结核分枝杆菌RD105区缺失介导的多重耐药调控机制研究

Tuberculosis (TB) remains a major global health problem. The emergence of drug-resistant strains of Mycobacterium tuberculosis, especially those that are multidrug resistant and extensively drug resistant, has posed a serious threat to global TB control program. Much progress has been made to identify gene mutations in specific loci of the M. tuberculosis genome as the molecular basis for TB drug resistance and as drug targets for the development of anti-TB drugs. However,the molecular mechanism of multidrug resistance is still less understood. Many regions of difference (RDs) of M. tuberculosis have been identified and RD105 deletion was exclusively found in all strains from Beijing lineage and is suggested to be a marker of this group. Some studies claimed that Beijing strains were more likely to develop MDR.However,the relation between the deletion of RD105 region and the development of multidrug resistance of Beijing strains is still less known. RD105 region includes a truncated C-terminal of Rv0071 and N-terminal Rv0074, and full-length of Rv0072 and Rv0073. The deletion of RD105 results in a new coding frame Rv0071/74 that lack Rv0072 and Rv0073, but includes truncated N-terminal of Rv0071 with a variable 9-bp sequence (gtggacccg) repeats and C-terminal of Rv0074. Our study revealed that Rv0071/74 fusion gene may encode a multidrug resistance gene and the variable repeat located 5′end of fusion gene regulates the developemtn of this drug resistance. Recombinant expressing in M. smegamtis proved that Rv0071/74 fusion gene with 9m (Rv0071/74-9m) is a novel multidrug resistance gene. Moreover, we observed that M. smegmatis expressing Rv0071/74-9m formed compact colonies with smooth edge on the bottom of well culture plates whereas the control groups containing only pVV16 vector loosely grouped on the bottom of culture plates and formed colonies with radiated edge. These results implied that the occurrence of multidrug resistance of Rv0071/74-9m may be regulated by the permeability of drug through the thickness of cell wall. In this research, we plan to reveal the regulating molecular mechanism of multidrug resistance of the fusion gene Rv0071/74 resulted by the deletion of RD105 region. The findings in this research will not only reveal a novel mechanism for the development of the MDR of TB, but also provide a clear pathway for the diagnosis or design of more effective drugs for the treatment of drug-resistant TB.

耐多药结核菌的出现使结核病防控形势更为严峻，但调控多重耐药的机制目前尚不清楚。RD105区缺失是结核菌北京家族菌株的特征标识。研究显示该家族菌株易产生多重耐药，但RD105区缺失是否参与多重耐药发生仍未知。课题组前期研究发现：（1）RD105缺失形成新融合基因，该基因5'端存在多态性位点；（2）含有不同等位融合基因的菌株具有不同药敏表型，提示融合基因可能是一种新耐药基因，且耐药表型受多态性位点调控；（3）重组表达实验证实融合基因是一种新耐药基因，介导了多重耐药的产生；（4）不同等位基因重组菌株产生的耐药表型不同；（5）具有多重耐药表型的重组菌株具有不同细菌形态。本项目拟在前期研究基础上，通过对融合蛋白的结构功能、重组菌细胞壁超微结构以及胞内药物浓度等展开研究，深入揭示RD105区缺失介导的多重耐药调控机制。本项目研究成果将为结核菌的耐药机制研究提供新思路，为抗结核药物研发提供更多理论依据。

耐多药结核菌的出现使结核病防控形势更为严峻，但调控多重耐药的机制目前尚不清楚。RD105区缺失是结核菌北京家族菌株的特征标识。研究显示该家族菌株易产生多重耐药，但RD105区缺失是否参与多重耐药发生仍未知。课题组前期研究发现：（1）RD105缺失形成了新的融合基因Rv0071/74，该基因5’端存在多态性位点（其串联数目为3-9m）；（2）含有不同等位融合基因的菌株具有不同药敏表型，提示融合基因可能是一种新耐药基因，且耐药表型受多态性位点调控；（3）重组表达实验证实融合基因是一种新耐药基因，介导了多重耐药的产生；（4）不同等位基因重组菌株产生的耐药表型不同；（5）具有多重耐药表型的重组菌株具有不同细菌形态。本项目在前期研究基础上深入揭示了RD105区缺失介导的多重耐药分子调控机制。结果显示：RD105区缺失产生的Rv0071/74融合基因表达产生的羧肽酶可参与结核菌细胞壁中肽聚糖的合成，其中Rv0071/74-9m亚型可显著提高细胞壁的厚度，增加药物进入胞内的难度，从而导致胞内药物浓度显著降低，难以达到有效的杀菌或抑菌浓度，最终导致了该亚型结核菌多重耐药表型的产生。本研究成果不仅证明了RD105缺失形成的Rv0071/74融合基因为一种新的多重耐药基因，也为结核菌的多重耐药机制研究提供了新思路。