An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, alpha-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the 'sequence template' approach which we have used to design potent artificial helical AMPs, to guide structure-activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides.

The solid-phase synthesis of peptides (SPPS) containing [60]fullerene-functionalized amino acids is reported. A new amino acid, fulleropyrrolidino-glutamic acid (Fgu), is used for the SPPS of a series of analogues of different length based on the natural Leu(5)-Enkephalin and on cationic antimicrobial peptides. These fullero-peptides were prepared on different solid supports to analyze the influence of the resin on the synthesis. Optimized protocols for the coupling and deprotection procedures were determined allowing the synthesis of highly pure peptides in sufficient quantities for evaluation of biological activities. In particular, to avoid side reactions of the fullerene moiety with bases and nucleophiles, the removal of the protecting groups was performed under inert conditions (nitrogen or argon in the dark). We have encountered serious problems with the recovery of the crude compounds, especially when Fgu was inserted in the proximity of the resin core as fullero-peptides tend to remain embedded inside the resin. Eventually, all of the fullero-peptides were easily purified, and the cationic peptides were tested for their antimicrobial activities. They displayed a specific activity against the Gram-positive bacterium S. aureus and also lysed erythrocytes. The availability of a fullero-amino acid easily useable in the SPPS of fullero-peptides may thus open the way to the synthesis of new types of biologically active oligomers.

We have investigated the molecular evolution of the gene coding for beta-defensin 3 (DEFB103) in 17 primate species including humans. Unlike the DEFB4 genes (coding for beta-defensin 2) [Boniotto, Tossi, Del Pero, Sgubin, Antcheva, Santon and Masters (2003) Genes Immun. 4, 251-257], DEFB103 shows a marked degree of conservation in humans, Great Apes and New and Old World monkeys. Only the Hylobates concolor defensin hcBD3 showed an amino acid variation Arg17-->Trp17 that could have a functional implication, as it disrupts an intramolecular salt bridge with Glu27, which locally decreases the charge and may favour dimerization in the human congener hBD3. This is thought to involve the formation of an intermolecular salt bridge between Glu28 and Lys32 on another monomer [Schibli, Hunter, Aseyev, Starner, Wiencek, McCray, Tack and Vogel (2002) J. Biol. Chem. 277, 8279-8289]. To test the role of dimerization in mediating biological activity, we synthesized hBD3, hcBD3 and an artificial peptide in which the Lys26-Glu27-Glu28 stretch was replaced by the equivalent Phe-Thr-Lys stretch from human beta-defensin 1 and we characterized their structure and anti-microbial activity. Although the structuring and dimerization of these peptides were found to differ significantly, this did not appear to affect markedly the anti-microbial potency, the broad spectrum of activity or the insensitivity of the anti-microbial action to the salinity of the medium.

A novel method, based on the rational and systematic modulation of macroscopic structural characteristics on a template originating from a large number of natural, cell-lytic, amphipathic alpha-helical peptides, was used to probe how the depths and shapes of hydrophobic and polar faces and the conformational stability affect antimicrobial activity and selectivity with respect to eukaryotic cells. A plausible mode of action explaining the peptides' behaviour in model membranes, bacteria and host cells is proposed. Cytotoxic activity, in general, correlated strongly with the hydrophobic sector depth, and required a majority of aliphatic residue side chains having more than two carbon atoms. It also correlated significantly with the size of polar sector residues, which determines the penetration depth of the peptide via the so-called snorkel effect. Both an oblique gradient of long to short aliphatic residues along the hydrophobic face and a stabilized helical structure increased activity against host cells but not against bacteria, as revealed by haemolysis, flow cytofluorimetric studies on lymphocytes and surface plasmon resonance studies with model phosphatidylcholine/cholesterol membranes. The mode of interaction changes radically for a peptide with a stable, preformed helical conformation compared with others that form a structure only on membrane binding. The close correlation between effects observed in biological and model systems suggests that the 'carpet model' correctly represents the type of peptides that are bacteria-selective, whereas the behaviour of those that lyse host cells is more complex.

No abstract

Cathelicidin genes homologous to the human CAMP gene, coding for the host defense peptide LL-37, have been sequenced and analyzed in 20 primate species, including Great Apes, hylobatidae, cercopithecidae, callithricidae, and cebidae. The region corresponding to the putative mature antimicrobial peptide is subject to a strong selective pressure for variation, with evidence for positive selection throughout the phylogenetic tree relating the peptides, which favors alterations in the charge while little affecting overall hydrophobicity or amphipathicity. Selected peptides were chemically synthesized and characterized, and two distinct types of behavior were observed. Macaque and leaf-eating monkey RL-37 peptides, like other helical antimicrobial peptides found in insect, frog, and mammalian species, were unstructured in bulk solution and had a potent, salt and medium independent antimicrobial activity in vitro, which may be the principal function also in vivo. Human LL-37 and the orangutan, hylobates, and callithrix homologues instead showed a salt-dependent structuring and likely aggregation in bulk solution that affected antimicrobial activity and its medium dependence. The two types of peptides differ also in their interaction with host cells. The evolution of these peptides has thus resulted in distinct mechanisms of action that affect the direct antimicrobial activity and may also modulate accessory antimicrobial functions due to interactions with host cells.

A large number of gene encoded host defence peptides (HDPs) has been described over the past two decades (many are collected in the AMSDb database at http:// www.bbcm.units.it/tossi/antimic.html), and it has become quite clear that they are used as a defence mechanism throughout the living world [1][2][3][4]. Microorganisms use them to antagonize competitors [5,6], plants and insects as the major effector molecules to prevent and combat microbial infections [7,8], while mammals use them to control commensal microorganisms and as a first line of defence against invading pathogens [9][10][11].Several families of HDPs contribute to host defence in mammals. Among them, a prominent role is played by the cysteine-rich a-and b-defensins [10] and by several other linear peptides belonging to the cathelicidins. This family includes a large and quite diverse group of HDPs, all deriving from propeptides with a wellconserved N-terminal proregion [11] among which the a-and b-defensins and cathelicidins play a prominent role. Mammalian HDPs can have both a direct antimicrobial activity and ⁄ or act as immunomodulatory molecules for cellular components of innate and adaptive immune responses. In the former case, they are thought to function principally at the level of bacterial membranes, to which they are drawn by their cationic nature, and into which they can insert by preformed or assumed amphipathic structures. These are based on PMAP-36 is a cathelicidin-derived host defence peptide originally deduced by a transcript from pig bone marrow RNA. The expression of the propeptide in leukocytes, and the structure, antimicrobial activity, and mechanism of action of the mature peptide were investigated. The proform is stored as a dimeric precursor of 38 kDa formed by a dimerization site at its C-terminal cysteine residue; it is likely that the mature peptide is dimeric when released. Monomeric and dimeric forms of PMAP-36 were chemically synthesized and their activity compared. Both forms assumed an amphipathic a-helical conformation and exhibited a potent and rapid microbicidal activity against a wide spectrum of microorganisms, mediated by their ability to permeabilize the microbial membranes rapidly. A shortened fragment localized the helical region to the N terminus, but showed a significantly lower potency and slower permeabilization kinetics, indicating an important role of the nonhelical C-terminal hydrophobic portion of this molecule. Dimerization modulated the effectiveness of the peptide in terms of killing and permeabilization kinetics, and reduced medium dependence. It allows the molecule to achieve an impressive charge density (+28 in 70 residues), although the significance of this feature with respect to biological activity has yet to be determined. Abbreviations

The evolution of orthologous genes coding for ␤-defensin 2 (BD2) in primates has been subject to positive selection during the divergence of the platyrrhines from the catarrhines and of the Cercopithecidae from the Hylobatidae, great apes, and humans. Three peptides have been selected for a functional analysis of the effects of sequence variations on the direct antimicrobial activity: human BD2 (hBD2), Macaca fascicularis BD2 (mfaBD2), and a variant of the human peptide lacking Asp 4 , (؊D)hBD2, which is characteristic only of the human/great ape peptides. hBD2 and mfaBD2 showed a significant difference in specificity, the former being more active towards Escherichia coli and the later towards Staphylococcus aureus and Candida albicans. Asp 4 in the human peptide appears to be important, as (؊D)hBD2 was less structured and had a markedly lower antimicrobial activity. The evolution of ␤-defensin 2 in primates may thus have been driven, at least in part, by different environmental pressures so as to modulate antimicrobial activity.Human ␤-defensin 2 (hBD2) is a host defense peptide originally isolated from the skin of psoriasis patients (6). The hBD2 gene is constitutively expressed in skin, lung, and trachea, being markedly upregulated by inflammatory stimuli, and its induced expression can also be detected in other tissues (5,7,11,13). The peptide has a relatively broad antimicrobial activity spectrum, ranging from bacteria to yeasts (6), and it has been hypothesized that this depends on the interaction of an oligomeric form with the negatively charged microbial surface via an electrostatic charge-based mechanism (10). Apart from this direct antimicrobial activity, hBD2 also plays other roles in host defense, as it is a chemoattractant for immature dendritic cells and memory T cells (15).Recently, the molecular evolution of the ␤-defensin 2 gene (originally DEFB2, now DEFB4) has been investigated in humans and 16 primate species (2) to obtain insights into how its evolution might have affected the structure of the protein and thus its function. Evidence of positive selection was indeed found during the divergence of the platyrrhines (New World monkeys) from the catarrhines (Old World monkeys) and during the divergence of the Cercopithecidae from the Hylobatidae, great apes, and humans. In contrast, the evolution of the DEFB1 gene orthologues in primates (coding for ␤-defensin 1 congeners) shows a random variation consistent with the neutral theory of molecular evolution (3), while DEFB103 gene orthologues (coding for ␤-defensin 3 congeners) are instead markedly conserved (1).At the amino acid level, the human and great ape BD2 peptides are effectively identical, while those from Hylobates vary only in a short N-terminal stretch (2). The positively selected variations in the Cercopithecidae and New World monkeys concern a more marked variation in the C-terminal region of the mature peptides. On the basis of these variation patterns, three peptides have been selected for chemical synthesis and functional analysis ...