Candidacidal Activities of Human Lactoferrin Peptides Derived from the N Terminus

Abstract

In light of the need for new antifungal agents, the candidacidal activities of human lactoferrin (hLF) and synthetic peptides representing the first, hLF(1-11), and second, hLF(21-31), cationic domains of its N terminus were compared. The results revealed that hLF(1-11) was more effective in killing fluconazole-resistant Candida albicans than hLF(21-31) and much more effective than lactoferrin, as determined microbiologically and by propidium iodide (PI) staining. By using hLF(1-11) and various derivatives, it was found that the second and third residues of the N terminus of hLF(1-11) were critical for its candidacidal activity. Detailed investigation to elucidate the mechanism of action of hLF(1-11) revealed a dose-dependent release of ATP by Candida upon exposure to hLF(1-11). Our observations that sodium azide reduced the PI uptake and candidacidal activity of hLF(1-11) and that, upon exposure to hLF(1-11), the fluorescent dye rhodamine 123 first accumulated inside the mitochondria and later was released into the cytoplasm indicate that the peptide triggers the energized mitochondrion. Furthermore, oxidized ATP, which interferes with the interaction of ATP with its extracellular receptors, blocked the candidacidal action of hLF(1-11), as measured microbiologically and by PI staining. Addition of ATP (or analogues) was not a sufficient stimulus to kill C. albicans or to act synergistically with suboptimal concentrations of the peptide. The main conclusions are that the first two arginines at the N terminus of hLF are critical in the candidacidal activity of hLF(1-11) and that extracellular ATP is essential but not sufficient for the peptide to exert its candidacidal activity.

Human antimicrobial peptides’ antifungal activity against Aspergillus fumigatus

“…The maximum hyphal damage by the various antimicrobial peptides amounted to 70-80%, which was similar to the values for ≥0.1 nM amphotericin B. Comparison of the EC 50 values for the various peptides revealed that dhvar5 (EC 50 = 3±1 μM) is the most potent peptide; the EC 50 values for hLF (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), hLF(21-31), dhvar4, UBI 18-35, and UBI 29-41 being 29±5 μM, 142±13 μM, 19±5 μM, 37±15 μM, and 91±19 μM, respectively. Notably, hLF(1-11) is 5-fold more effective than hLF(21-31), dhvar5 is 6-fold more potent than dhvar4, and UBI 18-35 is almost 3-fold more effective than UBI 29-41 in damaging A. fumigatus hyphae.…”

“…The results revealed a dose-dependent antifungal activity against A. fumigatus hyphae for hLF (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) (Fig. 1).…”

“…The synthetic peptides hLF(1-11) (GRRRRSVQWCA; Mw 1,374 Da), hLF(21-31) (FQWQRNMRKVR; Mw 1,567 Da), UBI 18-35 (KVAKQEKKKKKTGRAKRR; 2,169 Da), UBI 29-41 (TGRAKRRMQYNRR; 1,693 Da), and peptide 4 (part of gp120 from HIV-1; RPVVSTQLLLNGSLAEEEVV; 2,171 Da) were prepared and purified as described [7]. The peptides dhvar4 (KRLFKKLLFSLRKY; 1,841 Da) an d d hvar5 (LLLFLLKKRKKRKY; 1,848 Da) were a kind gift from Dr. W. van't Hof (Academic Centre for Dentistry, Department of Oral Biochemistry, Free University, Amsterdam, the Netherlands).…”

“…The purity of the various peptides ranged from 88-97%, as determined by reverse-phase highperformance liquid chromatography (RP-HPLC). The peptides were stored and used as previously described [7].…”

“…This antimicrobial protein contains two cationic domains in its N terminus. A recent study indicated that a synthetic peptide including the first cationic domain, further referred to as hLF (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), is more effective in killing Candida albicans than the native protein or a peptide, hLF(21-31), comprising the second cationic domain [7].…”