The Role Of Mycobacterium Tuberculosis Proteasome Accessory Factor C (PafC) In Moxifloxacin Resistance

Tuberculosis（TB） caused by Mycobacterium tuberculosis remains a serious global infectious disease. According to World Health Organization report,1/3of the world’s people latently infected with M. tuberculosis, and there are8.6million new infections and the global TB deaths was up to1.3millionjust in2012. The epidemic of TB is so serious, in addition to the unique pathogenic characteristics of M. tuberculosis, due to the emergence of multi-drug resistant TB （MDR-TB）, extensively drug-resistant TB （XDR-TB） and HIV-associated TB, which makes control the epidemic situation of TB more difficult.The current first-line treatment for TB, including isoniazid （INH）, rifampicin （RIF）, pyrazinamide （PZA） and ethambutol （EMB）, is invalid for the MDR-TB and XDR-TB patients. Therefore the second-line anti-TB drugs, aminoglycoside antibiotic, polypeptide antibiotic and fluoroquinolone（FLQ） antibiotics are the main drugs against MDR-TB and XDR-TB. With low toxicity and a large number of derivatives, the FLQ have attracted too much attention. However, continuous appearance of resistant to FLQ makes the battle against with infection worse.Therefore, we try to find the genes of Mycobacterium involving in intrinsic resistance to FLQ drugs and the related proteins that can be targeted by antibioticpotentiators, which provides support for coping strategy to the MDR-TB and XDR-TB. At early times, we got an ultra-sensitive to moxifloxacin（MXF） mutant strain M371by screening a transposon insertion library of Mycobacterium smegmatis and identified transposon insertion in MSMEG₃888. Based on our laboratory’s previous work, this research will further verify the mutant M371ultra-sensitive phenotype and uncover the mechanism under this ultra-sensitive phenotype, and explore the mutant instress responses.First of all, we treated M. smegmatis wild-type and mutant M371with8timesMIC of the wild-type M. smegmatis MXF, recorded CFU after processing for different time, and then calculated the survival rate and constructed the sterilization curve in order to verify the ultra-sensitive phenotype to MXF. Then, in order to study the effect of MSMEG₃888gene in M. smegmatis in intrinsic resistance to MXF, we constructed a complemental strain of mutant M371. The counterpart of MSMEG₃888in M. tuberculosis H37Rv is Rv2095c. So the gene of Rv2095c was cloned andligated to the shuttle plasmid pALACE. And the recombinant plasmid pALACE-Rv2095c was transformed into M371mutant, then we obtained the complemental strain. We detected expression of Rv2095c by Western-blot and sensitivity of the complemental strain to MXF by MIC experiment. Finally, we explored difference in surface properties of the wild strain, the mutant M371and the complement, and difference in stress responses of the three strains by treating them with hydrogen peroxide （H2O2） and twelve sodium alkyl sulfonate （SDS） in vitro to simulate alveolar environment.The experimental results show that:by MXF treatment, survival rate of the mutant strain M371was lower than that of the wild type M. smegmatis, we confirmed M371was ultra-sensitive to MXF. Validation of bacterium liquid PCR andgene sequencing proved we constructed the complemental strain M371-pALACE-Rv2095c successfully. Western-blot result showed heterologous expression of Rv2095c in M371was successful, and the complemental strain and wild type haved the same tolerance to MXF. This data suggested the gene MSMEG₃888involved in intrinsic resistance to MXF. In the biological nature of the surface,M. smegmatis wild-type, mutant M371and complemental strains, there was noremarkable difference among the three strains in the single colony morphology and sliding ability; but the difference in the lipid coefficient was obvious; compared to the wild type the mutant was significantly decreased. No significant difference among the three in SDS tolerance level, and there were no significant differences between wild-type and M371in hydrogen peroxide tolerance levelneither. No significant difference among the three strains in stress response.Therefore, this work shows MSMEG₃888gene involves in intrinsic resistanceof M. smegmatis, but does not affect the surface properties of M. smegmatis and stress response.