Modulation of In Vitro Fungicidal Activity of Human Lactoferrin againstCandida albicansby Extracellular Cation Concentration and Target Cell Metabolic Activity

Abstract

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...

How Does It Kill?: Understanding the Candidacidal Mechanism of Salivary Histatin 5

“…Two major processes are likely to account for these differences: (i) the dynamic turnover of salivary proteins balancing secretion with proteolytic degradation (64,65) and (ii) the phenomenon first described as "masking" (62) that is likely due to Hsts binding with salts and possibly with various metals, including Ca (66), Cu, and Zn in saliva. A similar phenomenon was also observed for the candidacidal activity of another antimicrobial salivary protein, lactoferrin (67). Studies have established that various transitional metals, such as Zn, Ni, Cu, and Fe, are intrinsically present in the saliva (68)(69)(70).…”

In vivo function of airway epithelial TLR2 in host defense against bacterial infection

“…Mice were killed 24 h later to examine lung Mp load. The doses of purified human LTF were selected according to the previous studies in humans (29) and were also consistent with several mouse studies (15,32).…”

“…Mp (4 ϫ 10 4 cfu/ml) was incubated with purified human LTF protein from milk at various concentrations (62.5, 125, 250, and 500 g/ml) or BSA (an irrelevant protein control) in a 96-well tissue culture plate for 2 h. The LTF doses were justified based on previous studies (12,15,29,32). The supernatants were plated on pleuropneumonia-like organis agar plates (Remel) and incubated at 37°C with 5% CO 2 for 7 days to qualify Mp.…”

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