Mycobacterium Tuberculosis Rifampin Resistance Associated Gene Mutation Detection And Rv2629 Gene 191a / C Mutation Function Of A Preliminary Study

Tuberculosis is one of the oldest recognized bacterial world-wide diseases with a third of the total world population(approximately 1.8 billion persons)infected with the causative bacterium,Mycobacterium tuberculosis(M.tb).According to WHO,There are eight to ten million new cases of clinical tuberculosis and 2-3 millions death per year.About 98% of all TB deaths occur in developing countries,where surveillance for resistance of M.tb isolates to anti-TB drugs is uncommon.China,as a developing country with huge population pressure,has a very high TB burden.In recent years,the epidemiology of tuberculosis has altered due to the emergence of HIV infection and the propagation of drug-resistance strains.The serious problem we are now facing is the prevalence of drug-resistant tuberculosis infections.Resistance to the four primary drugs rifampicin(RIF),isoniazid(INH), ethambutol(EMB)and streptomycin(STR)makes tuberculosis difficult to treat and severely threaten public health control efforts.Recently,a subset of MDR-TB strains has been identified as extensively(or extremely)drugresistant(XDR-TB).These are now defined as being resistant not only to isoniazid and rifampicin,but also to fluoroquinolones and to at least one of three injectable drugs usually employed in second-line therapy of MDRTB; capreomycin,kanamycin and amikacin.XDR-TB is virtually untreatable.Neither the standard drugs nor at least three of the six classes of more toxic and less-effective backup drugs are effective.Based on WHO and US CDC survey,the burden of XDR-TB is about 10% of the MDR isolates in a sample of 17,690 M.tb isolates from 49 countries from 2000-2004.Populationbased data were available from the United States,Latvia and South Korea,where 4%,19%,and 15% of MDR-TB cases, respectively,were XDR.In some cases,XDR-TB has been shown to represent a particularly aggressive form of TB,causing very high mortality and,at least in one setting,killing HIV-positive patients within an average of 25 days from diagnosis.Rifampicin is a major component of multidrug regiments used for treating tuberculosis.Over 95% of RIF-resistant was attributable to mutations within a conserved region consisting of 81-base pair(bp)situated near the center of theβ-subunit of RNA polymerase encoded by the rpoB gene.At the same time, Resistance to Rifampicin is a potential marker for multidrug-resistant tuberculosis as nearly 90% of rifampicin resistant strains are also resistant to isoniszid.Some 3%～10% of the RIF-resistant strains didn't show any mutations inside the 81-bp region.They could have novel resistance mechanism or an unknown mechanism.But these important questions remain unanswered.In an effort to identify the new resistance factors in the rifampicin resistant(RIF~r) M.tb,comparative proteome analysis and gene mutation assays were applied to identifythe differentially expressed proteins.These methods were used to detect the correlated gene mutations among the clinical RIF~r isolates lacking of rpoB mutations and the clinical RIF sensitive(RIF~s)isolates and compare to the laboratory H37Rv M. tb.The protein spots of interest were identified by mass spectrometry (MALDI-TOF-MS),and then genes of the identified proteins were amplified by PCR and cloned for sequencing.MALDI-TOF-MS revealed 9 differentially expressed protein spots.There were 5 spots of protein DevR,BfrB,Ag84,CysK and Rv2629 that were more than 2 fold upregulated in the clincial RIF~r isolates compared with the laboratory H37Rv strain and the RIF~s isolates.There were 4 spots of protein Ssb, Rv0927c,OpcA,and PPE51 that were 2 fold downregulated.PCR sequencing results showed 5 genes out of the 9 protein spots were relatively unchanged,and 4 genes were mutated when we compared the RIF~r to RIF~s isolates,and when we compared them to the H37Rv strain.The newly identified 191A/C mutation of Rv2629 gene, which converted 64Asp to 64Ala in the coding protein,was carried by 111 out of 112 clinical RIF~r isolates(99.1%),but was absent in the laboratory strain and in all of the clinical RIF~s isolates.Here,the common 191A allele is wild type and the rare C allele is mutant.The RIF sensitive M.smegmatis displayed RIF resistance only after being transformed with the mutated M.tb Rv2629 gene of 191C,but did not restore with the wild M.tb Rv2629 gene of 191A.These results indicate that the 191A/C mutation of Rv2629 gene,and the upexpressed Rv2629 protein may be associated with RIF resistance in M.tb.