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Objective To investigate the inhibitory effects of pyruvate-ferredoxin oxidoreductase(PFOR)by luteolin and its anti-Clostridium difficile effect.Methods The PFOR encoding sequence of Clostridium difficile was cloned into the expression vector pET-2a and transformed into competent Escherichia coli.The crude enzyme was prepared after induction with IPTG(Isopropyl β-D-Thiogalactoside).The inhibitory rate of the test compounds on PFOR was determined after an 8-hour anaerobic reaction between PFOR and 40 μmol·L-1 of test compounds at 25℃.The minimum inhibitory concentration(MIC)of PFOR inhibitors against C.difficile strains(ATCC BAA 1382 and ATCC BAA 1870)was determined by monitoring the OD600 of the bacterial culture.Molecular docking was performed to investigate the possible interaction mechanisms between PFOR and inhibitors.Results Among the tested compounds,the luteolin showed the strongest inhibitory activity against PFOR,with a single-point inhibition rate of approximately 33%,which is comparable to that observed with the positive inhibitor nitazoxanide(40%).Molecular docking revealed that luteolin could form hydrogen bonds with Asp428,Val431,Gly429,Asp456,Lys458,Lys459,and other residues in the PFOR domain.The MIC of luteolin against C.difficile was approximately 32 μg·mL-1.Conclusion Luteolin exhibits good activity against C.difficile,and PFOR may be a target for its antibacterial action.
RESUMEN
To evaluate whether iron concentration in TYM medium influence on hydrogenosomal enzyme gene expression and hydrogenosomal membrane potential of Trichomonas vaginalis, trophozoites were cultivated in irondepleted, normal and iron-supplemented TYM media. The mRNA of hydrogenosomal enzymes, such as pyruvate ferredoxin oxidoreductase (PFOR), hydrogenase, ferredoxin and malic enzyme, was increased with iron concentrations in T. vaginalis culture media, measured by RT-PCR. Hydrogenosomal membrane potentials measured with DiOC6 also showed similar tendency, e.g. T. vaginalis cultivated in iron-depleted and iron-supplemented media for 3 days showed a significantly reduced and enhanced hydrogenosomal membrane potential compared with that of normal TYM media, respectively. Therefore, it is suggested that iron may regulate hydrogenosomal activity through hydrogenosomal enzyme expression and hydrogenosomal membrane potential.