The Potentiation And Mechanisms Of Tetracyclines Activity With 2-methoxy-1,4-naphthoquinone

Tetracycline antibiotics including tetracycline,chlorotetracycline,oxytetracycline and doxycycline,as effective broad-spectrum antibacterial agents were substantially used to address enteric pathogens and parasites infections in veterinary clinic.Nowadays,the widespread use of tetracyclines contributes to the emergence and dissemination of multidrug-resistant bacteria at alarming rates.The antimicrobial activities of tetracyclines were commonly subject to both multiple efflux pumps families and ribosome protective proteins,with low-level resistance and low risk of transmission characteristics.Notably,the structural optimizations and modifications of tetracyclines substantially overcome the main tetracycline resistance mechanisms.Alarmingly,the recent emergence and worldwide dissemination of high-level mobile tigecycline resistance genes tet(X3)/tet(X4)posed an enormous threat to the public health and agricultural settings.Urgently,feasible strategies to screen and identify the Tet(X3)/Tet(X4)inhibitors to combat the drug-resistant pathogens infections were absolutely imperative.In addition to the acquired resistance mechanisms,intrinsic defense mechanisms of bacterium are highly diverse and perpetually evolving.Considerable evidences revealed that microbial changes caused by bacterial metabolism observably modulate antibiotic efficacy.Thus,modulating bacterial metabolism presents a potentially feasible strategy to potentiate antibiotic efficacy.In the present study,an active compound 2-methoxy-1,4-naphthoquinone(MNQ)was found to potentiate antibiotics activity by targeting inactivating bacterial enzymes Tet(X3)/Tet(X4)and bacterial metabolism.Our study clarified the multiple mechanisms underlying the potentiation of tetracyclines activities by MNQ and presented a potential lead compound and novel anti-infection therapy strategy to tackle the growing problem of tetracyclines resistance.To screen the efficient and safe tetracyclines adjuvants,the synergistic activities of quinones,including benzoquinone,naphthoquinone,phenanthrenequinone and anthraquinone,in combination with tetracyclines were determined by a modified checkerboard method-broth microdilution assay.Plumbagin,2-methoxy-1,4-naphthoquinone,2,3-dichloro-1,4-naphthoquinone and p-benzoquinone significantly potentiated tetracyclines activity(including tetracycline and tigecycline)against tet(X4)-positive Escherichia coli(E.coli)(FICI ≤ 0.50).The synergistic efficacy and application prospects of active compounds were further contrasted and analyzed.A highly active compound 2-methoxy-1,4-naphthoquinones with abundant pharmacological activities and extensive sources was employed to explore and elucidate the synergistic efficacy and mechanisms.MNQ as a broad-spectrum antibacterial synergist effectively potentiated the antibacterial activity of various antibiotics including tetracyclines against tet(X3)/tet(X4)-positive E.coli and Gram-positive Staphylococcus aureus(S.aureus)by checkerboard method-broth microdilution method and time-dependent killing curves assay.Additionally,subinhibitory concentrations of MNQ inflicted less selective pressure and thwarted the emergence of tigecyclines resistance.Furthermore,the differential metabolites of bacteria with or without MNQ treatment were identified by metabolomics,and enrichment analyses confirmed the involvement and changes of multiple effective metabolic pathways,such as TCA cycle.MNQ was found to significantly interfere with bacterial metabolism and energy homeostasis,and trigger the reactive oxygen species(ROS)-associated oxidative stress.Further,exposure to MNQ infiltrated and elicited membrane structural damages,enhanced membrane permeability,dramatically dissipated proton motive force(PMF)and the intracellular ATP was sequentially decreased,and thus modulated the antibiotics susceptibility.The present study preliminarily revealed synergistic mechanisms of MNQ.To further confirm the inhibitory efficacy of MNQ on Tet(X3)/Tet(X4)resistance enzyme activities and the molecular mechanisms,Tet(X3)and Tet(X4)recombinant proteins with biological activity were successfully expressed and purified using prokaryotic expression system.Enzyme inhibition assay demonstrated that MNQ significantly inhibited the catalytic activity of inactivating enzymes Tet(X3)and Tet(X4)(P<0.05).MNQ(128 μg/m L)addition was found to dramatically alter the secondary structures of Tet(X3)/Tet(X4)such as α-helix andβ-turn angle by circular dichroism detection.Molecular dynamics simulations revealed the binding domains and patterns of Tet(X3)/Tet(X4)proteins with MNQ,and the active residues LEU222,MET215 and PHE224 of Tet(X3)and LEU219 and ILE234 of Tet(X4)were further mutated using site-specific mutagenesis techniques.The fluorescence quenching assay revealed that compared with the mutants,MNQ exhibited an evidently stronger binding force to the wild-type Tet(X3)/Tet(X4)proteins.Consistently,the synergistic activity of MNQ combined with tetracycline against mutant strains were significantly decreased compared to the wild-type strains(FICI > 0.50).These findings indicated that the above amino acid active residues were involved in the inhibitory efficacy of MNQ on Tet(X3)/Tet(X4)enzyme activities.Collectively,MNQ potently inhibited the catalytic hydroxylation reaction by engaging with the catalytic pocket of Tet(X3)/Tet(X4).Further exploration indicated that the expression and transcription of resistance gene tet(X3)/tet(X4)were prominently inhibited by MNQ.The mice thigh infection model and E.coli systemic infection model were established to evaluate the in vivo-synergistic activity of MNQ,as evidenced by mouse survival rate,colonization analysis and pathological histological observation.MNQ obviously potentiated the therapeutic efficacy of tetracycline in the mice thigh infection model.Compared with the infection group or the tetracycline monotherapy group,the combination therapy effectively decreased the bacterial load on the mice thigh muscle(P<0.05).In addition,compared with the infection group or the methacycline monotherapy group,the survival of mice following combination therapy was observably increased by 25.0% and alleviated the bacterial burden and pathological injury(P<0.05)in the E.coli systemic infected mice.Overall,MNQ could notably potentiate the efficacy of tetracycline antibiotics in vivo.Collectively,the present study found that native compound MNQ evidently potentiated the tetracyclines activity against drug-resistant pathogens in vitro/in vivo by multi-target effects.Moreover,the current study presented a novel therapeutic strategy and a safe and lead compound to tackle clinically complex drug-resistant pathogens infections.