Two Pathways And Mechanisms Underlying Bactericidal Action Of Quinolone Antimicrobials

Bacterial resistance to antimicrobial therapy is a major challenge to the clinical management of infectious diseases.One approach for controlling the emergence of resistance is to find new ways to reduce bacterial tolerance to antimicrobials and to markedly improve bactericidal activity.The present proposal focuses on understanding how the quinolones kill bacteria.These broad-spectrum agents,which are highly effective and have an excellent safety record,are now among the most widely used antimicrobials.Since the invention of the first quinolone-class drug,nalidixic acid,in 1962,4 generations and hundreds of new derivatives have been made and bacterial type ?A topoisomerases(DNA gyrase and topoisomerase ?)have been identified as the targets of such antibacterial agents.Previous studies have shown that bacterial DNA replication,which requires the cooperation of DNA gyrase and topoisomerase ?,is a vital process for bacterial growth and survival.The widespread clinical application and huge success of type ?A topoisomerase-targeting agents in the past 50 years has also demonstrated the clinical relevance of this class of antimicrobials.However,misuse and over-use of quinolone class compounds have led to a steady increase in bacterial resistance.To preserve the usefulness of this proven antimicrobials for treatment efficacy,the molecuar mechanisms underlying quinolone-mediated killing is urgently needed.Our recent work reveals the existence of two distinct quinolone-mediated killing pathways,each of which is controlled by unidentified proteins.Mutations in genes encoding some of these unidentified proteins are expected to neither grow nor die when exposed to quinolones.Three such quinolone-toleratant mutants have already been obtained in our preliminary work.In the present application,we propose to dissect the two pathways of quinolone killing by obtaining drug tolerant mutants via enrichment and selection procedures that we recently developed.Mutants obtained will be subjected to whole-genome DNA sequence analysis to identity the genes encoding "lethal proteins"involved in the killing pathway that requires de novo in synthesis and the genes encoding"auxiliary proteins" contributing to the protein synthesis-insensitive pathway of killing.We expect to reveal molecular details on how these "lethal protein(s)" and "auxiliary protein(s)" interact with quinolone-topoisomerase-DNA ternary complex,process the complex,and release double-stranded DNA breaks from the complex.Such work will eventually elucidate mechanisms of quinolone lethality,which would in turn help devise high-throughout screening systems for identification of quinolone lethality potentiators and new quinolone derivatives that are highly active in killing non-growing,dormant pathogens.Successful completion of the present program wil guide new antimicrobial development and help control development of clinical resistance.