Comparison of inhibition of Escherichia coli topoisomerase IV by quinolones with DNA gyrase inhibition

Abstract

In order to examine the inhibitory activities of quinolones against topoisomerase IV, both subunits of this enzyme, ParC and ParE, were purified from Escherichia coli. The specific activity of topoisomerase IV decatenation was found to be more than five times greater than that of topoisomerase IV relaxation. Thus, the decatenation activity of topoisomerase IV seems the most relevant activity for use in studies of drug inhibition of this enzyme. Although topoisomerase IV was less sensitive to quinolones than DNA gyrase, the 50% inhibitory concentrations for decatenation were significantly lower than those for type I topoisomerases. Moreover, there was a positive correlation between the inhibitory activity against topoisomerase IV decatenation and that for DNA gyrase supercoiling. These results imply that topoisomerase IV could be a target for the quinolones in intact bacteria and that quinolones could inhibit not only supercoiling of DNA gyrase but also decatenation of topoisomerase IV when high concentrations of drug exist in bacterial cells.Four topoisomerases have been isolated from Escherichia coli so far: topoisomerase I (39), topoisomerase II (DNA gyrase) (9), topoisomerase III (34), and topoisomerase IV (16). Among these enzymes, topoisomerases I, II, and IV are implicated in the control of the intracellular supercoiling density of chromosomal or plasmid DNA, affecting the efficacy of DNA replication and transcription (4, 6-8, 20, 24, 29, 36, 37). In addition, some topoisomerases are required for the segregation of daughter chromosomes or plasmid DNA (1,21,35,40). Although all of these topoisomerases are capable of changing the topology of DNA by a cleaving and rejoining step, each enzyme appears to possess a favored reaction in vitro. Type I topoisomerases, topoisomerase I and topoisomerase III, show efficient relaxing and decatenating activity, respectively, in the absence of ATP (2,3,5,11,29,30,34 subunits of DNA gyrase), respectively (16). Similar sequences are located especially around the region of DNA gyrase known as the "quinolone resistance-determining region." The quinolone antibacterial agents, in particular the fluoroquinolones, strongly inhibit DNA gyrase, which results ultimately in bacterial death (32). However, the inhibitory activities of quinolones against type I topoisomerases, topoisomerase I (25) and topoisomerase III (2) in E. coli, are weak. The inhibitory activities of fluoroquinolones against topoisomerase IV have yet to be fully investigated. The similarity in amino acid sequence between DNA gyrase and topoisomerase IV, especially around sites in the GyrA protein of DNA gyrase at positions known to produce quinolone-resistant DNA gyrases (41), implies that quinolones may be capable of inhibiting the activity of topoisomerase IV as well as against DNA gyrase.In this study, in order to define the inhibitory activities of quinolones against topoisomerase IV, both subunits of topoisomerase IV, ParC and ParE, were purified, and optimum conditions for the decatenating activity of topoi...

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance

“…Recent studies have confirmed our original suggestion (5,10) that the Ser-833Trp (serine-to-tryptophan mutation at position 83) GyrA resistance mutation acts by diminishing binding of quinolones to the gyrase complex (37,39). Mutations in the related type II enzyme, DNA topoisomerase IV, a C 2 E 2 complex encoded by parC and parE genes and essential for chromosome segregation, are a secondary event (1,3,(17)(18)(19). Interestingly, in Staphylococcus aureus, mutations in the parC (grlA) gene confer low-level resistance and precede those in gyrA (7,8,34).…”

In vitro and in vivo antifungal activities of DU-6859a, a fluoroquinolone, in combination with amphotericin B and fluconazole against pathogenic fungi

“…The inhibitory activity of DU-6859a on bacterial gyrases from various bacteria has been proven to be much more potent than those of other fluoroquinolones, although its activity against mammalian topoisomerase II is negligible (14). In addition, recent studies suggest that fluoroquinolones including DU-6859a inhibit bacterial topoisomerase IV, a novel site of action for this class of antimicrobial agents (7,13). Along this line, more intensive and careful research on fungal topoisomerases might generate a new understanding of the antifungal action of DU-6859a.…”

“…The bactericidal activities of fluoroquinolones are largely based on their inhibitory effects on bacterial DNA gyrase (topoisomerase II) (23,24); however, some other effects have recently been suggested (7,13). There are reports that some fluoroquinolones potentiate the activities of antifungal agents (21,26,28).…”

Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus

“…It has also been recently determined that DNA topoisomerase IV is an in vivo target in E. coli by constructing isogenic mutants leading to a Ser-80-to-Leu substitution in the ParC subunit (18). Undisputable evidence that S. aureus DNA topoisomerase IV is a target of the quinolone drugs will be provided by biochemical analysis of the purified enzyme, as has been shown for E. coli DNA topoisomerase IV (12,29), and by reverse genetics.…”