Human lactoferrin (hLf) induced an apoptosis-like phenotype in Candida albicans cells, 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-Candida activity of the innate defense protein human lactoferrin was investigated. Lactoferrin displayed a clear fungicidal effect against Candida albicans only 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 (Ca 2؉ and Mg 2؉ at >4 mM). A slight cellular release of K ؉ , cytosolic acidification, and a change in the membrane potential were observed in C. albicans cells treated with lactoferrin, suggesting that this protein directly or indirectly interacts with the cytoplasmic membrane. Mitochondrial inhibitors (carbonyl cyanide m-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.Candida albicans is a commensal yeast that colonizes the oral mucosa of most healthy individuals (7), but it is also the major fungal pathogen of humans, causing frequent mucosal and systemic opportunistic infections (7,35,41). The colonization of the oral mucosa by C. albicans is modulated by specific and nonspecific defenses present in saliva, which contains proteins (i.e., lactoferrin, lactoperoxidase, and lysozyme) and peptides (i.e., histatins and ␤-defensins) with demonstrated anticandidal activities in vitro (11,42). These antimicrobial compounds exert a regulatory role on the microbiota, allowing the colonization of mucosal surfaces by microbial species evolutionarily adapted to the host but preventing the overgrowth of the microorganisms and the invasion of internal host tissues (9).Human lactoferrin (hLf) is a prominent host defense ironbinding glycoprotein (77 kDa) synthesized by polymorphonuclear neutrophils and acinar epithelial cells and is present at relatively high concentrations (0.2 to 2.2 mg/ml) in all mucosal bathing fluids (i.e., saliva), as well as in blood and milk, with the concentrations increasing significantly with various infections (21, 29). The antimicrobial mechanism of action of lactoferrin has not yet been totally elucidated. It is thought that lactoferrin exerts an antibacterial effect by limiting the availability of iron required for microbial growth (for a review, see reference 24) and/or by directly interacting with the bacterial surface (3), with subsequent damage to the outer membranes of gram-negative bacteria (12). It has also been hypothesized that the antimicrobial activity of this protein could be related to the interaction of the hLf amino acid sequences homologous to the antimicrobial hLf-derived peptides termed lactoferricin (residues 1 to 42) or kaliocin-1 (residues 153 to 183) with the bacterial membrane (5, 40). Although considerable attention has been focused on the antibacterial activity of lactoferrin (1, 13, 34), very little is known about its antifungal mechanism of acti...

Serum transferrin, ovotransferrin and lactoferrin constitute the most notable members of the transferrin family. Among their multiple biological functions, they possess an important antibacterial activity. These proteins can permeate the Escherichia coli outer membrane, reaching the inner membrane where they selectively cause permeation of ions, resulting in dissipation of the electrical potential without a¡ecting the pH gradient. Similar results were obtained using arti¢cial liposomes, suggesting a direct action of the proteins on the lipid bilayer, which was mediated by detectable conformational changes in their structures.

bHuman lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P 3A -type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H ؉ -ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H ؉ accumulation and subsequent K ؉ efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H ؉ -ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H ؉ -ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events. L actoferrin (Lf) is a 77-kDa iron-binding glycoprotein of the transferrin family of proteins and an important effector molecule of innate immunity with antimicrobial activity in mammalian mucosal fluids (reviewed in reference 1). We have previously reported that human lactoferrin (hLf) induces apoptosis-like cell death of the pathogenic opportunistic yeast pathogen Candida albicans, demonstrating also that K ϩ channel-mediated K ϩ efflux is a common apoptotic event in yeast and metazoan cells (2). At present, the mechanism of action by which lactoferrin and other antimicrobial peptides induce apoptosis-like processes in yeast remains unknown (reviewed in reference 3). However, we have previously identified in bacteria the proton-translocating ATPase (H ϩ -ATPase) as the molecular target of lactoferrin, suggesting that the subsequent perturbation of the proton gradient (⌬pH) and intracellular pH (pH i ) will lead to cell death in vitro (4). This finding revealed lactoferrin as a natural extracellular inhibitor of the proton translocation mediated by H ϩ -ATPases, suggesting that the possible inhibition of a similar target also explains its antifungal activity. In this hypothetical case, the antifungal effect of lactoferrin might be due to altered pH homeostasis, as reported for bacteria (4).In yeast, control of pH i is critical for cell survival and is regulated through a concerted moveme...

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.