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Analyzes of a new rifampicin resistant mechanism by acid-fast bacteria

抗酸细菌对利福平的新耐药机制分析

基本信息

批准号：
08670301
负责人：
MIKAMI Yuzuru
金额：
$ 1.54万
依托单位：
Chiba Univ.
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Rifampicin is avaluable chemotherapeutic agent. Its antimicrobial activity is due to inhibition of prokaryotic DNA-dependent RNA polymerases and most rifampin-resistant M.tuberculosis have been reported to have an alteration in the b-subunit of this enzyme. However, most rifampicin-resistant clinical isolates of M.avium and M.intracellulare do not have any mutations in the rpoB gene.During our studies on the mechanisms of rifampicin resistance in acid-fast bacteria, we found and reported that in activation of rifampicin due to the phosphorylation or glucosylation of 21-OH and 23-OH group is major mechanismsin these bacteria. In addition to these in activation, we found that M.smegmatishas an ability to inactivaterifampicin by ribosylation, the first reported case of such a mechanism. Gene disruption experiments showed that ribsylative inactivation of rifampicin is a major contributor to the low susceptibility of M.smegmatis and that this is the principal rifampicin inactivation mechanism in this bacterium. We have also found that many mycobacteria including M.chelonae, M.flavescens, M.vaccae, and M.parafortuitum strains inactivated rifampicin by ribosylation. Theribosylated antibiotic was purified from culturebroth of M.smegmatis carrying the cloned generesponsible. To study this inactivation process the gene was expressed off the/ac promoter in Escherichia coli. The cell homogenates generated a novel derivative RIP-TAs, determined to be 23- [O- (ADP-ribosyl) ] rifampicin. To our knowledge, this is the first case of ADP-ribosylation as a mechanism of antibiotic inactivation. Our results also indicated that RIP-TAs was an intermediate in the pathway leading to ribosylated-rifampicin and that the previously characterized gene was a mono (ADP-ribosyl) transferase which, however, showed no sequence similarity to other enzymes of this class.

利福平是一种有价值的化疗药物。它的抗菌活性是由于抑制原核DNA依赖性RNA聚合酶，据报道，大多数利福平耐药结核分枝杆菌的这种酶的b亚基发生了改变。然而，大多数耐利福平的鸟分枝杆菌和胞内分枝杆菌临床分离株不存在rpoB基因突变，我们在抗酸细菌耐利福平机制的研究中发现并报道，21-OH和23-OH的磷酸化或糖基化是这些细菌对利福平耐药的主要机制。除了这些激活作用外，我们还发现耻垢分枝杆菌具有通过核糖基化使利福平失活的能力，这是第一例报道的这种机制。基因破坏实验表明，利福平的核糖基化失活是导致耻垢分枝杆菌低敏感性的主要原因，这是该细菌中利福平失活的主要机制。我们还发现许多分枝杆菌，包括龟分枝杆菌、黄分枝杆菌、母牛分枝杆菌和副偶发分枝杆菌菌株，通过核糖基化使利福平失活。从携带克隆的相关基因的耻垢分枝杆菌培养液中纯化了硫代糖基化抗生素。为了研究这种失活过程，在大肠杆菌中表达了该基因的/ac启动子。细胞匀浆产生一种新的衍生物RIP-TA，确定为23- [O-（ADP-核糖基）]利福平。据我们所知，这是ADP-核糖基化作为抗生素失活机制的第一例。我们的研究结果还表明，RIP-TAs是一个中间体的途径，导致核糖基化利福平和先前表征的基因是一个单（ADP-核糖基）转移酶，然而，没有显示出序列相似性的其他酶这一类。