Ultrastructural effects of antimicrobial peptides from bovine lactoferrin on the membranes of Candida albicans and Escherichia coli.

Antimicrobial peptides allegedly exert their action on microbial membranes. Bovine lactoferrin enfold two antimicrobial domains, lactoferricin B (LFcin B) and lactoferrampin (LFampin). Effects of representative peptides thereof on the membranes of Candida albicans and Escherichia coli were investigated. Confocal laser scanning microscopy revealed that these peptides were internalized within a few minutes, concurrently with disrupting membrane integrity as indicated by freeze-fracture transmission electron microscopy. The most striking findings were induction of distinct vesicle-like structures in the membrane of C. albicans by the LFampin peptide, and detachment of the outer membrane and surface protrusions in E. coli by the LFcin B peptide.

Effect of amino acid substitutions on the candidacidal activity of LFampin 265-284.

LFampin 265-284, derived from bovine lactoferrin, has broad-spectrum antimicrobial activity against the yeast Candida albicans and several Gram-positive and Gram-negative bacteria. A glycine substitution scan was used to identify residues that are important for its candidacidal activity. Each single substitution of a positively charged residue led to considerable reduction in candidacidal activity, for each residue to a different extent. Substitution within the helix-facilitating N-terminal sequence DLIW had less severe effect; substitution of Ile and Trp led to a somewhat reduced potency. No substantial effects were found on the propensity to adopt a helical structure or to bind to C. albicans cells.

Lactoferrampin, an antimicrobial peptide of bovine lactoferrin, exerts its candidacidal activity by a cluster of positively charged residues at the C-terminus in combination with a helix-facilitating N-terminal part.

The antimicrobial activity of bovine lactoferrin (bLF) is attributed to lactoferricin, which is situated in the N1-domain of bLF. Recently, another antimicrobial domain consisting of residues 268-284, designated lactoferrampin (LFampin), has been identified in the N1-domain of bLF, which exhibited antimicrobial activity against Candida albicans and several bacteria. In the present study, the candidacidal activity of a series of peptides spanning this antimicrobial domain was investigated in relation to the charge and the capacity to form a helical conformation in hydrophobic environments. C-Terminal truncation of LFampin resulted in a drastic decrease in candidacidal activity. Positively charged residues clustered at the C-terminal side of the LFampin domain appeared to be crucial for the candidacidal activity. The ability to adopt helical conformations did not change when LFampin was truncated at the C-terminal side. N-Terminally truncated LFampin peptides, truncated up to the sequence 270-284, were more reluctant to adopt a helical conformation. Therefore, we conclude that the C-terminal part of LFampin 265-284, which is the most active peptide, is crucial for its candidacidal activity, due to the presence of clustered positive charges, and that the N-terminal part is essential for activity as it facilitates helix formation.

A peptide domain of bovine milk lactoferrin inhibits the interaction between streptococcal surface protein antigen and a salivary agglutinin peptide domain.

The peptide domain of salivary agglutinin responsible for its interaction with cell surface protein antigen (PAc) of Streptococcus mutans or bovine lactoferrin was found in the same peptide, scavenger receptor cysteine-rich domain peptide 2 (SRCRP2). Inhibition studies suggest that PAc and lactoferrin, of which residues 480 to 492 seem important, competitively bind to the SRCRP2 domain of salivary agglutinin.

Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin.

The antimicrobial activity of bovine lactoferrin is attributed to lactoferricin, situated in the N1-domain. Based on common features of antimicrobial peptides, a second putative antimicrobial domain was identified in the N1-domain of lactoferrin, designated lactoferrampin. This novel peptide exhibited candidacidal activity, which was substantially higher than the activity of lactoferrin. Furthermore, lactoferrampin was active against Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, but not against the fermenting bacteria Actinomyces naeslundii, Porphyromonas gingivalis, Streptococcus mutans and Streptococcus sanguis. Notably, lactoferrampin is located in the N1-domain in close proximity to lactoferricin, which plays a crucial role in membrane-mediated activities of lactoferrin.

Internalisation and degradation of histatin 5 by Candida albicans.

Histatins, salivary antimicrobial peptides, are susceptible to proteolytic degradation, often ascribed to host proteinases. In this study, we addressed the question whether proteolytic activity from microbial sources can contribute to this degradation. Candida albicans, an opportunistic yeast that is susceptible to the histatins, was used as target organism. The most potent histatin (histatin 5: sequence: DSHAKRHHGYKRKFHEKHHSHRGY), two histatin 5 fragments (dh-5: sequence: KRKFHEKHHSHRGY; P-113: sequence: AKRHHGYKRKFH) and an all-D isomer of the latter (P-113D) were used as model peptides. All L-peptides were susceptible to degradation by C. albicans. Cleavage was established at Lys5 and His19 of histatin 5, Lys11, Arg12, Phe14, Glu16, Lys17, His18 and Ser20 of dh-5 and Ala4 and Lys11 of P-113. In addition, it was found that secreted C. albicans enzymes are not involved in the degradation process and that blocking cell entry of the peptides greatly impedes degradation. Moreover, P-113D, which is biologically as active as P-113, was hardly susceptible to proteolysis. These data imply that proteolysis occurs mainly intracellularly and is not used as a protective mechanism against histatin activity. Together, our results suggest that, besides host proteinases, microbial enzymes play an important role in histatin degradation.

Effects of carbohydrate polymers applicable in saliva substitutes on the anti-Candida activity of a histatin-derived peptide.

The effects of polymers applicable in saliva substitutes on the anti-Candida activity of the cationic antimicrobial peptide dhvar1 were investigated. Dhvar1 is a derivative of the 14 C-terminal amino acids of histatin 5. The effects of the following polymers were tested: uncharged hydroxyethylcellulose (HEC), negatively charged xanthan (XG) and three types of negatively charged carboxymethylcellulose (CMC) of identical mass but different degrees of carboxylic acid-group substitution (DS). The effects were tested at pH 4.0, 7.0 and 8.5 in a killing assay. HEC had no effect at any pH tested; XG and the three types of CMC caused a decrease in activity at increasing concentrations. Within the CMC group, inhibition increased slightly with increasing DS. These results suggest that the reduction in activity associated with these polymers is the result of electrostatic interaction between the positively charged peptides and the negatively charged polymers. In the absence of polymers, no effect of pH was found on the activity of dhvar1. In the presence of the charged polymers XG and CMC, lowering the pH from 7.0 to 4.0 resulted in a decrease of dhvar1 activity. It was concluded that, with respect to the retention of activity, HEC is the most appropriate polymer for use in combination with dhvar1. However, for use in saliva substitutes XG seems more suitable because of its rheological properties. If XG or CMC are to be used, their reductive effect on the anti-Candida activity of dhvar1 should be compensated for by increasing the peptide dose.