Margherita Zanetti

Cathelicidins, multifunctional peptides of the innate immunity

Cathelicidins comprise a family of mammalian proteins containing a C‐terminal cationic antimicrobial domain that becomes active after being freed from the N‐terminal cathelin portion of the holoprotein. Many other members of this family have been identified since the first cathelicidin sequences were reported 10 years ago. The mature peptides generally show a wide spectrum of antimicrobial activity and, more recently, some of them have also been found to exert other biological activities. The human cathelicidin peptide LL‐37 is chemotactic for neutrophils, monocytes, mast cells, and T cells; induces degranulation of mast cells; alters transcriptional responses in macrophages; stimulates wound vascularization and re‐epithelialization of healing skin. The porcine PR‐39 has also been involved in a variety of processes, including promotion of wound repair, induction of angiogenesis, neutrophils chemotaxis, and inhibition of the phagocyte NADPH oxidase activity, whereas the bovine BMAP‐28 induces apoptosis in transformed cell lines and activated lymphocytes and may thus help with clearance of unwanted cells at inflammation sites. These multiple actions provide evidence for active participation of cathelicidin peptides in the regulation of the antimicrobial host defenses.

Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain.

A novel protein family, showing a conserved proregion and a variable C‐terminal antimicrobial domain, and named0 cathelicidin, has been identified in mammalian myeloid cells. The conserved proregion shows sequence similarity to members of the cystatin superfamily of cysteine proteinase inhibitors. Cathelicidins are stored in the cytoplasmic granules of neutrophil leukocytes and release the antimicrobial peptides upon leukocyte activation. Some of these peptides can assume an α‐helical conformation, others contain one or two disulfide bonds, still others are Pro‐ and Arg‐rich, or Trp‐rich. In addition to bacterial killing, some of these peptides exert additional functions related to host defense such as LPS‐neutralization and promotion of wound healing.

Structural features and biological activities of the cathelicidin-derived antimicrobial peptides

Cathelicidins are a numerous group of mammalian proteins that carry diverse antimicrobial peptides at the C-terminus of a highly conserved preproregion. These peptides, which become active when released from the proregion, display a remarkable variety of sizes, sequences, and structures, and in fact comprise representatives of all the structural groups in which the known antimicrobial peptides have been classified. Most of the cathelicidin-derived peptides exert a broad spectrum and potent antimicrobial activity and also bind to lipopolysaccharide and neutralize its effects. In addition, some of them have recently been shown to exert other activities and might participate in host defense also by virtue of their ability to induce expression of molecules involved in a variety of biological processes. This review is aimed at providing a general overview of the cathelicidins and of the peptides derived therefrom, with emphasis on aspects such as structure, biological activities in vitro and in vivo, and structure/activity relationship studies.

Biological Characterization of Two Novel Cathelicidin-derived Peptides and Identification of Structural Requirements for Their Antimicrobial and Cell Lytic Activities

Cathelicidins are a family of myeloid antimicrobial peptide precursors that have been identified in several mammalian species (Zanetti, M., Gennaro, R., and Romeo, D. (1995) FEBS Lett. 374, 1-5). Two novel bovine congeners have been deduced from cDNA. Their C-terminal sequences of 27 and 28 residues correspond to putative antimicrobial peptides with a cationic N-terminal region predicted to assume an amphipathic α-helical conformation followed by a hydrophobic C-terminal tail. Peptides corresponding to these sequences have been chemically synthesized and shown to exert a potent antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, and fungi. Both peptides are also cytotoxic to human erythrocytes and neutrophils, although at higher than microbicidal concentrations. The target selectivity has been improved by synthesizing truncated analogues, comprising only the 18 N-terminal residues, which show a great reduction in cytotoxic, but not in antimicrobial activity. The involvement of the C-terminal hydrophobic tail in the cytotoxic activity has been further demonstrated by inducing a major loss of activity in an analogue after replacing highly hydrophobic residues with more hydrophilic ones.

SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes

SMAP‐29 is a cathelicidin‐derived peptide deduced from sheep myeloid mRNA. The C‐terminally amidated form of this peptide was chemically synthesized and shown to exert a potent antimicrobial activity. Antibiotic‐resistant clinical isolates highly susceptible to this peptide include MRSA and VREF isolates, that are a major worldwide problem, and mucoid Pseudomonas aeruginosa associated with chronic respiratory inflammation in CF patients. In addition, SMAP‐29 is also active against fungi, including Cryptococcus neoformans isolated from immunocompromised patients. SMAP‐29 causes significant morphological alterations of the bacterial surfaces, as shown by scanning electron microscopy, and is also hemolytic against human, but not sheep erythrocytes. Its potent antimicrobial activity suggests that this peptide is an excellent candidate as a lead compound for the development of novel antiinfective agents.

Structure and Biology of Cathelicidins

Cathelicidins are a recently discovered family of effector molecules in innate immunity. In the past few years, a great deal of investigations have elucidated several aspects of their biology, such as the gene structure and activation mechanism. Despite substantial progress in the field, several issues remain to be clarified, including the biological role of the conserved proregion and the molecular mechanisms responsible for diversification of the peptide domain. The cathelicidin-derived peptides have been deeply investigated with respect to structure, spectrum of activity and mechanism of action. In general, they show a potent in vitro activity against antibiotic-resistant microorganisms. The widespread diffusion of multi-resistant strains has highlighted their potential as lead compounds for the development of novel antiinfective agents. Indeed, some of these peptides, or analogs, are already under advanced clinical trials for the treatment of topical infections. Finally, several reports suggesting that cathelicidin peptides may play additional roles in host defense, such as wound healing and chemotactic activity, have opened new fields of investigations. Further studies however are required to clearly establish the physiological relevance of the observed effects.

BMAP-28, an Antibiotic Peptide of Innate Immunity, Induces Cell Death through Opening of the Mitochondrial Permeability Transition Pore

ABSTRACT BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca2+ and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.

Pro-rich antimicrobial peptides from animals: Structure, biological functions and mechanism of action

Pro-rich antimicrobial peptides are a group of linear peptides of innate immunity isolated from mammals and invertebrates, and characterised by a high content of proline residues (up to 50%). Members of this group are predominantly active against Gram-negative bacterial species which they kill by a non-lytic mechanism, at variance with the majority of the known antimicrobial peptides. Evidence is accumulating that the Pro-rich peptides enter the cells without membrane lysis and, once in the cytoplasm, bind to, and inhibit the activity of specific molecular targets essential to bacterial growth, thereby causing cell death. This mode of action makes these peptides suitable for drug development efforts. In addition to antibacterial action, PR-39, one of the better characterised Pro-rich peptides from mammals, exerts other potentially exploitable biological activities, such as induction of syndecan expression in mesenchymal cells and inhibition of the NADPH oxidase activity of neutrophils, suggesting a role of this peptide in wound repair and inflammation. PR-39 also exerts a protective effect in various animal models of ischemia-reperfusion injury, preventing the post-ischemic oxidant production, and is a potent inducer of angiogenesis both in vitro and in vivo. Although the physiological relevance of all these effects has not yet been established, the above observations underscore the therapeutic potential of this peptide in a number of complex processes such as inflammation, wound repair, ischemia-reperfusion injury, and angiogenesis.

LL-37 Protects Rats against Lethal Sepsis Caused by Gram-Negative Bacteria

ABSTRACT We investigated the efficacy of LL-37, the C-terminal part of the only cathelicidin in humans identified to date (termed human cationic antimicrobial protein), in three experimental rat models of gram-negative sepsis. Adult male Wistar rats (i) were given an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS, (ii) were given 2 × 1010 CFU of Escherichia coli ATCC 25922, or (iii) had intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intravenously isotonic sodium chloride solution, 1-mg/kg LL-37, 1-mg/kg polymyxin B, 20-mg/kg imipenem, or 60-mg/kg piperacillin. Lethality; growth of bacteria in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; and endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma were evaluated. All compounds reduced lethality compared to levels in controls. Endotoxin and TNF-α plasma levels were significantly higher in conventional antibiotic-treated rats than in LL-37- and polymyxin B-treated animals. All drugs tested significantly reduced bacterial growth compared to saline treatment. No statistically significant differences between LL-37 and polymyxin B were noted for antimicrobial and antiendotoxin activities. LL-37 and imipenem proved to be the most effective treatments in reducing all variables measured. Due to its multifunctional properties, LL-37 may become an important future consideration for the treatment of sepsis.

cDNA sequences of three sheep myeloid cathelicidins

Several myeloid antimicrobial peptide precursors have been shown to consist of a N‐terminal proregion similar to a protein named cathelin and a structurally varied C‐terminal antimicrobial domain. Proteins with these features have been named cathelicidins. In this paper we report the cDNA sequences of three ovine cathelicidins of 155, 160 and 190 residues, respectively, with cationic C‐terminal sequences corresponding to putative antimicrobial domains. These are structurally varied and include a Cys‐rich sequence of 12 residues, which is similar to the bovine antimicrobial cyclic dodecapeptide, a novel 29 residue sequence named SMAP‐29 with a possible α‐helical conformation, and a 60 residue sequence named Bac7.5, which appears to be a new member of the Pro‐ and Arg‐rich group of mammalian antimicrobial peptides.