Wim van 't Hof

Antimicrobial Peptides: Properties and Applicability

Abstract All organisms need protection against microorganisms, e. g. bacteria, viruses and fungi. For many years, attention has been focused on adaptive immunity as the main antimicrobial defense system. However, the adaptive immune system, with its network of humoral and cellular responses is only found in higher animals, while innate immunity is encountered in all living creatures. The turning point in the appreciation of the innate immunity was the discovery of antimicrobial peptides in the early eighties. In general these peptides act by disrupting the structural integrity of the microbial membranes. It has become clear that membraneactive peptides and proteins play a crucial role in both the innate and the adaptive immune system as antimicrobial agents. This review is focused on the functional and structural features of the naturally occurring antimicrobial peptides, and discusses their potential as therapeutics.

Identification of the Bacteria-binding Peptide Domain on Salivary Agglutinin (gp-340/DMBT1), a Member of the Scavenger Receptor Cysteine-rich Superfamily

Salivary agglutinin is encoded byDMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300–400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans.To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutansand a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.

Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process.

The salivary lipocalin Von Ebner's gland protein is a cysteine proteinase inhibitor

The lipocalins make up a heterogeneous superfamily of proteins. Although showing almost no sequence homology, they share very similar secondary and tertiary structures. Their ability to bind hydrophobic ligands is well established, but the physiological function of most lipocalins remains unclear. The lipocalin from the human Von Ebners Gland of the tongue (VEGh) contains three sequence motifs corresponding with the papain-binding domains of cystatins, a family of naturally occurring cysteine proteinase inhibitors. We found that VEGh inhibited papain activity to a similar extent as salivary cystatin S. Furthermore, synthetic peptides derived from VEGh and cystatin C, comprising these three motifs, inhibited papain, too. We conclude that VEGh is a physiological inhibitor of cysteine proteinases and therefore can play a role in the control of inflammatory processes in oral and ocular tissues.

A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides

The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and Candida albicans cells in phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC50/IC50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC50=0.6 μM) and had the lowest therapeutic index (HC50/IC50=0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.

Epitope mapping of the Dermatophagoides pteronyssinus house dust mite major allergen Der p II using overlapping synthetic peptides

Fourteen synthetic peptides of 15 amino acid residues length, overlapping by five residues and spanning the entire sequence of the major allergen Der p II from the house dust mite Dermatophagoides pteronyssinus were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope mapping studies using human IgE antisera. These antibodies bound predominantly to the peptide comprising residues 65-78, the binding of which was inhibited by native Der p II. In addition these antisera bound, to a lesser extent, to the peptide that comprised residues 1-15, which binding was not inhibited by native Der p II. Thus, we found one sequential epitope for a number of IgE sera.

Energy Depletion Protects Candida albicans against Antimicrobial Peptides by Rigidifying Its Cell Membrane

Inhibitors of the energy metabolism, such as sodium azide and valinomycin, render yeast cells completely resistant against the killing action of a number of cationic antimicrobial peptides, including the salivary antimicrobial peptide Histatin 5. In this study the Histatin 5-mediated killing of the opportunistic yeast Candida albicans was used as a model system to comprehensively investigate the molecular basis underlying this phenomenon. Using confocal and electron microscopy it was demonstrated that the energy poison azide reversibly blocked the entry of Histatin 5 at the level of the yeast cell wall. Azide treatment hardly induced depolarization of the yeast cell membrane potential, excluding it as a cause of the lowered sensitivity. In contrast, the diminished sensitivity to Histatin 5 of energy-depleted C. albicans was restored by increasing the fluidity of the membrane using the membrane fluidizer benzyl alcohol. Furthermore, rigidification of the membrane by incubation at low temperature or in the presence of the membrane rigidifier Me2SO increased the resistance against Histatin 5, while not affecting the energy charge of the cell. In line, azide induced alterations in the physical state of the interior of the lipid bilayer. These data demonstrate that changes in the physical state of the membrane underlie the increased resistance to antimicrobial peptides.

IgE epitopes on the cat (Felis domesticus) major allergen Fel d I: A study with overlapping synthetic peptides

BACKGROUND The major cat allergen Fel d I is composed of two disulfide-linked polypeptide chains, chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Reduction and alkylation of Fel d I eliminates almost all antigenic and allergenic activity, and detection of linear epitopes with synthetic peptides is therefore not expected. METHODS We synthesized synthetic peptides of both chains of about 14 amino acid residues, overlapping by 7 residues. The peptides were coupled to Sepharose (Pharmacia, Uppsala, Sweden) and tested with sera of patients with cat allergy. RESULTS Three peptides showed specific binding of human IgE, residues 25-38 and 46-59 of chain 1 and residue 15-28 of chain 2. IgE binding was inhibited by Fel d I and the corresponding peptide. Of 61 patients with cat allergy tested, 65% showed IgE binding to at least one of the peptides; 46% showed IgE binding to peptide 25-38, 11% to peptide 46-59, and 28% to peptide 15-28. Each peptide was recognized by only one of the 78 patients with negative RAST results. By affinity chromatography with peptide-Sepharose anti-Fel d I antibodies were isolated, also confirming the specificity of IgE binding to the peptides. The percentage of IgE antibodies against Fel d I reactive with the peptides varied with the serum and the peptide-Sepharose used and ranged from 2% to 55%. CONCLUSIONS Because the affinity of IgE binding to the peptides was very low and only serum samples with high titers of Fel d I-specific IgE antibodies (RAST 4+/5+) showed significant binding, these peptides are not suitable for diagnostic purposes. However, the peptides are useful tools for comparing IgE and IgG responses and for studying the relationship to the T-cell epitopes on this molecule.

Reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5.

The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an indicator for ROS. With both peptides, a substantial enhancement of fluorescence was observed. However, TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a cell-permeant ROS scavenger, did not have an inhibitory effect on killing or on the enhancement of fluorescence. Furthermore, antimycin and azide, which have been reported to induce ROS in vitro, were not able to enhance the dihydroethidium fluorescence, while chlorhexidine, a non-specific antiseptic agent, enhanced dihydroethidium fluorescence to the same extent as did the peptides. Fluorescence microscopy showed the fluorescence enhancement to be a consequence of the release of unbound preformed ethidium from the mitochondrial matrix within the cell. It is concluded that ROS do not play a role in the histatin 5-mediated killing of C. albicans.

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.