katG基因突變在結核分枝桿菌之異菸鹼醯胼抗藥性中的角色

Abstract

結核病由結核分枝桿菌（Mycobacterium tuberculosis）所引發，不僅名列全球十大死因之一，也是國內法定傳染病中每年確診數及死亡數最多的傳染病。結核病的療程需至少六個月的規律服藥，而抗藥性的產生更是其治療難度增加的主要原因。 本研究以衛生福利部疾病管制署所提供自台灣臨床分離的抗藥性菌株作為研究主題，藉由定序得知其中四株異菸鹼醯胼（Isoniazid）抗藥性菌株的KatG 蛋白分別帶有R146P、W341R、P375L 及L398P 的氨基酸序列突變，而在過去研究中上述突變皆未被記載與抗藥性有關。由藥物敏感性測試結果可得知，周教授實驗室所提供在KatG 蛋白分別帶有R146P、W341R及L398P突變的抗藥性菌株其異菸鹼醯胼最低抑制濃度都不低於128 μg/mL。 本研究藉由pMN437 載體在抗藥性菌株中表現結核分枝桿菌H37Rv 參考菌株katG 基因去探討上述基因突變與抗藥性產生的關聯。在帶有L398P氨基酸序列突變的抗藥性菌株中表現結核分枝桿菌H37Rv 參考菌株katG 基因會產生至少八倍的藥物感受性的回復；而於KatG W341R 突變株中表現結核分枝桿菌H37Rv 參考菌株katG 基因亦能看到藥物感受性的部分的回復。 為了檢測抗藥性是否因上述位點突變而有所影響。將H37Rv的katG 基因置換成katG L398P 的同源染色體置換菌株已建構完成，並將進行藥物敏感性測試確認KatG 突變是否與抗藥性的機制有關。

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis which is not only one of the ten major causes of death worldwide, but also recorded as the highest incidence and mortality rate among all communicable diseases in Taiwan. Since the disease takes at least 6 months to treat, the emergence of drug-resistant TB increases the difficulty of therapies. In this study, we focus on clinically isolated drug-resistant M. tuberculosis in Taiwan, provided by the Taiwan Centers for Disease Control. The sequence of the strains was analyzed and amino acid substitutions of KatG at R146P, W341R, P375L and L398P were found in four isoniazid-resistant strains, which have not yet been reported to be related to drug-resistance. The drug susceptibility test showed that above-mentioned mutations had minimum inhibitory concentration (MIC), no less than 128 μg/mL. In this study, plasmid pMN437-katG was used to express H37Rv KatG in drug-resistant strains to investigate the correlation between the drug-resistant M. tb strains and the katG gene mutations mentioned above. The results revealed that overexpression of KatG in KatG L398P mutant strain restored drug sensitivity by more than 8 times, and KatG overexpression in KatG W341R mutant strain has partially restored the sensitivity of INH. To reconfirm the association between isoniazid-resistant phenotype and katG gene point mutations, the unmark replacement mutant substituting katG of H37Rv with katG L398P mutant was successfully constructed for further drug susceptibility test in order to study the novel katG mutation in drug resistance mechanism.