Mutational analysis of the rifampicin glycosyl-transferase (rgt) inactivation protein from Nocardia brasiliensis and its relationship to the vancomycin resistance of this organism

[摘要] Rifampicin is a chemotherapeutic agent used to combat mycobacterial and nocardialinfections. Four enzymatic inactivation mechanisms have been identified which arepartially responsible for the increasing number of rifampicin resistant strains. These areADP-ribosylation, phosphorylation, decomposition and glucosylation. The gene encodingthe latter, rgt, has been cloned and characterized from the opportunistic pathogenNocardia brasiliensis. However, as of yet nothing is known of these inactivationenzymes. Thus in order to study the properties of the mechanism it is necessary toobserve structure-function relationships through the characterization of mutants.Furthermore, the rgt gene confers a small yet reproducible increase to the vancomycinMIC. This has indicated that there may be other enzymatic mechanisms which areinvolved in the inactivation of vancomycin. Vancomycin is an important antibiotic as it isused to treat gram-positive infections by multi-drug resistant strains. Hitherto, nomechanisms of enzymatic inactivation have been identified for vancomycin. Thus inorder to identify regions of DNA which may play a role in the high level resistance tovancomycin as observed in N. brasiliensis it was necessary to screen a genomic library ofthis organism. This was performed in a gram-positive background. No clones wereidentified in this study that had an increased resistance to vancomycin, indicating that theDNA involved in the phenotype is greater than that of the average insert size of thelibrary, 1.9 kb.Future work will thus involve the generation of a genomic library with larger fragmentsand the subsequent screening of this. Additionally, performing a mutational analysis onthe rgt gene may provide further insight into the specifics of the inactivation enzymes andthus will contribute to combating infection by opportunistic and other pathogens.

[发布机构] University of the Witwatersrand