A novel protein family, showing a conserved proregion and a variable C-terminal antimicrobial domain, and named cathelieidin, has been identified in mammalian myeloid cells. The conserved proregion shows sequence similarity to members of the cystatin snperfamily 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 a-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.

Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species. Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and activate mesenchymal cells during wound repair. To permit further study of these proteins a reverse transcriptase-polymerase chain reaction approach was used to identify potential mouse homologs. A full-length 562-base pair cDNA clone was obtained encoding an NH 2 -terminal prepro domain homologous to other cathelicidins and a unique COOH-terminal peptide. This gene, named Cramp for cathelin-related antimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins have been mapped in pig and man. Northern blot analysis detected a 1-kilobase transcript that was expressed in adult bone marrow and during embryogenesis as early as E12, the earliest stage of blood development. Reverse transcriptase-polymerase chain reaction also detected CRAMP expression in adult testis, spleen, stomach, and intestine but not in brain, liver, heart, or skeletal muscle. To evaluate further the expression and function of CRAMP, a peptide corresponding to the predicted COOH-terminal region was synthesized. CD spectral analysis showed that CRAMP will form an amphipathic ␣-helix similar to other antimicrobial peptides. Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of bacterial strains (minimum inhibitory concentrations 0.5-8.0 M) and by permeabilizing the inner membrane of Escherichia coli directly at 1 M. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils. Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse. These data suggest that inflammatory cells in the mouse can use a nonoxidative mechanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair.

SummaryIn contrast to many antimicrobial peptides, members of the proline-rich group of antimicrobial peptides inactivate Gram-negative bacteria by a non-lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline-rich Bac7(1-35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptide's mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA-null mutants, in fact showed resistance to several proline-rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1-35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline-rich antimicrobial peptides of eukaryotic origin.

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.

In this work we study (i) the formation and stabilization of silver nanoparticles in a bioactive chitosan-derived polysaccharide solution, (ii) the antimicrobial properties, either in solution or in 3D hydrogel structures, obtained by mixtures with the polysaccharide alginate, and (iii) the cytotoxicity of the latter nanocomposite materials on different eukaryotic cell lines. Antimicrobial results show that these nanocomposite systems display a very effective bactericidal activity toward both Gram+ and Gram- bacteria. However, the hydrogel does not show any cytotoxic effect toward three different eukaryotic cell lines. This is due to the fact that the nanoparticles, immobilized in the gel matrix, can exert their antimicrobial activity by simple contact with the bacterial membrane, while they can not be uptaken and internalized by eukaryotic cells. This novel finding could advantageously contribute to responding to the growing concerns on the toxicity of nanoparticles and facilitate the use of silver-biopolymer composites in the preparation of biomaterials.

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 ␣-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 Grampositive 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.Research on naturally occurring antimicrobial peptides has resulted in the recognition of a surprisingly large number of peptides that are produced by animals and plants so as to fight infections (1, 2). In recent years, an additional impulse to the identification of novel antimicrobial peptides has come from the demand for new drugs ensuing from the emergence of multidrug resistant pathogens (3). Accordingly, the conversion of naturally occurring antimicrobial peptides into drugs is a goal that is currently being pursued by several research groups and biotechnology companies.We and others have recently described a number of novel mammalian antimicrobial peptides that are derived from a group of precursors named cathelicidins (4). Precursors of this family are stored in the neutrophil granules of various mammalian species. They show a highly conserved N-terminal prosequence and a markedly varied C-terminal domain that exhibits antimicrobial activity after the propiece has been removed. Cathelicidin-derived antimicrobial peptides range in length from 12 to about 100 residues, and include ␣-helical peptides, e.g. human LL-37/hCAP18 (5-7) and pig PMAP-37 (8); linear peptides with one or two predominant amino acids, e.g. the bovine Pro-and Arg-rich Bac5 and Bac7 (9) and the Trp-rich indolicidin (10); and peptides with one or two disulfide bonds, e.g. bovine cyclic dodecapeptide (11) and pig protegrins (12).A characteristic feature of cathelicidins is the extensive conservation of their mRNAs in the 5Ј region corresponding to the 5Ј noncoding region, the signal peptide and the prosequence. This feature has allowed identification of the mRNAs of several novel congeners in various mammalian species. At present, more than 20 cathelicidins with molecular masses of 16 ...

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.

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