Novel 5-Nitrofuran-Activating Reductase in Escherichia coli

Abstract

The global spread of multidrug-resistant enterobacteria warrants new strategies to combat these pathogens. One possible approach is the reconsideration of “old” antimicrobials, which remain effective after decades of use. Synthetic 5-nitrofurans such as furazolidone, nitrofurantoin, and nitrofurazone are such a class of antimicrobial drugs. Recent epidemiological data showed a very low prevalence of resistance to this antimicrobial class among clinical Escherichia coli isolates in various parts of the world, forecasting the increasing importance of its uses to battle antibiotic-resistant enterobacteria. However, although they have had a long history of clinical use, a detailed understanding of the 5-nitrofurans’ mechanisms of action remains limited. Nitrofurans are known as prodrugs that are activated in E. coli by reduction catalyzed by two redundant nitroreductases, NfsA and NfsB. Furazolidone, nevertheless, retains relatively significant antibacterial activity in the nitroreductase-deficient ΔnfsA ΔnfsB E. coli strain, indicating the presence of additional activating enzymes and/or antibacterial activity of the unreduced form. Using genome sequencing, genetic, biochemical, and bioinformatic approaches, we discovered a novel 5-nitrofuran-activating enzyme, AhpF, in E. coli. The discovery of a new nitrofuran-reducing enzyme opens new avenues for overcoming 5-nitrofuran resistance, such as designing nitrofuran analogues with higher affinity for AhpF or screening for adjuvants that enhance AhpF expression.

In vitro synergy between sodium deoxycholate and furazolidone against enterobacteria

“…To investigate the role of these two enzymes in the DOC-FZ synergy, we examined the interaction between DOC and FZ in the ΔnfsA ΔnfsB E. coli strain lacking both of these enzymes. In agreement with the FZ activation role of NfsA/NfsB, disruption of these two genes led to an increase in the MIC causing 50% growth inhibition by a factor of 8 [12]. Nonetheless, the synergy between DOC and FZ still remained significant in the ΔnfsA ΔnfsB genetic background, with the FICI at 50% growth inhibition as low as 0.3125 (Fig.…”

“…We recently identified a third FZactivating enzyme in E. coli, AhpF, which contributes to this prodrug activation in the ΔnfsA ΔnfsB genetic background [12]. Nevertheless, FZ was still effective against the ΔnfsA ΔnfsB ΔahpF mutant with the MIC 50% being 10-fold increased over the wild-type parent, suggesting the presence of additional 5-nitrofuran-activating enzymes in this organism and/or activation-independent mechanisms of action [12]. Here we analyzed the DOC-FZ synergy in the triple ΔnfsA ΔnfsB ΔahpF mutant and showed that the FICI value was close to that of the wildtype parent and double mutant (Fig.…”

“…FZ is used to treat bacterial diarrhea, giardiasis and as a component in combinatorial therapy for Helicobacter pylori infections; NIT and NFZ are used for urinary tract infections and topical applications, respectively [11]. They are prodrugs which require reductive activation, which is mediated in E. coli largely by two type-I oxygen-insensitive nitroreductases, NfsA and NfsB, and in their absence by type II oxygen-sensitive nitroreductase, AhpF [12]. NfsA and NfsB perform stepwise 2-electron reduction of the nitro moiety of the compound into two redox-reactive nitroso and hydroxylamino intermediates and biologically inactive amino-substituted product [13,14].…”

Nitrofurans: Revival of an “old” drug class in the fight against antibiotic resistance

“…The identity of enzymes catalyzing the oxygen-sensitive nitrofuran reduction has not been revealed until 2019, when we identified a novel nitrofuran-activating enzyme by selecting for furazolidone-resistant mutants in an nfsA nfsB E. coli double knock-out strain. A total of 15 independently isolated mutants resistant to bactericidal concentration of furazolidone contained mutations in the ahpF gene, which encodes a component of the antioxidant alkyl hydroperoxide reductase [9]. Subsequent enzymatic assays of purified AhpF protein determined that this enzyme is a type II oxygen-sensitive nitroreductase [9].…”

“…In this spirit, a possible novel nitrofuran resistance mechanism(s), besides well-known nfsA/nfsB mutations, must receive due attention and resources in order to sustain the utility of this drug class. While the prevalence of nitrofuran resistance among E. coli clinical isolates in recent epidemiology surveys around the world is still low [9], nitrofuran-hyperresistant isolates with the MIC higher than 128 μg/mL have been encountered [15,16]. This high level of resistance cannot solely be explained by mutations in the nfsA, nfsB, and ahpF genes and points to unknown resistance determinants that are already circulating in pathogenic strains.…”