Jasper Groenink

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.

Effects of histatin 5 and derived peptides on Candida albicans.

Three anti-microbial peptides were compared with respect to their killing activity against Candida albicans and their ability to disturb its cellular and internal membranes. Histatin 5 is an anti-fungal peptide occurring naturally in human saliva, while dhvar4 and dhvar5 are variants of its active domain, with increased anti-microbial activity. dhvar4 has increased amphipathicity compared with histatin 5, whereas dhvar5 has amphipathicity comparable with that of histatin 5. All three peptides caused depolarization of the cytoplasmic and/or mitochondrial membrane, indicating membranolytic activity. For the variant peptides both depolarization and killing occurred at a faster rate. With FITC-labelled peptides, no association with the cytoplasmic membrane was observed, contradicting the formation of permanent transmembrane multimeric peptide pores. Instead, the peptides were internalized and act on internal membranes, as demonstrated with mitochondrion- and vacuole-specific markers. In comparison with histatin 5, the variant peptides showed a more destructive effect on mitochondria. Entry of the peptides and subsequent killing were dependent on the metabolic state of the cells. Blocking of the mitochondrial activity led to complete protection against histatin 5 activity, whereas that of dhvar4 was hardly affected and that of dhvar5 was affected only intermediately.

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.

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

Abstract 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.

Detection and Quantification of MUC7 in Submandibular, Sublingual, Palatine, and Labial Saliva by Anti-peptide Antiserum

The large carbohydrate moiety of low-Mr salivary mucin MUC7 (originally referred to as MG2) is subject to variations. Biochemical analysis and quantification of MUC7 in saliva samples require recognition tools that are independent of the carbohydrate moiety. Therefore, we have evoked three antisera to synthetic peptides of MUC7. One of these (CpMG2), raised against the C-terminal peptide, recognized native MUC7 in saliva and was characterized further. Recognition of MUC7 by CpMG2 turned out to be specific, resistant to dissociating and reductive treatments, and independent of glycosylation differences, as indicated by Western analysis and ELISA. The antiserum could be used to monitor MUC7 during purification procedures. MUC7 was demonstrated in small volumes of saliva from all (sero)mucous glands, including the palate and lip. Analysis with antibodies and lectins indicated large variations in amount as well as in glycosylation of MUC7. An ELISA was developed to determine the relative quantity of MUC7 in the glandular salivas: mean values of approximately 220, 980, and 100 μg mucin per mL were found in submandibular, sublingual, and palatine saliva, respectively.

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.

Histatin 5 and derivatives: Their localization and effects on the ultra-structural level

Histatins, a family of cationic peptides present in saliva, are active against the opportunistic yeast Candida albicans. The mechanism of action is still unclear. Histatin 5 and more potent synthetic variants, dhvar4 and dhvar5, were used to study localization and effects on morphology on the ultra-structural level. Although all peptides induced leakage, no association with the plasma membrane, indicative for permanent pores, was observed with immuno-gold-labeling. Freeze-fracturing showed severe changes of the plasma membrane. Together with, for the dhvars, the loss of intracellular integrity, this suggests that leakage may be a secondary effect rather than an effect of formation of permanent pores.

Internalisation and degradation of histatin 5 by Candida albicans

Abstract 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.

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.

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

ABSTRACT 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.