A novel inhibitor of monooxygenase reversed the activity of tetracyclines against tet(X3)/tet(X4)-positive bacteria

BACKGROUND: Tigecycline is one of the few last-resort antibiotics for the treatment of carbapenem-resistant Enterobacteriaceae infection, the incidence of which has been rapidly increasing. However, the emergence and spread of tigecycline resistance genes tet(X) (including tet(X3) and tet(X4)) has largely compromised the efficient usage of tetracyclines in the clinical settings. METHODS: The synergistic effect was determined by a checkerboard minimum inhibitory concentration (MIC) assay, a time-killing assay and scanning electron microscopy (SEM) analysis. In-depth mechanisms were defined using an enzyme inhibition assay, western blotting, RT-PCR analysis, molecular dynamics (MD) simulations, biolayer interferometry (BLI) assay and metabolomics analysis. FINDINGS: Herein, our work identified a natural compound, plumbagin, as an effective broad-spectrum inhibitor of Tet(X) (also known as monooxygenase) by simultaneously inhibiting the activity and the production of Tet(X3)/Tet(X4). Plumbagin in combination with tetracyclines showed a synergistic bactericidal effect against Tet(X3)/Tet(X4)-producing bacteria. Mechanistic studies revealed that direct engagement of plumbagin with the catalytic pocket of Tet(X3)/Tet(X4) induced an alternation in its secondary structure to inhibit the activity of these monooxygenases. As a consequence, monotherapy or combination therapy with plumbagin increases the oxidative stress and metabolism in bacteria. Moreover, in a mouse systemic infection model of tet(X4)-positive E. coli, the combination of plumbagin and methacycline exhibited remarkable treatment benefits, as shown by a reduced bacterial load and the alleviation of pathological injury. INTERPRETATION: Plumbagin, as an inhibitor of Tet(X3)/Tet(X4), represents a promising lead drug, as well as an adjunct with tetracyclines to treat bacterial infections, especially for extensively drug-resistant bacteria harbouring Tet(X3)/Tet(X4). FUNDING: The National Natural Science Foundation of China.