结核分枝杆菌耐吡嗪酰胺分子机制研究

Molecular basis of Mycobacterium tuberculosis to pyrazinamide resistance

吡嗪酰胺(PZA)是结核病短程化疗中的一线抗结核药物,由吡嗪酰胺酶转换成为活性形式吡嗪酸而生效。吡嗪酰胺酶由pncA基因编码,pncA基因突变会导致该酶活性丧失,与PZA耐药性产生有关。为了进一步明确PZA耐药性产生的基因学基础和PZA耐药株的pncA基因突变率,对中国100株结核分枝杆菌临床分离株进行了DNA序列测定,其中85株为PZA耐药株,15株为PZA敏感株。PZA耐药株有27%(23/85)发生了pncA基因突变,从而导致吡嗪酰胺酶基本氨基酸序列的改变,突变分布在pncA基因开读框架17-546位的核苷酸。其中有一株突变位于pncA基因的调节区域-11位处。同时发现20%(3/15)pncA敏感株也发生了pncA基因突变。敏感株发生突变可能是由于PZA敏感性实验不准确或存在其它耐药机制。实验表明,pncA基因突变是PZA耐药的主要机制之一,中国PZA耐药临床分离株尚存在其它耐药分子机制。

Pyrazinamide （PZA） is an important component in short-course tuberculosis chemotherapy. While the mode of action of PZA is not fully understood, there is substantial evidence that the drug must first be converted to pyrazlnoic acid （POA） by the enzyme pyrazinamidase （PZase） for its antimycobacterial activity. To further define the genetic basis of PZA resistance and determine the frequency ofpncA mutations in PZA-resistant strains, we sequenced the pncA gene from a panel of 100 clinical isolates, including 85 PZA-resistant strains and 15 PZA-susceptible strains. The results showed that 27% （ 23/85 ） PZA-resistant isolates had pncA mutations which altered the primary amino acid sequence of pyrazinamidase. Mutations within the open reading frame were distributed in the region from nuclcotide 17 to nucleotide 546. One putative regulatory mutation was identified. Meanwhile 20% （ 3/15 ） PZA-susceptible isolates also had pncA gene mutations, and it is not clear whether this result is due to incorrect PZA susceptibility testing or an additional mechanism of resistance existing. We conclude that mutation in pncA is a major mechanism in PZA-resistance and the other molecular mechanisms exist in PZA-resistant clinical isolates in China.