Antimicrobial mechanism of action of transferrins : Selective inhibition of H + - ATPase 1 2 María

26 27 Two bacterial species, Pseudomonas aeruginosa and Lactococcus lactis, with different 28 metabolic features, were used as a comparative experimental model to investigate the antimicrobial 29 target and mechanism of transferrins. In anaerobiosis, P. aeruginosa cells were not susceptible to 30 lactoferrin (hLf) or transferrin (hTf). In aerobiosis, the cells were susceptible but O2-consumption 31 was not modified, indicating that components of the electron transport chain (ETC) were not 32 targeted. However, the respiratory chain inhibitor piericidin A significantly reduced the killing 33 activity of both proteins. Moreover, 2,6-dichlorophenolindophenol (DCIP), a reducing agent that 34 accepts electrons from the ETC coupled to H + -extrusion, made P. aeruginosa susceptible to hLf and 35 hTf in anaerobiosis. These results indicated that an active cooperation of the cell was indispensable 36 for the antimicrobial effect. In L. lactis cells lacking an ETC, the absence of a detectable 37 transmembrane electrical potential in hLf-treated cells suggested a loss of the H + -ATPase activity. 38 Furthermore, the inhibition of ATPase activity and H + -translocation (inverted membrane vesicles) 39 provided direct evidence of the ability of hLf to inhibit H + -ATPase in L. lactis. Based on these data, 40 we propose that hLf and hTf also inhibit the H + -ATPase of respiring P. aeruginosa cells. Such 41 inhibition thereby interferes with re-entry of H + from the periplasmic space to the cytoplasm, 42 resulting in perturbation of intracellular pH and the transmembrane proton gradient. Consistent with 43 this hypothesis, periplasmic H + -accumulation was prevented by anaerobiosis or by piericidin A, or 44 induced by DCIP in anaerobiosis. Collectively, these results indicate that transferrins target H + 45 ATPase and interfere with H + -translocation, yielding a lethal effect in vitro. 46 47 48 49 50