Characterization Of A Quinolone-resistant Gene Of Mycobacterium Tuberculosis

Tuberculosis is the world's leading cause of mortality owing to an infectious bacterial agent, Mycobacterium tuberculosis. The HIV/AIDS pandemic has been attributed to the global resurgence of TB, with increased mobility owing to mass movement of population and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB). The emergence of MDR-TB is one of the major causes of the treatment failure. The complete genome sequence and annotation of Mycobacterium tuberculosis H37Rv was published in 1998.This has provided researchers important information to gain insight into the mechanism of the drug resistance.The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in an increased interest in the second-line antituberculosis agents, and Quinolones, one class of the synthetic antibacterial agents, are being used in selected tuberculosis patients. Quinolones have possessed the activity of antituberculosis since the third generation. The DNA gyrase composed of two A and two B subunits is the only target of quinolones in mycobacteria. Mutation in the target often confers quinolones resistance. Mutation in a particular region of gyrA,which encodes the gyrase A subunit, has been associated with a high level of quinolone resistance. Mutations conferring low-level resistance have also been found in gyrB, which encodes the B subunit of the DNA gyrase. However, these mutations do not account for all resistant strains, indicating that other mechanisms have contributed to the resistance in mycobacteria.Notably, most quinolones are only moderately active against mycobacteria, and unfortunately, quinolone-resisitant clinical isolates of Mycobacterium tuberculosis have already appeared. If more can be learned about what determines the effectiveness of a particular quinolone against the mycobacteria and the mechanisms by which the resistance develops, new agents or strategies may be designed to prevent or circumvent this resistance.In this study, using bioinfomatics method to analyze the genome of Mycobacterium tuberculosis, we discovered that the genome of Mycobacterium tuberculosis H37Rv has 20 open reading frames encoding putative efflux proteins. Most of them associate with resistance. Furthermore, Rv2994 gene may be the efflux bump of the quinolones.First, we amplified the gene Rv2994 from the genome of Mycobacterium tuberculosis H37Rv to make an attempt to investigate the function of it. A recombinant fused expression vector pGEX-2994 was constructed. The recombinant plasmid could express the fusion protein GST-Rv2994 stably, thus provided the basis for the further study of the gene Rv2994.Then, a second recombinant plasmid bearing Rv2994 was constructed with the shuttle vector pMV261 and electroporated into avirulent Mycobacterium smegmatis me2155. The transformants were induced toexpress a 47KDa protein of Rv2994. The MIC and accumulation of ofloxacin for the strains of mc2155, mc2155-261 and mc2155-2994 are determined with and without the membrane deenergizer carbonyl cyanide m-chlorophenylhydrazone (CCCP) to clarify the Rv2994 gene function.Finally, the study of quinolone-resistant genes in Sichuan Province confirmed a strong correlation between the quinolone-resistance and the mutation of gyrA gene, which might be a major molecular mechanism of quinolone-resistance in Mycobacterium tuberculosis .In summary, the majority of the results in this study support our hypothesis that the protein encoded by Rv2994 gene is a drug efflux bump, conferring resistance to ofloxacin. The fact that the Rv2994 protein could export drugs and confer multidrug-resistance extends a promising field for future works on Mycobacterium tuberculosis.