four candidate sRNAs and the severity of iron deficiency,three groups of iron-deficiency culture media were designed in this study.Real-time quantitative PCR showed that the expression of ASssr was significantly up-regulated in severe iron-deficient cultures,while mild iron deficiency was up-regulated but not significantly different;ncRv1354 was up-regulated in iron-deficient environments and had significant differences by spearman correlation test.It was confirmed that only the expression of ASssr was highly positively correlated with the degree of iron deficiency.We compared the results of PCR with genomic and cDNA templates and then confirmed that the ASssr co-transcribes with the upstream gene Rv3098A.To study the function of Asssr,we constructed an overexpression strain of ASssr,named H37Rv-pMV261-ASssr.We studied the growth traits of the over-expressed strains.In the normal culture environment,there was no difference with the control.In the iron-deficient environment,the control grew slowly,and the over-expressed strain was more sensitive to the environment and basically did not grow.We used real-time quantitative PCR to analyze the transcriptome of the ASssr overexpressing strain and found that 14 iron metabolism related genes,21 predicted target genes,and 3 distantly unrelated genes were significantly downregulated,indicating that ASssr could down-regulation of gene expression after overexpression is universal.Through the minimum inhibitory concentration experiment results,we found that the minimal inhibitory concentration of Streptomycin,Isoniazid,and Rifampicin on the overexpressed strain generally decreased,especially the Rifampicin minimal inhibitory concentration decreased by 8 times.We hypothesize that ASssr has a general down-regulation of M.tuberculosis gene expression,reduces the tolerance of M.tuberculosis to iron deficiency stress environments,increases the susceptibility of M.tuberculosis to anti-tuberculosis drugs,and may become a treatment for tuberculosis in particular.It is a target for multi-drug resistant Mycobacterium tuberculosis?MDR-TB?. |
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