pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis
pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis
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DOI：
10.1016/j.tube.2013.03.005
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发表时间：
2013-09-01
期刊：
影响因子：
3.2
通讯作者：
Zimic, Mirko
中科院分区：
文献类型：
--
作者：
Sheen, Patricia;Lozano, Katherine;Zimic, Mirko
吡嗪酰胺酶（PZAse）编码基因pncA的突变被认为是结核分枝杆菌吡嗪酰胺（PZA）耐药的主要原因。然而，最近的研究表明，对PZA的耐药机制并不单一。吡嗪酸（POA）外排率是PZA敏感性韦恩试验的基础，其定量测量已被发现是PZA耐药性的高度敏感和特异性预测因子。从生物学角度考虑，PZAse活性、pncA表达水平和POA外排泵系统的效率直接决定了POA外排速率，本研究分析了PZAse活性、pncA表达和POA外排速率对PZA抗性的个体和校正贡献。分析具有已知微生物PZA敏感性或抗性的结核菌株。对于每种菌株，重组产生PZAse并测量其酶活性。用定量RT-PCR检测pncA mRNA的表达水平，并测定POA外排率。通过DNA测序检测pncA启动子中的突变。所有因素通过多元回归分析进行评估，以确定其对PZA耐药水平的调整效果。在pncA野生型耐药菌株中观察到与pncA启动子区突变相关的低水平pncA表达。在调整其他因素后，POA外排率是最好的预测因子，其次是PZAse活性。这些结果表明，依赖于pncA突变或PZAse活性的测试可能比测量POA流出率的测试更少预测真实的PZA抗性。这应该根据确定PZA耐药性的替代检测方法的发展进行进一步分析。(c)2013爱思唯尔有限公司保留所有权利。
Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system.This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance.Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance.Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance. (c) 2013 Elsevier Ltd. All rights reserved.