Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells     

Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei

Three novel glycine-rich peptides, named ctenidin 1–3, with activity against the Gram-negative bacterium E. coli, were isolated and characterized from hemocytes of the spider Cupiennius salei. Ctenidins have a high glycine content (>70%), similarly to other glycine-rich peptides, the acanthoscurrins, from another spider, Acanthoscurria gomesiana. A combination of mass spectrometry, Edman degradation, and cDNA cloning revealed the presence of three isoforms of ctenidin, at least two of them originating from simple, intronless genes. The full-length sequences of the ctenidins consist of a 19 amino acid residues signal peptide followed by the mature peptides of 109, 119, or 120 amino acid residues. The mature peptides are post-translationally modified by the cleavage of one or two C-terminal cationic amino acid residue(s) and amidation of the newly created mature C-terminus. Tissue expression analysis revealed that ctenidins are constitutively expressed in hemocytes and to a small extent also in the subesophageal nerve mass.     

Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics

Gramicidin S and polymyxins are small cationic cyclic peptides and act as potent antibiotics against Gram-negative and Gram-positive bacteria by perturbing integrity of the bacterial membranes. Screening of a natural antibiotics library with bacterial membrane vesicles identified gramicidin S as an inhibitor of cytochrome bd quinol oxidase and an alternative NADH dehydrogenase (NDH-2) and polymyxin B as an inhibitor of NDH-2 and malate: quinone oxidoreductase. Our studies showed that cationic cyclic peptide antibiotics have novel molecular targets in the membrane and interfere ligand binding on the hydrophobic surface of enzymes. Improvement of the toxicity and optimization of the structures and clinical uses are urgently needed for their effective application in combating drug-resistant bacteria.     

Peptides and epithelial growth regulation

Summary There is now considerable evidence implicating several peptides in the control of gastrointestinal epithelial cell proliferation and cell renewal. While some of these may act directly, many may be involved in regulating the powerful trophic effects of the intake and digestion of foold on the gut epithelium.—Several peptides have been associated with the regulation of intestinal cell proliferation. There is little doubt that gastrin is trophic to the stomach, but, its role in the rest of the gastrointestinal tract is debatable. Enteroglucagon has often been associated with increased intestinal epithelial proliferation, but at the moment all the evidence for this is circumstantial. The effects of peptide YY and bombesin warrant further study. The availability of recombinant epidermal growth factor (EGF) has recently enabled us to demonstrate a powerful trophic response to infused EGF throughout the gastrointestinal tract. The increasing availability of peptides will eventually allow the rigorous in vivo evaluation of the trophic role of these potentially very important peptides.     

Peptides and epithelial growth regulation

There is now considerable evidence implicating several peptides in the control of gastrointestinal epithelial cell proliferation and cell renewal. While some of these may act directly, many may be involved in regulating the powerful trophic effects of the intake and digestion of food on the gut epithelium. Several peptides have been associated with the regulation of intestinal cell proliferation. There is little doubt that gastrin is trophic to the stomach, but, its role in the rest of the gastrointestinal tract is debatable. Enteroglucagon has often been associated with increased intestinal epithelial proliferation, but at the moment all the evidence for this is circumstantial. The effects of peptide YY and bombesin warrant further study. The availability of recombinant epidermal growth factor (EGF) has recently enabled us to demonstrate a powerful trophic response to infused EGF throughout the gastrointestinal tract. The increasing availability of peptides will eventually allow the rigorous in vivo evaluation of the trophic role of these potentially very important peptides.     

Peptide antigens for gamma/delta T cells

γδ T cells express adaptive antigen receptors encoded by rearranging genes. Their diversity is highest in the small region of TCR V–J junctions, especially in the δ chain, which should enable the γδ TCRs to distinguish differences in small epitopes. Indeed, recognition of small molecules, and of an epitope on a larger protein has been reported. Responses to small non-peptides known as phospho-antigens are multi-clonal yet limited to a single γδ T cell subset in humans and non-human primates. Responses to small peptides are multi-clonal or oligo-clonal, include more than one subset of γδ T cells, and occur in rodents and primates. However, less effort has been devoted to investigate the peptide responses. To settle the questions of whether peptides can be ligands for the γδ TCRs, and whether responses to small peptides might occur normally, peptide binding will have to be demonstrated, and natural peptide ligands identified.     

Relaxin family peptide systems and the central nervous system

Since its discovery in the 1920s, relaxin has enjoyed a reputation as a peptide hormone of pregnancy. However, relaxin and other relaxin family peptides are now associated with numerous non-reproductive physiologies and disease states. The new millennium bought with it the sequence of the human genome and subsequently new directions for relaxin research. In 2002, the ancestral relaxin gene RLN3 was identified from genome databases. The relaxin-3 peptide is highly expressed in a small region of the brain and in species from teleost to primates and has both conserved sequence and sites of expression. Combined with the discovery of the relaxin family peptide receptors, interest in the role of the relaxin family peptides in the central nervous system has been reignited. This review explores the relaxin family peptides that are expressed in or act upon the brain, the receptors that mediate their actions, and what is currently known of their functions.     

Esculentin(1-21), an amphibian skin membrane-active peptide with potent activity on both planktonic and biofilm cells of the bacterial pathogen Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that forms sessile communities, named biofilms. The non-motile forms are very difficult to eradicate and are often associated with the establishment of persistent infections, especially in patients with cystic fibrosis. The resistance of P. aeruginosa to conventional antibiotics has become a growing health concern worldwide and has prompted the search for new anti-infective agents with new modes of action. Naturally occurring antimicrobial peptides (AMPs) represent promising future template candidates. Here we report on the potent activity and membrane-perturbing effects of the amphibian AMP esculentin(1-21), on both the free-living and sessile forms of P. aeruginosa, as a possible mechanism for biofilm disruption. Furthermore, the findings that esculentin(1-21) is able to prolong survival of animals in models of sepsis and pulmonary infection indicate that this peptide can be a promising template for the generation of new antibiotic formulations to advance care of infections caused by P. aeruginosa.     

Anti-Candida activity of 1–18 fragment of the frog skin peptide esculentin-1b: in vitro and in vivo studies in a Caenorhabditis elegans infection model

Candida albicans represents one of the most prevalent species causing life-threatening fungal infections. Current treatments to defeat Candida albicans have become quite difficult, due to their toxic side effects and the emergence of resistant strains. Antimicrobial peptides (AMPs) are fascinating molecules with a potential role as novel anti-infective agents. However, only a few studies have been performed on their efficacy towards the most virulent hyphal phenotype of this pathogen. The purpose of this work is to evaluate the anti-Candida activity of the N-terminal 1–18 fragment of the frog skin AMP esculentin-1b, Esc(1–18), under both in vitro and in vivo conditions using Caenorhabditis elegans as a simple host model for microbial infections. Our results demonstrate that Esc(1–18) caused a rapid reduction in the number of viable yeast cells and killing of the hyphal population. Esc(1–18) revealed a membrane perturbing effect which is likely the basis of its mode of action. To the best of our knowledge, this is the first report showing the ability of a frog skin AMP-derived peptide (1) to kill both growing stages of Candida; (2) to promote survival of Candida-infected living organisms and (3) to inhibit transition of these fungal cells from the roundish yeast shape to the more dangerous hyphal form at sub-inhibitory concentrations.     

Antimicrobial peptides: natural templates for synthetic membrane-active compounds

The innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (AMPs) to resist microbial invasion. Crafted by evolution into an extremely diversified array of sequences and folds, AMPs do share a common amphiphilic 3-D arrangement. This feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. This minireview reports on current understanding of the modes of interaction of AMPs with biological and model membranes, especially focusing on recent insights into the folding and oligomerization requirements of peptides to bind and insert into lipid membranes and exert their antibiotic effects. Given the potential of AMPs to be developed into a new class of anti-infective agents, emphasis is placed on how the information on peptide-membrane interactions could direct the design and selection of improved biomimetic synthetic peptides with antibiotic properties.     

Tumor cell membrane-targeting cationic antimicrobial peptides: novel insights into mechanisms of action and therapeutic prospects

There is an ongoing need for effective and targeted cancer treatments that can overcome the detrimental side effects presented by current treatment options. One class of novel anticancer molecules with therapeutic potential currently under investigation are cationic antimicrobial peptides (CAPs). CAPs are small innate immunity peptides found ubiquitously throughout nature that are typically membrane-active against a wide range of pathogenic microbes. A number of CAPs can also target mammalian cells and often display selective activity towards tumor cells, making them attractive candidates as novel anticancer agents warranting further investigation. This current and comprehensive review describes key examples of naturally occurring membrane-targeting CAPs and their modified derivatives that have demonstrated anticancer activity, across multiple species of origin and structural subfamilies. In addition, we address recent advances made in the field and the ongoing challenges faced in translating experimental findings into clinically relevant treatments.     

Anti herpes simplex virus activity of lactoferrin/lactoferricin – an example of antiviral activity of antimicrobial protein/peptide

One of the most common viral infections in humans is caused by the herpes simplex virus (HSV). It was first effectively treated in the 1970s with the introduction of acyclovir, which is still the most commonly used treatment. Naturally occurring antimicrobial proteins and peptides have also been shown to possess antiviral activity against HSV. This review will focus on the anti-HSV activity of one such protein, lactoferrin, and a small peptide fragment from its N-terminal domain, lactoferricin. Both components have been shown to effectively block entry of HSV into the host cell. In addition to blocking HSV entry, the peptides appear to have immune stimulatory activity, although this is still somewhat controversial. Mode of action studies and knowledge about the anti-HSV activity of lactoferricin have also been successfully employed in the design of new, more specific HSV blockers. Received 25 May 2005; received after revision 24 August 2005; accepted 6 September 2005     

Role of host-defence peptides in eye diseases

The eye and its associated tissues including the lacrimal system and lids have evolved several defence mechanisms to prevent microbial invasion. Included among this armory are several host-defence peptides. These multifunctional molecules are being studied not only for their endogenous antimicrobial properties but also for their potential therapeutic effects. Here the current knowledge of host-defence peptide expression in the eye will be summarised. The role of these peptides in eye disease will be discussed with the primary focus being on infectious keratitis, inflammatory conditions including dry eye and wound healing. Finally the potential of using host-defence peptides and their mimetics/derivatives for the treatment and prevention of eye diseases is addressed.     

Protecting the boundary: the sentinel role of host defense peptides in the skin

The skin is our primary shield against microbial pathogens and has evolved innate and adaptive strategies to enhance immunity in response to injury or microbial insult. The study of antimicrobial peptide (AMP) production in mammalian skin has revealed several of the elegant strategies that AMPs use to prevent infection. AMPs are inducible by both infection and injury and protect the host by directly killing pathogens and/or acting as multifunctional effector molecules that trigger cellular responses to aid in the anti-infective and repair response. Depending on the specific AMP, these molecules can influence cytokine production, cell migration, cell proliferation, differentiation, angiogenesis and wound healing. Abnormal production of AMPs has been associated with the pathogenesis of several cutaneous diseases and plays a role in determining a patient’s susceptibility to pathogens. This review will discuss current research on the regulation and function of AMPs in the skin and in skin disorders.     

Structure and function of eukaryotic peptide transporters

The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.     

Antifungal proteins: targets,mechanisms and prospective applications

All organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. Higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. A second defence system, innate immunity, was discovered in the early 1980s, consisting of small cationic peptides with a broad antimicrobial spectrum. These proteins act immediately at sites of infection or inflammation. The production of proteins with antimicrobial activity was not limited to higher organisms but was also found in insects, plants and microorganisms. During the last 2decades a broad range of proteins with very different structural features have been isolated and characterised from differing organisms ranging from bacteria to human beings. Over 500cationic membrane-acting proteins with antimicrobial and antifungal activities have been identified to date. Apart from these proteins, a very large number of antifungal proteins active on the fungal cell wall, on enzymes of the cell wall synthesis machinery, the plasma membrane and on intracellular targets have been characterised.Received 17 June 2003; received after revision 4 August 2003; accepted 18 August 2003     

Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics

Naturally occurring polymers of N-acetylneuraminic acid (polysialic acids) are biodegradable, highly hydrophilic and have no known receptors in the body. Following intravenous injection, polysialic acids exhibit long half-lives in the blood circulation and have therefore been proposed as carriers of short-lived drugs and small peptides. In addition, shorter-chain polysialic acids can be used as a means to increase the circulatory half-life of proteins and thus serve as an alternative to the nonbiodegradable monomethoxypoly(ethylene glycol). Recent work has shown that covalent coupling of a low molecular weight polysialic acid (colominic acid) to catalase and asparaginase leads to a considerable increase of enzyme stability in the presence of proteolytic enzymes or blood plasma. Comparative studies in vivo with polysialylated and intact asparaginase revealed that polysialylation significantly increases the half-life of the enzyme. The highly hydrophilic and innocuous nature of polysialic acids renders them suitable as a means to prolong the circulation of peptides and proteins.     

Penaeidins, a family of antimicrobial peptides from penaeid shrimp (Crustacea, Decapoda)

The production of antimicrobial peptides represents a first-line host defense mechanism of innate immunity that is widespread in nature. Only recently such effectors were isolated in crustacean species, whereas numerous antimicrobial peptides have been characterized from other arthropods, both insects and chelicerates. This review presents findings on a family of antimicrobial peptides, named penaeidins, isolated from the shrimp Penaeus vannamei. Their structure and antimicrobial properties as well as their immune function will be discussed through analyses of penaeidin gene expression and peptide distribution upon microbial challenge. Received 21 January 2000; received after revision 10 March 2000; accepted 10 March 2000     

Spleen antibacterial peptides: high levels of PR-39 and presence of two forms of NK-lysin

Antibacterial peptides were isolated from porcine spleen by acetic acid extraction, ion exchange chromatography and reverse-phase high-performance liquid chromatography. C-terminal ladder sequence analysis of a bioactive peptide with matrix-assisted laser desorption/ionization mass spectrometry after digestion with carboxypeptidases P and Y showed that it is identical to the antibacterial proline/arginine-rich intestinal peptide PR-39. It is present at high levels in granulocytes of the spleen, and peptides with C-terminal proline amide and internal adjacent Pro residues can be analyzed with this method. In addition, two forms of NK-lysin (NKL) were found. One, NKLi, is identical to that isolated from pig intestine, and the other, NKLbw, to a mature peptide deduced from a clone from a porcine bone marrow cDNA library.     

Peptidergic co-transmission in Aplysia: functional implications for rhythmic behaviors.

Despite their ubiquitous presence in the central and peripheral nervous systems, the behavioral functions of peptide co-transmitters remain to be elucidated. The marine mollusc Aplysia, whose simple nervous system facilitates the study of the neural basis of behavior, was used to investigate the role of peptidergic co-transmission in feeding behavior. Several novel modulatory neuropeptides were purified, and localized to identified cholinergic motorneurons. Physiological and biochemical studies demonstrated that these peptides are released when the motorneurons fire at frequencies that occur during normal behavior, and that the peptides modify the relationship between muscle contraction amplitude and relaxation rate so as to maintain optimal motor output when the intensity and frequency of feeding behavior change.