The human cathelicidin hCAP18/LL-37 has become a paradigm for the pleiotropic roles of peptides in host defence. It has a remarkably wide functional repertoire that includes direct antimicrobial activities against various types of microorganisms, the role of 'alarmin' that helps to orchestrate the immune response to infection, the capacity to locally modulate inflammation both enhancing it to aid in combating infection and limiting it to prevent damage to infected tissues, the promotion of angiogenesis and wound healing, and possibly also the elimination of abnormal cells. LL-37 manages to carry out all its reported activities with a small and simple, amphipathic, helical structure. In this review we consider how different aspects of its primary and secondary structures, as well as its marked tendency to form oligomers under physiological solution conditions and then bind to molecular surfaces as such, explain some of its cytotoxic and immunomodulatory effects. We consider its modes of interaction with bacterial membranes and capacity to act as a pore-forming toxin directed by our organism against bacterial cells, contrasting this with the mode of action of related peptides from other species. We also consider its different membrane-dependent effects on our own cells, which underlie many of its other activities in host defence. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

LL-37 is a multifunctional component of innate immunity, with a membrane-directed antimicrobial activity and receptor-mediated pleiotropic effects on host cells. Sequence variations in its primate orthologues suggest that two types of functional features have evolved; human LL-37-like peptides form amphipathic helical structures and self-assemble under physiological conditions, whereas rhesus RL-37-like peptides only adopt this structure in the presence of bacterial membranes. The first type of peptide has a lower and more medium-sensitive antimicrobial activity than the second type, but an increased capacity to stimulate host cells. Oligomerization strongly affects the mode of interaction with biological membranes and, consequently, both cytotoxicity and receptor-mediated activities. In the present study we explored the effects of LL-37 self-association by using obligate disulfide-linked dimers with either parallel or antiparallel orientations. These had an increased propensity to form stacked helices in bulk solution and when in contact with either anionic or neutral model membranes. The antimicrobial activity against Gram-positive or Gram-negative bacteria, as well as the cytotoxic effects on host cells, strongly depended on the type of dimerization. To investigate the extent of native oligomerization we replaced Phe5 with the photoactive residue Bpa (p-benzoyl-L-phenylalanine), which, upon UV irradiation, enabled covalent cross-linking and allowed us to assess the extent of oligomerization in both physiological solution and in model membranes.

The human cathelicidin peptide LL-37 is an important effector of our innate immune system and contributes to host defence with direct antimicrobial activity and immunomodulatory properties, and by stimulating wound healing. Its sequence has evolved to confer specific structural characteristics that strongly affect these biological activities, and differentiate it from orthologues of other primate species. In the present paper we report a detailed study of the folding and self-assembly of this peptide in comparison with rhesus monkey peptide RL-37, taking into account the different stages of its trajectory from bulk solution to contact with, and insertion into, biological membranes. Phenylalanine residues in different positions throughout the native sequences of LL-37 and RL-37 were systematically replaced with the non-invasive fluorescent and IR probe p-cyanophenylalanine. Steady-state and time-resolved fluorescence studies showed that LL-37, in contrast to RL-37, forms oligomers with a loose hydrophobic core in physiological solutions, which persist in the presence of biological membranes. Fourier transform IR and surface plasmon resonance studies also indicated different modes of interaction for LL-37 and RL-37 with anionic and neutral membranes. This correlated with a distinctly different mode of bacterial membrane permeabilization, as determined using a flow cytometric method involving impermeant fluorescent dyes linked to polymers of defined sizes.

A challenge when designing membrane-active peptide antibiotics with therapeutic potential is how to ensure a useful antibacterial activity whilst avoiding unacceptable cytotoxicity for host cells. Understanding their mode of interaction with membranes and the reasons underlying their ability to distinguish between bacterial and eukaryotic cytoplasmic cells is crucial for any rational attempt to improve this selectivity. We have approached this problem by analysing natural helical antimicrobial peptides of anuran origin, using a structure-activity database to determine an antimicrobial selectivity index (SI) relating the minimal inhibitory concentration against Escherichia coli to the haemolytic activity (SI=HC(50)/MIC). A parameter that correlated strongly with SI, derived from the lengthwise asymmetry of the peptides' hydrophobicity (sequence moment), was then used in the "Designer" algorithm to propose novel, highly selective peptides. Amongst these are the 'adepantins', peptides rich in glycines and lysines that are highly selective for Gram-negative bacteria, have an exceptionally low haemolytic activity, and are less than 50% homologous to any other natural or synthetic antimicrobial peptide. In particular, they showed a very high SI for E. coli (up to 400) whilst maintaining an antimicrobial activity in the 0.5-4μM range. Experiments with monomeric, dimeric and fluorescently labelled versions of the adepantins, using different bacterial strains, host cells and model membrane systems provided insight into their mechanism of action.

LL‐37 is an innate immune peptide derived from the human cathelicidin, which exerts pleiotropic roles in host defense and healing. These activities in part depend on its capacity to adopt an amphipathic helical structure in physiological solutions and then oligomerizing. Orthologues from other primates, such as rhesus RL‐37, remain monomeric and disordered under the same conditions. Intramolecular salt‐bridges, arising from appropriately spaced anionic and cationic residues in its sequence, may play a relevant role in determining the particular structure adopted by LL‐37. To probe this, we have effected minimal, targeted residue variations such as replacement of a single residue (K15→G), or inversion of one or both sets of two residues (E10 K11→ K10 E11 or E16 K18→ K16 E18). This could alter the pattern of intramolecular salt bridging without affecting other functionally relevant parameters such as overall hydrophobicity, helix amphipathicity or charge. The structural and functional effects were analyzed using CD spectroscopy, surface plasmon resonance, antimicrobial activity assays, and bacterial membrane permeabilization to fluorescent probes of increasing sizes, using flow cytometry. Analogs were functionally different from both LL‐37 and RL‐37, so it was not possible to switch from the function of one to that of the other simply by altering the salt‐bridging pattern in this manner. This indicates that the particular structure/function characteristics of LL‐37 likely depend quite subtly, and in a precise and complex manner, on a complex pattern of intramolecular interactions.