Sun Z, Zhang Y
Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Antimicrob Agents Chemother. 1999 Mar;43(3):537-42. doi: 10.1128/AAC.43.3.537.
Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug that requires conversion to the bactericidal compound pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) activity of nicotinamidase to show activity against Mycobacterium tuberculosis. Mutations leading to a loss of PZase activity cause PZA resistance in M. tuberculosis. M. kansasii is naturally resistant to PZA and has reduced PZase activity along with an apparently detectable nicotinamidase activity. The role of the reduction in PZase activity in the natural PZA resistance of M. kansasii is unknown. The MICs of PZA and POA for M. kansasii were determined to be 500 and 125 micrograms/ml, respectively. Using [14C]PZA and [14C]nicotinamide, we found that M. kansasii had about 5-fold-less PZase activity and about 25-fold-less nicotinamidase activity than M. tuberculosis. The M. kansasii pncA gene was cloned on a 1.8-kb BamHI DNA fragment, using M. avium pncA probe. Sequence analysis showed that the M. kansasii pncA gene encoded a protein with homology to its counterparts from M. tuberculosis (69.9%), M. avium (65.6%), and Escherichia coli (28.5%). Transformation of naturally PZA-resistant M. bovis BCG with M. kansasii pncA conferred partial PZA susceptibility. Transformation of M. kansasii with M. avium pncA caused functional expression of PZase and high-level susceptibility to PZA, indicating that the natural PZA resistance in M. kansasii results from a reduced PZase activity. Like M. tuberculosis, M. kansasii accumulated POA in the cells at an acidic pH; however, due to its highly active POA efflux pump, the naturally PZA-resistant species M. smegmatis did not. These findings suggest the existence of a weak POA efflux mechanism in M. kansasii.
吡嗪酰胺（PZA）是烟酰胺的类似物，是一种前体药物，需要通过烟酰胺酶的细菌吡嗪酰胺酶（PZase）活性转化为杀菌化合物吡嗪酸（POA），才能显示出对结核分枝杆菌的活性。导致PZase活性丧失的突变会导致结核分枝杆菌对PZA产生耐药性。堪萨斯分枝杆菌天然对PZA耐药，其PZase活性降低，同时烟酰胺酶活性明显可检测到。PZase活性降低在堪萨斯分枝杆菌天然PZA耐药性中的作用尚不清楚。堪萨斯分枝杆菌对PZA和POA的最低抑菌浓度分别测定为500和125微克/毫升。使用[14C]PZA和[14C]烟酰胺，我们发现堪萨斯分枝杆菌的PZase活性比结核分枝杆菌低约5倍，烟酰胺酶活性低约25倍。使用鸟分枝杆菌pncA探针，将堪萨斯分枝杆菌pncA基因克隆到一个1.8 kb的BamHI DNA片段上。序列分析表明，堪萨斯分枝杆菌pncA基因编码的蛋白质与其来自结核分枝杆菌（69.9%）、鸟分枝杆菌（65.6%）和大肠杆菌（28.5%）的对应物具有同源性。用堪萨斯分枝杆菌pncA转化天然对PZA耐药的牛分枝杆菌卡介苗可使其对PZA产生部分敏感性。用鸟分枝杆菌pncA转化堪萨斯分枝杆菌导致PZase的功能性表达和对PZA的高度敏感性，表明堪萨斯分枝杆菌的天然PZA耐药性是由于PZase活性降低所致。与结核分枝杆菌一样，堪萨斯分枝杆菌在酸性pH值下在细胞内积累POA；然而，由于其高度活跃的POA外排泵，天然对PZA耐药的耻垢分枝杆菌则不会。这些发现表明堪萨斯分枝杆菌存在一种较弱的POA外排机制。
