[Encyclopedia.of.Life.Sciences].Antimicrobial.Proteins.and.Peptides

Antimicrobial Proteins and Peptides Tomas Ganz, University of California, Los Angeles, California, USA Robert I Lehrer, University of California, Los Angeles, California, USA Antimicrobial proteins and peptides are important effectors of the innate and adaptive arms of the immune system. They interfere with microbial integrity or metabolism by targeting structures or nutrients specific to microbes. Introduction The production of polypeptides that disrupt microbial structures or interfere with microbial metabolism is a common means of host defence in animals and plants. Although microbial and host structures share many features, antimicrobial polypeptides achieve specificity by targeting components that host cells lack, by exploiting differences between corresponding host and microbial structures, and by selectively concentrating polypeptides on microbial surfaces. Some host peptides release or activate latent lytic enzymes (autolysins) of their microbial targets, and thereby potentiate their antimicrobial effects. Specific mechanisms to protect bystander host cells from damage exist, and include cell-associated or soluble macromolecules that bind and detoxify antimicrobial polypeptides. Infection or injury induces production of some anti- microbial polypeptides. Fruit fly (Drosophila melanoga- ster) mutants unable to induce antimicrobial peptide synthesis succumb to bacterial or fungal infections. Even more direct demonstrations of the importance of anti- microbial polypeptides for host defence have been achieved in plants, where transferring antimicrobial peptide genes from one species to another can produce resistance to specific pathogens. Mucosal Host Defence Initial encounters between microbes and multicellular animals often take place on moist surfaces (mucosae). These are coatedwith glycoproteins (mucins) that interfere with microbial attachment. Epithelia constitute physical barriers to microbial penetration. When ciliated, they mechanically remove microbes by transporting them in a blanket of mucus. Mucosal cells secrete antimicrobial peptides and proteins that damage microbes (Table 1). In some organs, such as the lungs, resident host defence macrophages patrol the epithelial surfaces, ingesting microbial trespassers. When surface-adherent microbes multiply or breach the epithelial barrier, affected host tissues produce signalling molecules (cytokines) that may induce the synthesis of additional antimicrobial polypep- tides. Concurrently, various microbial or host-derived molecules trigger an influx of mobile blood cells into the infected area. Phagocytosis Phagocytes are specializedhost defence cells that can ingest microbes and expose them to antimicrobial substances. Twogeneral types of phagocytes exist: the potentially long- lived macrophages and the short-lived granulocytes. Mature granulocytes are smaller than macrophages, manifest considerably less protein synthesis, and contain abundant cytoplasmic granules that are a repository of antimicrobial proteins and peptides and of hydrolytic enzymes. Phagocytes recognize their targets by phagocytic receptors coupled to the cellular machinery required for motility and ingestion. Certain receptors, such as the mannose receptor on macrophages, bind microbial sur- faces directly, while others bind via intermediate adapter molecules (opsonins) deposited onto microbial surfaces from the surrounding fluid. Specific antibodies (especially immunoglobulin G (IgG) class), complement fragments C3bandC3bi, andplasma lectins such asmannose-binding protein are particularly effective opsonins. Opsonins bound to microbial surfaces interact with Fcg receptors, C3b receptors (complement receptor 1, CR1), C3bi receptors (complement receptor 3, CR3), and other phagocytic receptors, causing the plasmamembrane of the phagocyte to gradually creep around the target microbe and to form a vacuole called the phagosome. Engagement of phagocytic receptors triggers cytoskeletal rearrange- ments, and fusion of granules or transport vesicles containing antimicrobial polypeptides to the phagosomal vacuole. Concurrently, NADPH oxidase complexes are Article Contents Introductory article . Introduction . Mucosal Host Defence . Phagocytosis . Complement . Defensins . Bactericidal Permeability-increasing Protein (BPI) . Proteases . Secretory Phospholipase A2 . Lactoferrin . Cathelicidins . Other Antimicrobial Polypeptides 1ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net assembled and activated in the phagosomal membrane, allowing copious production of superoxide anions. These undergo further reactions that generate hydrogen per- oxide, and (in concert with myeloperoxidase) more potent antimicrobial oxidants. In some species, a phagocyte enzyme that generates nitric oxide is also induced. The combined action of oxidants and antimicrobial polypep- tides destroys most ingested microbes within minutes. The sequestration of microbial targets in phagocytic vacuoles exposes microbes to high concentrations of cytocidal substances while minimizing collateral damage to the host cell. The most abundant granulocyte in human blood is the polymorphonucleated neutrophil (called PMN, ‘poly’, or neutrophil, for short). Although the cytoplasmic granules of human neutrophils are heterogeneous, one of its granule subsets (the primary or azurophilic granule) functions to deliver antimicrobial components to the phagosome. Other granule subsets – called secondary (or specific) and tertiary granules – secrete their contents into the extra- cellular fluid or into the plasma membrane.The primary and secondary granules of human neutrophils both contain antimicrobial polypeptides. Lysozyme Lysozyme (also calledmuramidase) is a widely distributed, cationic (i.e. positively charged) enzyme that cleaves the b 1,4-glycosidic linkage between N-acetyl muramate and N- acetyl glucosamine. It was first described by Sir Alexander Fleming in 1922, who called attention to its remarkable bacteriolytic properties. Because of its small size, abun- dance and easy purification, its crystal structure and catalytic mechanism have been extensively investigated. The principal target of lysozyme is peptidoglycan, a structural glycosidic polymer that gives bacteria shape stability and osmotic resistance. Disruption of the pepti- doglycan layer suffices to kill some bacteria and renders others more susceptible to osmotic stress. Enzymes with lysozyme activity are found in the digestive organs ofmany animals and also in host defence settings. Chitinases are functionally and structurally related enzymes that degrade chitin, an exoskeletal polymer of fungi and insects. High concentrations of lysozyme occur in avian egg white, and in mammalian tears, milk, respiratory secre- tions, granulocytes and macrophages. In human granulo- cytes, lysozyme is highly abundant in both primary and secondary granules, and it is a principal secreted protein of macrophages. Organisms susceptible to low concentra- tions of lysozyme include certain nonpathogenic Gram- positive bacteria (e.g. Micrococcus lysodeikticus and Bacillus species). Gram-negative bacteria are more resis- tant because their outer membranes hinder the access of lysozyme to peptidoglycan. At high but still realistic concentrations of lysozyme, nonenzymatic antimicrobial activity that depends on the highly cationic nature of this Table 1 Antimicrobial peptides and proteins secreted by mucosal cells Polypeptide Mass (kDa) Distribution Peroxidases 150 Myeloperoxidase Neutrophils, monocytes Eosinophil peroxidase Eosinophils Lactoperoxidase Epithelia (milk, saliva) Lactoferrin 80 Neutrophils Various epithelia Bactericidal permeability-increasing protein (BPI) 55–60 Neutrophils Serprocidins (cathepsin G, elastase, proteinase 3, azurocidin/CAP37) 25–30 Neutrophils Lysozyme 14 Neutrophils, monocytes, macrophages Various epithelia Phospholipase A2 14 Neutrophils, macrophages, platelets Paneth cells Many other tissues Secretory leucoprotease inhibitor (SLPI) 12 Macrophages Various epithelia Cathelicidins 1–18 Neutrophils (secondary granules) Inflamed skin Testes Defensins 4 Neutrophils Various epithelia Antimicrobial Proteins and Peptides 2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net molecule is also observed. The abundance and wide distribution of lysozyme suggest that it is of considerable importance. A single gene on chromosome 12 encodes the human lysozyme enzyme. Themouse has two lysozyme genes, one of which (lysozyme M) is expressed in macrophages and granulocytes, while the other (lysozyme P) is expressed by Paneth cells of the small intestine. In other animals, the number of lysozyme genes and their tissue-specific patterns of expression are highly variable. Complement The direct microbicidal effect of blood plasma on many microbial species is mediated by complement. The key lethal event is the assembly of the membrane attack complex (MAC) consisting of components C5b, C6, C7, C8 with two or more associated C9 molecules in the membranes of target microbes. The insertion of the initiating C5b fragment is the end result of complement activation via one of three complement cascades. The classical pathway is initiated by the binding of C1q molecules to specific antibody clustered on microbial surfaces, the alternative pathway is triggered by the interaction of C3b with the microbial surface, while the lectin pathway depends on contact of C1q-like mannose- binding protein with specific repeating glycans. The formation of the membrane attack complexes disrupts the ionic homeostasis of the microbes, killing susceptible microbes. Many pathogenic microbes have evolved thick cell walls or capsules that make them partially or completely resistant to complement. The importance of the direct microbicidal activity of comple- ment is illustrated by the predisposition of patients with a deficiency of C6, C7 or C8 to recurrent infections with Neisseria gonorrhoeae and N. meningitidis. Complement- like proteolytic cascades appeared early in the evolution of multicellular animals and have a documented antimicro- bial function already in arthropods (e.g. horseshoe crabs). Defensins Defensins are 3–4-kDa cationic antimicrobial peptides with three intramolecular cystine disulfide bonds and a largely b sheet structure. The vertebrate defensin family contains two branches, designated a- and b-defensins. Although similar in shape to a-defensins, b-defensins are slightly larger and differ in the placement and connectivity of their six conserved cysteine residues. Humans produce at least six different a-defensins and two b-defensins. Three human a-defensins (human neutrophil peptides: HNP-1, HNP-2 and HNP-3) constitute about 30% of the protein content of phagocytic granules of neutrophilic granulo- cytes. Trace amounts of a fourth defensin, HNP-4, are also found in the same compartment. HNP-1 and HNP-4 are encoded by genes designated DEFA1 and DEFA4, respectively. HNP-3 arises from a single nucleotide substitution in one of the several DEFA1 gene copies. HNP-2 results from posttranslational removal of the N- terminal residue fromHNP-1 and/or HNP-3. The remain- ing a-defensins HD-5 and HD-6 are expressed by human small-intestinal Paneth cells and are products of the DEFA5 and DEFA6 genes. Paneth cells are specialized, granule-laden secretory epithelial cells located at the bottom of crypts in the small intestine. Human b-defensins HBD-1 and HBD-2 are produced in various epithelia, and are encoded by theDEFB1 andDEFB2 genes, respectively. The DEFB1, DEFB2 gene and the human a-defensin gene cluster are neighbours, occupying a region of about 400– 600 kb on chromosome 8p23. Unlike the a-defensins of neutrophils or Paneth cells, epithelial b-defensins are not stored in cytoplasmic granules. Consequently, their local concentration may be governed primarily by their synth- esis and secretion rates. Other members of the defensin family of peptides have been found as abundant constituents of granulocytes in rabbits, rats, hamsters, guinea pigs, cows, sheep, chickens and turkeys but are absent from granulocytes of mice, pigs and horses. Epithelial defensins have been found in Paneth cells ofmice and rats, and in various epithelia of cows, pigs, sheep and mice. The production of tracheal and lingual antimicrobial peptides (TAP and LAP), as well as that of human epithelial defensin, HBD-2, is induced by inflam- matory stimuli. Inducible antimicrobial cationic peptides with six cysteines have been identified in the haemolymph of many insects and other invertebrates and have been called ‘insect’ defensins but their evolutionary relationship to vertebrate defensins is uncertain. Peptides of similar structure and composition are found in host defence roles in plants where they are referred to as plant defensins. Defensinsmanifest broad-spectrum antimicrobial activ- ity that is characteristically but variably inhibited by increasing salt concentrations. Many bacteria, fungi and some enveloped viruses are inactivated by defensins. The initial interaction of defensins with bacteria or fungi involves electrostatic forces, and is followed by permeabi- lization ofmicrobial membranes and the entry of defensins into the target cell. Preferential interaction of defensins with microbes as opposed to host cells is probably due to the microbial membrane composition: rich in anionic phospholipids and lacking cholesterol. Patients with ‘specific granule deficiency’ also have a deficiency of granulocyte defensins, and suffer from recurrent bacterial infections. Antimicrobial Proteins and Peptides 3ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Bactericidal Permeability-increasing Protein (BPI) Bactericidal permeability-increasing protein (BPI) is a highly cationic 55–60-kDa protein, abundant in human and other mammalian neutrophils. It is structurally related to the lipopolysaccharide-binding protein (LBP) in plasma, and likeLBP, binds to lipopolysaccharidewith high affinity. It kills many Gram-negative bacteria by a process that initially involves the permeabilization of their outer membranes, but is inactive against Gram-positive bacteria. BPI is an effective scavenger of lipopolysaccharide and recombinant BPI has promise as an experimental treatment inmeningococcal and other forms of Gram-negative sepsis. Human BPI is a highly elongated molecule formed by two domains of similar size connected by a proline-rich linker. An N-terminal domain of BPI expresses the bactericidal and endotoxin-neutralizing activities of BPI while the C-terminal domain may be required for binding events that facilitate phagocytosis of BPI-coated bacteria. The human genes encoding BPI, LBP and several other lipid-binding proteins are in close proximity on chromo- some 20 (q11.23–q12), suggesting a common ancestral origin. Despite their similarity in overall structural and functional design, the biological activities of BPI and LBP are strikingly different: whereas BPI is bactericidal toward Gram-negative bacteria and inhibits endotoxin signalling, LBP does not kill bacteria and greatly enhances the pro- inflammatory action of low doses of LPS. Initial interactions between BPI and target bacteria probably involve electrostatic attraction between the cationic regions of BPI and the anionic moieties clustered near the highly conserved lipid A region of LPS. In the outer membrane of intact bacteria, the insertion of BPI triggers the rearrangement of outer membrane lipids (LPS andphospholipids) anddisplacement ofLPS-boundMg2+ and Ca2+, resulting in increased permeability to small hydrophobic molecules, increased susceptibility of phos- pholipids to phospholipase A attack, and bacterial growth inhibition. In a rabbit model of peritoneal inflammation, extracellular BPI secreted by granulocytes into inflamma- tory exudates confers potent bactericidal activity against many Gram-negative bacteria. Some defensins, cathelici- dins and low concentrations of complement potentiate the activity of BPI. Gram-negative bacteria with long lipopo- lysaccharide chains (‘Smooth’ colony type) tend to be relatively resistant to BPI. Proteases Human and other mammalian granulocytes contain elastase, cathepsin G and proteinase 3, all of which are highly cationic 25–30-kDa serine proteases with antibac- terial properties (serprocidins). Azurocidin/CAP37 is another bactericidal, but enzymatically inactive, member of the same protein family. Azurocidin/CAP37, neutrophil elastase and proteinase 3 are encoded by a cluster of genes located at the tip of the short arm of chromosome 19. Cathepsin G gene is located in another cluster of serine protease genes on chromosome 14q11.2. Compared to the active serine proteases, azurocidin/CAP37 contains amino acid substitutions in two residues essential for catalytic activity (serine and histidine). In granulocytes, serproci- dins are located in the phagocytic (primary, azurophil) granules. Their collective abundance in humanneutrophils is similar to the other major granule proteins (lactoferrin, lysozyme, myeloperoxidase and defensins). The antibac- terial properties of serprocidins can often be dissociated from their proteolytic activity, and are competitively inhibited by increasing ionic strength, indicating the importance of charge interactions in their mechanism of action. Although neutrophil elastase is modestly micro- bicidal, it also converts inactive procathelicidins into highly microbicidal cathelicidins. Transgenic mice lacking neutrophil elastase are susceptible to sepsis and death from intraperitoneal infections with Gram-negative bacteria. Secretory Phospholipase A2 Human group II phospholipase A2 (PLA2) is a potent 14- kDa enzyme encoded by a gene on chromosome 1p35. The enzymebelongs to a large family of secretoryPLA2 enzymes that share similar structure and catalytic machinery, but differ in their preferred substrates. Human group II phospholipase A2 preferentially acts on the ester linkages of phospholipids found in bacterial membranes. The antimicrobial activity of this phospholipase is not shared by other phospholipases even though their catalytic mechanisms are very similar. Group II PLA2 is found in granulocytes, Paneth cells, epithelial secretions, and inflam- matory fluids. Against many strains and species of Gram- positive bacteria, purified group II PLA2 is active at nanomolar concentrations. Bacterial killing by PLA2 depends on binding to the bacterial surface (Ca2+- independent, presumably to sites in the cell wall), penetra- tion of the cell wall and Ca2+-dependent degradation of membrane phospholipids.AgainstGram-negative bacteria, independent antibacterial activity of mammalian group II PLA2 requires millimolar concentrations and is augmented by complement. Moreover, other host defence substances (e.g. bactericidal permeability-increasing protein, BPI) potentiate the action of nanogram per millilitre amounts of the PLA2 by disrupting the bacterial outer membrane. Lactoferrin Similar to the iron-binding plasma protein, transferrin, and encoded by a gene in the same region of chromosome Antimicrobial Proteins and Peptides 4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 3q21–23, lactoferrin is an iron-chelating 80-kDa protein found in the secretory (specific or secondary) granules of neutrophilic granulocytes and in epithelial secretions such as airway fluid, cervical mucus and breast milk. The molecule contains two similar lobes, each of which can bind a ferric cation. Lactoferrin is bacteriostatic by sequestering essential iron from many bacterial species but other species (e.g. Neisseria gonorrhoeae) have developed mechanisms for pilfering iron from lactoferrin. Naturally occurring limited proteolysis of lactoferrin yields a cationic bactericidal peptide, lactoferricin, whose ac