| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1996: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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.
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