Target-Mediated Fluoroquinolone Resistance in Neisseria gonorrhoeae: Actions of Ciprofloxacin against Gyrase and Topoisomerase IV

Abstract

Fluoroquinolones make up a critically important class of antibacterials administered worldwide to treat human infections. However, their clinical utility has been curtailed by target-mediated resistance, which is caused by mutations in the fluoroquinolone targets, gyrase and topoisomerase IV. An important pathogen that has been affected by this resistance is Neisseria gonorrhoeae, the causative agent of gonorrhea. Over 82 million new cases of this sexually transmitted infection were reported globally in 2020. Despite the impact of fluoroquinolone resistance on gonorrhea treatment, little is known about the interactions of this drug class with its targets in this bacterium. Therefore, we investigated the effects of the fluoroquinolone ciprofloxacin on the catalytic and DNA cleavage activities of wildtype gyrase and topoisomerase IV and the corresponding enzymes that harbor mutations associated with cellular and clinical resistance to fluoroquinolones. Results indicate that ciprofloxacin interacts with both gyrase (its primary target) and topoisomerase IV (its secondary target) through a water−metal ion bridge that has been described in other species. Moreover, mutations in amino acid residues that anchor this bridge diminish the susceptibility of the enzymes for the drug, leading to fluoroquinolone resistance. Results further suggest that ciprofloxacin primarily induces its cytotoxic effects by enhancing gyrase-mediated DNA cleavage as opposed to inhibiting the DNA supercoiling activity of the enzyme. In conclusion, this work links the effects of ciprofloxacin on wildtype and resistant gyrase to results reported for cellular and clinical studies and provides a mechanistic explanation for the targeting and resistance of fluoroquinolones in N. gonorrhoeae.

Gyrase and Topoisomerase IV: Recycling Old Targets for New Antibacterials to Combat Fluoroquinolone Resistance

“…The water–metal-ion bridge has been shown to play a critical role in mediating the actions of fluoroquinolones against gyrase and topoisomerase IV in every species examined to date. ,,− Although this bridge is the primary conduit between the drug and the type II topoisomerases, its architecture is nuanced and differs from enzyme to enzyme. In some cases, both the serine and the acidic residue are important for bridge formation, − ,, while in others, only one of the amino acids serves as the primary bridge anchor. , For example, the serine residue has been replaced by an alanine residue in M. tuberculosis gyrase. , As a result, the acidic residue is the major anchor for the water–metal-ion bridge . This finding explains the reduced potency of most fluoroquinolones against this enzyme. ,− …”

“…The function of the water–metal-ion bridge is dynamic and varies across type II enzymes. , In the majority of enzymes examined, the most important role of the bridge is to serve as a vehicle for fluoroquinolone binding, as evidenced by a coordinate loss of catalytic inhibition and DNA cleavage enhancement. ,,, However, with some enzymes, disruption of the bridge has little effect on fluoroquinolone binding, as evidenced by sustained catalytic inhibition, but undermines the ability of the drug to enhance DNA cleavage. , For the latter, it is assumed that the role of the bridge is to position the fluoroquinolone in the enzyme–DNA complex. In these cases, the fact that resistance tracks with the loss of DNA cleavage implies that the mechanism of drug toxicity must also be due to the induction of enzyme-generated double-stranded DNA breaks as opposed to the loss of essential enzyme activities. ,,,, Thus, understanding the bridge function can provide important insights into the mechanism of fluoroquinolone cytotoxicity.…”

“… 1 , 3 , 160 − 165 Although this bridge is the primary conduit between the drug and the type II topoisomerases, its architecture is nuanced and differs from enzyme to enzyme. In some cases, both the serine and the acidic residue are important for bridge formation, 160 − 162 , 165 , 167 while in others, only one of the amino acids serves as the primary bridge anchor. 163 , 164 For example, the serine residue has been replaced by an alanine residue in M. tuberculosis gyrase.…”

“… 1 , 3 In the majority of enzymes examined, the most important role of the bridge is to serve as a vehicle for fluoroquinolone binding, as evidenced by a coordinate loss of catalytic inhibition and DNA cleavage enhancement. 160 , 161 , 163 , 167 However, with some enzymes, disruption of the bridge has little effect on fluoroquinolone binding, as evidenced by sustained catalytic inhibition, but undermines the ability of the drug to enhance DNA cleavage. 162 , 167 For the latter, it is assumed that the role of the bridge is to position the fluoroquinolone in the enzyme–DNA complex.…”

“… 160 , 161 , 163 , 167 However, with some enzymes, disruption of the bridge has little effect on fluoroquinolone binding, as evidenced by sustained catalytic inhibition, but undermines the ability of the drug to enhance DNA cleavage. 162 , 167 For the latter, it is assumed that the role of the bridge is to position the fluoroquinolone in the enzyme–DNA complex. In these cases, the fact that resistance tracks with the loss of DNA cleavage implies that the mechanism of drug toxicity must also be due to the induction of enzyme-generated double-stranded DNA breaks as opposed to the loss of essential enzyme activities.…”