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

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生物dna依赖的RNA聚合酶，大多数利福平耐药结核分枝杆菌的这种酶的b亚基发生改变。然而，大多数利福平耐药的临床分离株鸟分枝杆菌和胞内分枝杆菌在rpoB基因上没有任何突变。在抗酸菌对利福平耐药机制的研究中，我们发现并报道了21-OH和23-OH基团磷酸化或糖基化激活利福平是这些细菌的主要机制。除了这些激活外，我们还发现smgmatis具有通过核糖基化使ifampicin失活的能力，这是首次报道这种机制的病例。基因破坏实验表明，利福平的核糖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M.chelonae， M.flavescens, m .母牛和M.parafortuitum菌株通过核糖基化灭活利福平。从携带克隆病菌的耻垢分枝杆菌培养液中纯化了基化抗生素。为了研究该基因在大肠杆菌/ac启动子上的失活过程。细胞匀浆产生一种新的衍生物RIP-TAs，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。我们的结果还表明，RIP-TAs是导致核糖化利福平途径的中间产物，并且先前表征的基因是单（adp -核糖化）转移酶，但与同类其他酶没有序列相似性。

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.