Barbara Skerlavaj

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.

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.

Cathelicidin Peptides as Candidates for a Novel Class of Antimicrobials

Cathelicidin peptides are a numerous group of mammalian cationic antimicrobial peptides. Despite a common evolutionary origin of their genes, peptides display a remarkable variety of sizes, sequences and structures. Their spectra of antimicrobial activity are varied and cover a range of organisms that includes bacteria, fungi and enveloped viruses. In addition, they bind to and neutralize the effects of endotoxin. These features make this family of peptides good candidates in view of a therapeutic use. The most promising ones are currently under evaluation as leads for the development of novel anti-infectives, and synthetic variants are in an advanced stage of development for specific clinical applications. This review focuses on recent studies on the structure and in vitro and in vivo biological activities of these peptides.

The Human Cathelicidin LL-37 Modulates the Activities of the P2X7 Receptor in a Structure-dependent Manner

Extracellular ATP, released at sites of inflammation or tissue damage, activates the P2X7 receptor, which in turn triggers a range of responses also including cell proliferation. In this study the ability of the human cathelicidin LL-37 to stimulate fibroblast growth was inhibited by commonly used P2X7 blockers. We investigated the structural requirements of the growth-promoting activity of LL-37 and found that it did not depend on helix sense (the all-d analog was active) but did require a strong helix-forming propensity in aqueous solution (a scrambled analog and primate LL-37 orthologs devoid of this property were inactive). The involvement of P2X7 was analyzed using P2X7-expressing HEK293 cells. LL-37 induced proliferation of these cells, triggered Ca2+ influx, promoted ethidium bromide uptake, and synergized with benzoyl ATP to enhance the pore and channel functions of P2X7. The activity of LL-37 had an absolute requirement for P2X7 expression as it was blocked by the P2X7 inhibitor KN-62, was absent in cells lacking P2X7, and was restored by P2X7 transfection. Of particular interest, LL-37 led to pore-forming activity in cells expressing a truncated P2X7 receptor unable to generate the non-selective pore typical of the full-length receptor. Our results indicate that P2X7 is involved in the proliferative cell response to LL-37 and that the structural/aggregational properties of LL-37 determine its capacity to modulate the activation state of P2X7.

Identification and characterization of a primary antibacterial domain in CAP18, a lipopolysaccharide binding protein from rabbit leukocytes

Secondary structure prediction studies on CAP18, a lipopolysaccharide binding protein from rabbit granulocytes, identified a highly cationic, 21‐residue sequence with the tendency to adopt an amphipathic α‐helical conformation, as observed in many antimicrobial peptides. The corresponding peptide was chemically synthesized and shown to exert a potent bacterieidal activity against both Gram‐negative and Gram‐positive bacteria, and a rapid permeabilization of the inner membrane of Escherichia coli. Five analogues were synthesized to elucidate structure/activity relationships. It was found that helix disruption virtually eliminates antibacterial activity, while the degree of amphipathicity and the presence of an aromatic residue greatly affect the kinetics of bacterial inner membrane permeabilization.

Production of a recombinant antimicrobial peptide in transgenic plants using a modified VMA intein expression system

Tobacco plants were engineered to express SMAP‐29, a mammalian antimicrobial peptide of innate immunity, as fusion protein with modified vacuolar membrane ATPase intein. The peptide was purified taking advantage of the intein‐mediated self‐cleaving mechanism. SMAP‐29 was immunologically detected in the chromatographic eluate and appeared tightly bound to copurified plant proteins. Electrophoretic separation under disaggregating conditions indicated that the recombinant peptide was cleaved off by intein at the expected site and an overlay gel assay demonstrated that the peptide retained antimicrobial activity. These results indicate that a modified intein expression system can be used to produce pharmaceutical peptides in transgenic plants.

Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates

Ten peptides from 13 to 35 residues in length and covering the whole sequence of the Pro-rich peptide Bac7 were synthesized to identify the domain responsible for its antimicrobial activity. At least 16 residues of the highly cationic N-terminal sequence were required to maintain the activity against Gram-negative bacteria. The fragments Bac7(1-35) and, to a lesser extent, Bac7(1-16) proved active against a panel of antibiotic-resistant clinical isolates of Gram-negative bacteria, with the notable exception of Burkholderia cepacia. In addition, when tested against fungi, the longer fragment was also active against collection strains and clinical isolates of Cryptococcus neoformans, but not towards clinical isolates of Candida albicans.