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Host Defense Peptide: Roles and Application
Guest Editor: Alessandro Tossi

Editorial: Host Defense Peptides: Roles and Applications
Alessandro Tossi
[Editorial In PDF]

Foreword
Ben Dunn
[Foreword In PDF]

Primate β-defensins – Structure, Function and Evolution Pp.7-21
Sergio Crovella, Nikolinka Antcheva, Igor Zelezetsky, Michele Boniotto, Sabrina Pacor,Maria Vittoria Verga Falzacappa and Alessandro Tossi
[Abstract] [Full text article]

The Cathelicidins – Structure, Function and Evolution Pp.23-34
L. Tomasinsig and M. Zanetti
[Abstract] [Full text article]

A Re-evaluation of the Role of Host Defence Peptides in Mammalian Immunity Pp.35-51
Dawn M.E. Bowdish, Donald J. Davidson and Robert. E.W. Hancock
[Abstract] [Full text article]

Defensins - Non-antibiotic Use for Vaccine Development Pp.53-60
Arya Biragyn
[Abstract] [Full text article]

Bacterial Lantibiotics: Strategies to Improve Therapeutic Potential Pp.61-75
Paul D. Cotter, Colin Hill and R. Paul Ross
[Abstract] [Full text article]

Enterococcal Cytolysin: A Novel Two Component Peptide System that Serves as a Bacterial Defense Against Eukaryotic and Prokaryotic Cells Pp.77-84
Christopher R. Cox, Phillip S. Coburn and Michael S. Gilmore
[Abstract] [Full text article]

Defensins – Components of the Innate Immune System in Plants Pp.85-101
F.T. Lay and M.A. Anderson
[Abstract] [Full text article]

Membrane Interactions of Host-defense Peptides Studied in Model Systems Pp.103-114
Raz Jelinek and Sofiya Kolusheva
[Abstract] [Full text article]

Abstracts

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Editorial: Host Defense Peptides: Roles and Applications
Alessandro Tossi
[Editorial In PDF]

Peptides play a primary role in host defense. To most researchers, they immediately call to mind protein fragments as antigens, or signaling molecules between the cellular components of innate and/or adaptive immunity. However, all life forms also produce peptides with a direct capacity to inactivate micro organisms, as a central component of innate defense. These antimicrobial peptides (AMPs) have an ancient lineage and have likely participated in the relentless battle between host and pathogen throughout evolution, so helping to shape the relationship between host and microbe, be it commensal or pathogen.

The term AMP covers a wide selection of gene encoded, and ribosomally produced molecules that display a considerable diversity in size and structure [1], but with the common general capacity to inactivate bacteria, and in some cases also fungi and viruses. Bacteria themselves produce such peptides, presumably to antagonize rival strains and so clear ecological niches. In eukaryotes, they have been identified in effectively all tested species, ranging from moulds to mammals. The antimicrobial activities of the bacterial peptides were observed quite early in the last century [2], and indications that a rapid, cell free defense system was active in plants, insects and vertebrates began to emerge in the 60’s and late 70’s [3-5]. This stimulated a systematic search for peptides from diverse sources, such as plant tissues, insect hemolymph, amphibian skin mucosa and mammalian neutrophils. The 80’s saw an enormous effort being made in the structural and functional characterization of these peptides, also with a view of developing novel potent antibiotics, which could help solve the increasing problem of bacterial resistance. Yet AMPs did not quite get the recognition they deserved as defense molecules, by the general scientific community. There are probably several reasons for this, and to name but a few, i) a tendency to discount the obvious and potent defensive properties of the non-mammalian peptides in our anthropocentric view, while those of mammalian origin were caught between the hammer of the active oxygen innate defense systems, then in their heyday, and the burgeoning anvil of the adaptive responses, ii) an excessive obsession with the direct antimicrobial properties of the peptides, centred mainly on their membranolytic properties - an over simplistic view that failed to completely explain the observed biological phenomena and iii) the failure to bring an AMP or analogue to clinical use as a novel therapeutic agent. Rational design strategies based too narrowly on optimization of membrane interactions were quite effective in increasing the bactericidal efficiency, but simultaneously resulted in an increased general toxicity that considerably narrowed the therapeutic applicability of AMP derived antibiotics. A first candidate drug remained demonstrably suitable for specific topical uses, but ran against a lack of understanding of its real potential by the FDA [6].

The wind started changing in the mid 90’s, when evidence began to mount that several AMPs could also act as signaling molecules for cellular effectors of both innate and adaptive immunity. Mammalian peptides in particular began to show different immunomodulatory functions, including chemotactic activities on dendritic and T-cells, induction of proinflammatory cytokines or chemokines, regulation of host gene expression, suppression of sepsis and promotion of wound healing or angiogenesis. The multifunctional role of these peptides, and the possibility that indirect forms of antimicrobial action may in some cases be as relevant, if not more, than their direct antibiotic activity, are now commonly accepted, so that the more generic term “Host Defense Peptide” (HDP) is starting to replace AMP, which has a narrower meaning. Firm evidence has also started to mount on the protective role of these peptides in vivo, in reducing susceptibility to infection, thus attracting the attention of medical researchers. In this new century, research on HDPs is consequently far more varied and dynamic, and I had no qualms in accepting to care for a hot topics issue on the subject. It only remained to decide what the hot topics could be, and my participation at the latest Gordon Research Conference on Antimicrobial peptides helped point me in the right direction.

A first topic of much current interest is the evolution of HDPs, and their phylogenetic relationships. Their involvement in interactions between organisms (host ad pathogen) makes them evolutionarily quite active and they have continued to respond to the varying challenges faced by animal, plant and microbial species throughout their evolution. HDPs from quite different species are based on common structural scaffolds that can tolerate a high degree of sequence variation, so that their persistence during evolutionary divergence of sequences from those of remote common ancestors may indicate that they are a requirement for antibiotic and/or signaling functions. It cannot however be excluded that they may have arisen independently from functiondriven structural convergence. These are not just academic considerations – understanding these relationships is key to understanding how HDPs work and why they are so effective, and could be very useful in helping to design novel multifunctional antibiotics. A second topic of current, and sometimes heated, debate is what the role of HDPs really is. Is the direct antimicrobial activity observed in vitro, especially in the case of mammalian HDPs, a red herring that has distracted us from their real immunomodulatory functions, or does it remain fundamental for their activity? A third and related topic is what are their modes of action. Studies in this area are somewhat stagnating at the moment, whereas it is vital that new insights be found and the enormous body of previous work be reinterpreted. The capacity to interact with the microbial cell wall in general, and membranes in particular, appears to be a definitive feature of many antimicrobial peptides, but does this mean that, the membrane and its compromising is their final goal or is it only a step on the way to the real target(s)? Another question is the relevance of membrane interactions for immunomodulatory functions. A final topic of constant general interest is how close are we to seeing a biomedical or biotechnological application of a HDP or its derivatives? Many hopes have been raised in the past only to be dashed, as this would demonstrate the added value of studying these peptides.

I have attempted to ensure that all these topics are covered in this issue, asking other participants at the Conference to provide their expert opinion.

In the opening review by Crovella et al. the evolution of an important family of HDPs, the b-defensins, is analyzed in man and primates, and in particular, their surprisingly varied patterns of evolutionary variation are considered in terms of possible genetics, structural or functional implications, and to observed variations on the antimicrobial activity in vitro, as well as their role in determining susceptibility to human diseases.

Another important and quite unique family of HDPs are the cathelicidins, which display a remarkable molecular diversity in the antimicrobial peptides carried on a relatively conserved pro-region. Tomasinsig and Zanetti provide an updated catalogue of these peptides, which are now known from primitive vertebrates to mammals, including man. They suggest that in mammals, repeated gene duplication events and subsequent divergence have produced the array of distinct family members, and that the diverse nature of these peptides may account for distinct functions.

While most workers are now willing to admit that HDPs are more than just antimicrobial, some are taking a more radical view, and suggest that for the mammalian peptides at least, this function is not principal. Bowdish et al. review the concentrations of host defense peptides and ions reported throughout the body and compare this information with the concentrations and conditions in which HDPs have antimicrobial or immunomodulatory functions in vitro. This leads them to re-evaluate and formulate new hypotheses on their antimicrobial roles.

Regarding the applicative potential of HDPs, the recognition of immunomodulatory capacities opens up new scenarios for the development of useful therapeutic agents. In his fascinating brief review, Biragyn analyzes the biological meaning of the immunomodulatory and immuno-enhancing features of defensins, and in particular their capacity to orchestrate chemotaxis and activation of effector immune cells, including immature dendritic cells, to utilize them for the development of novel DNA vaccines to combat cancer and clinically relevant diseases.

Cotter et al. review the lantibiotics, ribosomally-synthesized antimicrobial peptides produced by Gram-positive bacteria, with unusual post-translationally modified amino acids, of which Nisin is the only current example of an antimicrobial with a wide commercial use as a food preservative. They describe the studies that have permitted the implementation of rational mutagenesis strategies (‘intelligenetics’), which may allow in the near future to generate tailor-made lantibiotics and facilitate the widespread therapeutic application of these novel antimicrobial agents.

The paper by Cox et al. instead deals bacterial peptides with a darker side, the cytolysin, associated with acutely terminal enterococcal infection in humans. They suggest that the concept of Host Defense is relative, and that the cytotoxic activities of these peptides may have originally evolved to defend the producer bacteria from eukaryotic cell predation. These peptides are broadly similar in size to the mammalian defensins, and their expression is tightly controlled in a manner involving their signaling capacities, so that in a sense they can be considered as a sort of contradefensin.

Lay and Andersen provide a detailed and fascinating view of the plant defensins, ubiquitous peptides that display a diverse array of biological activities, but with a common function in plant host defense. Their review describes the distribution, biosynthesis, structure, function and mode of action of plant defensins, and considers the potential agribiotechnological and pharmaceutical applications of these peptides, in particular with respect to their exploitation in transgenic plants.

Finally, the paper by Jelinek and Kolusheva summarizes the state of the art in techniques and model systems used to probe peptide-membrane interactions and their relationships with the peptides’ biological actions. Different artificial models have facilitated examination of specific biological or chemical parameters affecting peptide action, and the strengths and limitations of the various approaches are considered.

I hope that this special issue of Current Protein and Peptide Science can be useful to those readers already active in the study of Host Defense, stimulating reflection and discussion, but principally serve to attract new researchers from other fields. The role of Host Defense Peptides in immunity is a complex one, and their understanding will benefit greatly from as wide a multi-disciplinary approach as possible. It only remains for me to indicate a few online tools which provide information on Host Defense Peptides, and which can be useful supplements to the above reviews.

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Foreword
Ben Dunn
[Foreword In PDF]

With this issue, we begin the sixth year of publication of Current Protein and Peptide Science. The past year has seen several important developments for our journal. We have achieved a first-time citation index impact factor of 1.79, which is very good for a new journal. Authors can be assured that their manuscripts are reaching a wide audience and that they will be cited in other publications. In order to achieve a citation index, all papers must be formatted by the publisher and submitted to Medline for indexing. I want to thank the staff of Bentham Science Publishing for their efforts to handle this effort and to put procedures in place to insure that this will continue.

In addition, we published three special Hot Topics issues last year: “Supermolecular Machines and Assemblies”, Volume 5, issue #2, edited by Katherine L.B. Borden and Paul S. Freemont (note that this is the second special issue edited by Dr. Borden); “Ubiquitin-Proteosome Pathway”, Volume 5, issue #3, edited by A. Jennifer Rivett, which assumes special importance with the 2004 Nobel Prize in Chemistry going to Professors Rose, Ciechanover, and Hersko for their seminal work on the Ubiquitin-mediated protein degradation pathway, and “Circular Peptides and Proteins”, Volume 5, issue #5, edited by David J. Craik. All three issues were of very high quality and, combined with the excellent papers published in the three “general” issues, are certain to boost the impact factor further. We plan to publish three more special Hot Topics issues this year, beginning with this issue, organized by Professor Alessandro Tossi on “Host Defense Peptides: Roles and Applications”. We will alternate these special issues and the general issues during 2005.

As always, I welcome suggestions for special Hot Topic issues and volunteers to organize them. At the same time, we will continue to call for submissions of regular reviews on topics of interest to protein and peptide scientists. I thank all contributors to the first five volumes of CPPS and look forward to working with many new authors in the future.

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Primate β-defensins – Structure, Function and Evolution
Sergio Crovella, Nikolinka Antcheva, Igor Zelezetsky, Michele Boniotto, Sabrina Pacor,Maria Vittoria Verga Falzacappa and Alessandro Tossi
[Full text article]

Host defense peptides (HDPs) are endogenous antibiotics that play a multifunctional role in the innate immunity of mammals. Among these, β-defensins contribute to mucosal and epithelial defense, also acting as signal molecules for cellular components of innate and adaptive immunity. Numerous members of this family have been identified in mammalian and avian species, and genomic studies in human and mouse indicate a considerable complexity in their gene organization. Recent reports on the evolution of primate and rodent members of this family indicate quite a complex pattern of variation. In this review we briefly discuss the evolution of mammalian β-defensins in relation to other types of defensins, and then concentrate on the evolution of β-defensins 1 to 4 in primates. In particular, the surprisingly varied patterns of evolution, which range from neutral or weakly purifying, to positive selection to a high level of conservation are analyzed in terms of possible genetics, structural or functional implications, as well as to observed variations on the antimicrobial activity in vitro. The role of polymorphisms in the genes encoding for these host defense peptides in determining susceptibility to human diseases are also briefly considered.

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The Cathelicidins – Structure, Function and Evolution
L. Tomasinsig and M. Zanetti
[Full text article]

The cathelicidin family of host defense peptides includes a group of cationic and usually amphipathic peptides that display a variety of activities related to host defense functions, among which the most acknowledged is a direct antimicrobial activity against various microbial pathogens. All members of this family are synthesized as precursors characterized by an N-terminal cathelin-like domain which is relatively well conserved also in evolutionary distant vertebrates. By contrast, the C-terminal region, which carries the active peptide, appears to be a focus for genetic mechanisms that have selectively generated a considerable sequence diversity. This process is particularly striking in Cetartiodactyls, where repeated gene duplication events and subsequent divergence have produced an array of distinct family members. The corresponding mature cathelicidin peptides are considerably diverse in length, amino acid sequence and structure, variously adopting α-helical, elongated or β-hairpin conformations. The diverse nature of these peptides may account for distinct functions and for a diverse spectrum of activity and/or antimicrobial potency.

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A Re-evaluation of the Role of Host Defence Peptides in Mammalian Immunity
Dawn M.E. Bowdish, Donald J. Davidson and Robert. E.W. Hancock
[Full text article]

Host defence peptides are found in all classes of life and are a fundamental component of the innate immune response. Initially it was believed that their sole role in innate immunity was to kill invading microorganisms, thus providing direct defence against infection. Evidence now suggests that these peptides play diverse and complex roles in the immune response and that, in higher animals, their functions are not restricted to the innate immune response. In in vitro experiments certain host defence peptides have been demonstrated to be potent antimicrobial agents at modest concentrations, although their antimicrobial activity is often strongly reduced or ablated in the presence of physiological concentrations of ions such as Na⁺ and Mg²⁺. In contrast, in experiments done in standard tissue culture media, the composition of which more accurately represents physiological levels of ions, mammalian host defence peptides have been demonstrated to have a number of immunomodulatory functions including altering host gene expression, acting as chemokines and/or inducing chemokine production, inhibiting lipopolysaccharide induced pro-inflammatory cytokine production, promoting wound healing, and modulating the responses of dendritic cells and cells of the adaptive immune response. Animal models indicate that host defence peptides are crucial for both prevention and clearance of infection. As interest in the in vivo functions of host defence peptides is increasing, it is important to consider whether in mammals the direct antimicrobial and immunomodulatory properties observed in vitro are physiologically relevant, especially since many of these activities are concentration dependent. In this review we summarize the concentrations of host defence peptides and ions reported throughout the body and compare that information with the concentrations of peptides that are known have antimicrobial or immunomodulatory functions in vitro.

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Defensins - Non-antibiotic Use for Vaccine Development
Arya Biragyn
[Full text article]

Vaccines should elicit protective and long lasting immune memory, which depends on well choreographed responses between innate and acquired immunity. Defensins are small host defense peptides of innate immunity hitherto reported to have antimicrobial activity, which also orchestrate chemotaxis and activation of effector immune cells, including immature dendritic cells. This review analyzes the biological meaning of the immunomodulatory and immunoenhancing features of defensins and their use for the development of novel vaccines to combat cancer and clinically relevant diseases.

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Bacterial Lantibiotics: Strategies to Improve Therapeutic Potential
Paul D. Cotter, Colin Hill and R. Paul Ross
[Full text article]

Lantibiotics are ribosomally-synthesised antimicrobial peptides produced by Gram-positive bacteria that are characterised by the presence of lanthionine and/or methyllanthionine residues. Other unusual post-translationally modified amino acids, most frequently dehydroalanine and dehydrobutyrine, can also be present. While it has been frequently suggested that these peptides have the potential to be utilised in a wide range of medical applications, to date no actual therapeutic applications have been convincingly described. More recently, however, they have been the focus of much attention as a consequence of improved biotechnological capabilities, an improved understanding of lantibiotic biosynthesis and mode of action, and their high specific activity against multi-drug resistant bacteria. This review concerns the fundamental analyses that have revealed the importance of individual amino acids in these peptides and has permitted the implementation of rational mutagenesis strategies (‘intelligenetics’) to alter individual residues with a view to ultimately widening the active pH range, improve stability, and enhance binding to cell wall targets with the ultimate aim of optimising their antimicrobial activity. It is hoped that as a consequence of this improved knowledge the most suitable application of individual lantibiotics will become apparent. It should also prove possible, in the near future, to generate tailor-made lantibiotics and utilise biosynthetic enzymes to incorporate modified amino acids into non-lantibiotic peptides. In the shorter term, the extensive characterisation of lantibiotics will be instrumental in reassuring drug industry regulators of their safety and facilitate the widespread application of these novel antimicrobial agents in medicine.

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Enterococcal Cytolysin: A Novel Two Component Peptide System that Serves as a Bacterial Defense Against Eukaryotic and Prokaryotic Cells
Christopher R. Cox, Phillip S. Coburn and Michael S. Gilmore
[Full text article]

The cytolysin is a novel, two-peptide lytic toxin produced by some strains of Enterococcus faecalis. It is toxic in animal models of enterococcal infection, and associated with acutely terminal outcome in human infection. The cytolysin exerts activity against a broad spectrum of cell types including a wide range of gram positive bacteria, eukaryotic cells such as human, bovine and horse erythrocytes, retinal cells, polymorphonuclear leukocytes, and human intestinal epithelial cells. The cytolysin likely originated as a bacteriocin involved with niche control in the complex microbial ecologies associated with eukaryotic hosts. However, additional anti-eukaryotic activities may have been selected for as enterococci adapted to eukaryotic cell predation in water or soil ecologies. Cytolytic activity requires two unique peptides that possess modifications characteristic of the lantibiotic bacteriocins, and these peptides are broadly similar in size to most cationic eukaryotic defensins. Expression of the cytolysin is tightly controlled by a novel mode of gene regulation in which the smaller peptide signals high-level expression of the cytolysin gene cluster. This complex regulation of cytolysin expression may have evolved to balance defense against eukaryotic predators with stealth.

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Defensins – Components of the Innate Immune System in Plants
F.T. Lay and M.A. Anderson
[Full text article]

Plant defensins are small (c.a. 5 kDa), basic, cysteine-rich proteins with antimicrobial activities. They are ubiquitous in plants and form part of the innate immunity arsenal. Plant defensins are encoded by small multigene families and are expressed in various plant tissues, but are best characterized in seeds. They are typically produced as preproteins, however, a small subset are produced as larger precursors with C-terminal prodomains.

To date, the three-dimensional solution structures of seven seed- and two floral-derived defensins have been elucidated by ¹H-NMR spectroscopy. Despite limited amino acid sequence identities, these defensins have comparable global folds with features that are characteristic of the cysteine-stabilized αβ (CSα/β ) motif. Interestingly, their structures are remarkably similar to those of insect defensins and scorpion toxins. Functionally, these proteins exhibit a diverse array of biological activities, although they all serve a common function as defenders of their hosts.

This review describes the distribution, biosynthesis, structure, function and mode of action of plant defensins and reflects on their potential in agribiotechnological applications.

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Membrane Interactions of Host-defense Peptides Studied in Model Systems
Raz Jelinek and Sofiya Kolusheva
[Full text article]

Host-defense, antibiotic peptides are believed to generate their cytolytic effects by interacting with the membranes of bacterial cells. Direct analyses of peptide interactions with real cellular membranes are difficult, however, due to the high complexity of physiological membranes. This review summarizes experimental work aiming to understand peptide-membrane interactions and their relationships with the peptides' biological actions using specific model systems. Varied model assemblies have been constructed that generally aim to mimic the fundamental lipid bilayer organization of the membrane. The model systems we will describe include multilamellar and unilamellar vesicles, planar lipid bilayers, lipid monolayers and micelles, and colorimetric biomimetic membranes. The different artificial models have facilitated examination of specific biological or chemical parameters affecting peptide action, for example the effect of membrane lipid composition on peptide affinities and membrane penetration, the relationship between membrane fluidity and peptide interactions, the conformations of active peptides, and other factors. We evaluate the strengths and limitations of the various approaches, and point to future directions in the field.