Human lactoferrin (hLf) induced an apoptosis-like phenotype inCandida albicanscells, which includes phosphatidylserine externalization, nuclear chromatin condensation, DNA degradation, and increased reactive oxygen species (ROS) production. Intracellular ROS accumulation was seen to correlate with candidacidal activity in hLf-treated cells. Mitochondrial activity was involved as indicated by mitochondrial depolarization and increased hLf resistance of cells preincubated with sordarin or erythromycin, the latter of which inhibits protein synthesis in mitoribosomes. Interestingly, Cl⁻- and K⁺-channel blockers prevented the hLf antimicrobial activity, but only when cells were pretreated with the blocking agent (tetraethylammonium) prior to the hLf-induced K⁺-release period. These results indicate for the first time that K⁺-channel-mediated K⁺efflux is required for the progression of apoptosis-like process in yeast, suggesting that this essential apoptotic event of higher eukaryotes has been evolutionary conserved among species ranging from yeasts to humans.

The anti-Candidaactivity of the innate defense protein human lactoferrin was investigated. Lactoferrin displayed a clear fungicidal effect againstCandida albicansonly under low-strength conditions. This candidacidal activity was inversely correlated with the extracellular concentration of the monovalent cations and was prevented by Na⁺and K⁺(≥30 mM) and by divalent cations (Ca²⁺and Mg²⁺at ≥4 mM). A slight cellular release of K⁺, cytosolic acidification, and a change in the membrane potential were observed inC. albicanscells treated with lactoferrin, suggesting that this protein directly or indirectly interacts with the cytoplasmic membrane. Mitochondrial inhibitors (carbonyl cyanidem-chlorophenylhydrazone, 2,4-dinitrophenol, azide, and antimycin) as well as anaerobic conditions significantly reduced the killing effect of lactoferrin. These results suggest that low-strength conditions and the cellular metabolic state may modulate the candidacidal activity of human lactoferrin.

The synthetic peptides Lfpep and kaliocin-1 include the sequences from positions 18 to 40 and 153 to 183 of human lactoferrin, respectively. Lfpep is a cationic peptide with bactericidal and giardicidal effects, whereas kaliocin-1 is a novel bactericidal peptide that corresponds to a highly homologous sequence present in the transferrin family of proteins. Both peptides presented fungicidal activity against Candida spp., including fluconazole- and amphotericin B-resistant clinical isolates. Lfpep exhibited higher antifungal activity (8- to 30-fold) and salt resistance than kaliocin-1. The killing activity of Lfpep was mediated by its permeabilizing activity on Candida albicans cells, whereas kaliocin-1 was unable to disrupt the cytoplasmic membrane, as indicated by its inability to allow permeation of propidium iodide and the small amount of K ⁺ released. The amino acid sequence of kaliocin-1 includes the “multidimensional antimicrobial signature” conserved in disulfide-containing antimicrobial peptides and a striking similarity to brevinin-1Sa, an antimicrobial peptide from frog skin secretions, exhibiting a “ Rana box”-like sequence. These features may be of interest in the design of new antifungals.

The susceptibilities of 143 Porphyromonas gingivalis , Prevotella intermedia , and Prevotella nigrescens isolates to 18 antimicrobial agents were tested. All P. gingivalis isolates were susceptible. In contrast, some Prevotella spp. (17%) were resistant to β-lactams, erythromycin, clindamycin, or tetracycline and carried resistance genes, ermF or tetQ , or β-lactamases.

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