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

Abstract

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