Mechanism Of A SRNA MrsI Regulating The Resistance Of Mycobacterium To Isoniazid

Noncoding RNA（nc RNA）is a class of RNA that does not encode a protein,also called small RNA（s RNA）.It plays an important role in sensing environmental changes and regulates the expression of related genes.Previous studies have shown that s RNA is involved in regulating the drug resistance of many bacteria,such as Escherichia coli,Salmonella typhimurium,Pseudomonas aeruginosa,and Staphylococcus aureus.Tuberculosis（TB）is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis infection,which is a serious threat to human health.In the20^th century,with the discovery of many anti-tuberculosis drugs such as streptomycin,isoniazid and rifampicin,the treatment of tuberculosis achieved good results.However,in recent years,the incidence of drug-resistant has increased year by year due to the irrational use of anti-tuberculosis drugs,making the treatment of tuberculosis more difficult.M.smegmatis is widely used as a model strain of mycobacteria because of its rapid reproduction and no pathogenicity.Presently,many s RNAs have been successfully identified in Mycobacterium,but their function is not clear.In this thesis,M.smegmatis was used to investigate the mechanism of drug resistance of mycobacteria at the level of non-coding RNA.The research results are as follows:（1）Investigated drug resistance of s RNA overexpressing strains and systematically revealed the relationship between s RNA and drug resistance regulation in mycobacteria.By analyzing different transcriptome data in M.smegmatis,we identified 35 novel trans-acting s RNAs and verified them by RT-PCR.In addition to the 44 already reported,79 trans-acting s RNAs have now been found in M.smegmatis.We selected 40 of them to overexpress and examined the resistance of these s RNA overexpressing strains to four first-line anti-tuberculosis drugs（isoniazid,rifampicin,streptomycin and ethambutol）.The results indicated that most of s RNA overexpressing strains did not exhibit different resistance to those drugs.A small number of s RNA overexpressing strains have weak changes in drug resistance.However,the overexpression of s RNA nc Ms13628Ac（Mrs I）prominently enhanced the resistance of M.smegmatis to isoniazid,but did not affect its resistance to the other three antibiotics.In the following,we mainly studied the regulation targets of Mrs I and its mechanism in mediating isoniazid resistance of mycobacteria.（2）Screenned the direct target of Mrs I by GRIL-seq.We established the GRIL-seq（Global small non-coding RNA target identification by ligation and sequencing）method in M.smegmatis,and used this method to identify the direct targets of Mrs I.GRIL-seq data was significantly enriched in the 3’-UTR of fur A3（fur A3＿3’-UTR）,suggesting that fur A3＿3’-UTR as a potential regulatory target of Mrs I.Through sequence alignment analysis,we found that the seed region of Mrs I has a perfectly complementary set of 10 nucleotides with fur A3＿3’-UTR.Furthermore,we confirmed that Mrs I can directly act on fur A3＿3’-UTR in vivo and in vitro through dual-plasmid reporter experiment and RNA:RNA EMSA（Electrophoretic Mobility Shift Assay）,respectively.（3）Revealed the molecular mechanism of Mrs I regulating mycobacterium resistance to isoniazid.Real-time PCR,Western blot and antibiotic tolerance experiments showed that Mrs I binding to fur A3＿3’-UTR did not affect the expression of fur A3,nor did this participate in the resistance regulation of M.smegmatis to isoniazid.By analyzing the sequences of both kat G1 and kat G2,we found that the 5’-UTR of the two genes are very similar,and six nucleotides consensus motif（CAGCCC）of the two 5’-UTRs are perfectly complementary to the seed region of Mrs I.Dual plasmid reporter assay and peroxidase activity experiments showed that Mrs I can directly bind to the 5’-UTR of kat G1 and kat G2 to inhibit the expression of their genes.Moreover,by mutating the onsensus motif（CAGCCC）of kat G1 and kat G2 5’-UTRs in the genome,we found that the inhibition of kat G1 and kat G2 by Mrs I is completely dependent on the target region in their 5’-UTR.Furthermore,it was confirmed that the inhibition of kat G1 expression by Mrs I binding to kat G1＿5’-UTR is the main pathway mediating M.smegmatis resistance to isoniazidFinally,to investigate whether Mrs I affects isoniazid resistance only by regulating Kat G（General name for all three Kat G）;we constructed kat G1 mutant,kat G1kat G2double mutant,and kat G1kat G2kat G3 triple mutant.Phenotypic experiments showed that overexpression of mrs I in the three mutant strains still conferred isoniazid resistance.This indicated that Mrs I regulates the resistance of mycobacteria to isoniazid not only through Kat G,but also has other unknown targets.In summary,this thesis identified a s RNA（Mrs I）that can regulate the resistance of mycobacteria to isoniazid,and identified direct targets of Mrs I,revealing the multiple regulatory mechanism of Mrs I-mediated isoniazid resistance of mycobacteria.