Natural flavonoids disrupt bacterial iron homeostasis to potentiate colistin efficacy

Abstract

In the face of the alarming rise in global antimicrobial resistance, only a handful of novel antibiotics have been developed in recent decades, necessitating innovations in therapeutic strategies to fill the void of antibiotic discovery. Here, we established a screening platform mimicking the host milieu to select antibiotic adjuvants and found three catechol-type flavonoids-7,8-dihydroxyflavone, myricetin, and luteolin-prominently potentiating the efficacy of colistin. Further mechanistic analysis demonstrated that these flavonoids are able to disrupt bacterial iron homeostasis through converting ferric iron to ferrous form. The excessive intracellular ferrous iron modulated the membrane charge of bacteria via interfering the two-component system pmrA/pmrB, thereby promoting the colistin binding and subsequent membrane damage. The potentiation of these flavonoids was further confirmed in an in vivo infection model. Collectively, the current study provided three flavonoids as colistin adjuvant to replenish our arsenals for combating bacterial infections and shed the light on the bacterial iron signaling as a promising target for antibacterial therapies.

Fig. 1.. Primary screening identified three catechol-type flavonoids as colistin adjuvants.

(A) Schematic illustration of the screening procedure. (B) Three flavonoids were identified as potent colistin adjuvants from the phytochemical collection. (C) Structure-activity relationship (SAR) analysis demonstrated the presence of catechol moiety on flavonoids as a prerequisite for observed synergy. (D) Fractional inhibitory concentration indices (FICIs) showing the combination between colistin and catechol-type/non–catechol-type flavonoids.

Fig. 2.. Candidate flavonoids restore the colistin activity against colistin-resistant bacteria and reduce the emergence of resistance.

(A) Isobolograms of the combination of colistin (CS) and 7,8-DHF against different colistin-resistant isolates (17ES, mcr-positive S. typhimurium; 2012FS, mcr-positive E. coli; CMG, mcr-positive K. pneumoniae; ZJ18-19, mgrB-disrupted K. pneumoniae). (B) Time-dependent killing of colistin-resistant isolates by the combination of colistin and 7,8-DHF. (C) The addition of candidate flavonoids prevented the development of colistin resistance in vitro.

Fig. 3.. Candidate flavonoids target bacterial iron homeostasis to potentiate colistin activity.

(A) Gene Ontology (GO) annotation analysis of the DEGs in bacteria treated by the combination of colistin and 7,8-DHF. ABC, ATP-binding cassette. (B) Expression level of genes responsible for bacterial iron acquisition. (C) Isobolograms of the combination of colistin and candidate flavonoids against tonB-/feoB-deficient mutants. (D) Candidate flavonoids reduced bacterial intracellular iron contents. (E) Candidate flavonoids rapidly converted intracellular iron from ferric form to ferrous form. (F) Catechol-type flavonoids strongly reduced iron to the ferrous form. (G) The addition of exogenous ferric iron abolished the synergy between colistin with candidate flavonoids.

Fig. 4.. Candidate flavonoids dysregulate bacterial iron status to inactivate pmrA phosphorylation.

(A) Pathway enrichment of DEGs in bacteria treated by the combination of colistin and 7,8-DHF. (B) Scheme of bacterial ion-responsive two-component systems (TCSs). (C) Transcriptional reporter assay demonstrated that the two genes modulating LPS modification under pmrA/pmrB control were suppressed by candidate flavonoids. (D) Candidate flavonoids reduced the phosphorylation of regulator PmrA. (E) Synergism between colistin and candidate flavonoids was abolished in the mutants lacking pmrA and pmrB. (F) Candidate flavonoids enhanced colistin binding on bacterial membrane by modulating the pmrA/pmrB TCS. ppb, parts per billion. (G) Membrane permeability of bacterial cells after treatment with colistin with or without 7,8-DHF, MYR, and LUT. ns, not significant. (H) Accumulation of ROS in bacterial cells after treatment with colistin with or without 7,8-DHF, MYR, and LUT. DCF, Dichlorofluorescein.

Fig. 5.. Candidate flavonoids enhance colistin efficacy in vivo.

(A) Schematic illustration of experimental protocol for the animal trial. (B) Survival curve of infected mice with different treatments. (C) Bacterial load in the liver of infected mice with different treatments. (D) Bacterial load in the spleen of infected mice with different treatments. (E) Bacterial load in feces of infected mice with different treatments.

Fig. 6.. Mechanistic insight into synergistic interaction between colistin and candidate flavonoids.