238 | MARCH 2005 | VOLUME 3 www.nature.com/reviews/micro R E V I E W S Department of Periodontics and Dows Institute for Dental Research, College of Dentistry, The University of Iowa, Iowa City,Iowa 52242,USA. e-mail: kim-brogden@uiowa.edu doi:10.1038/nrmicro1098 Published online 10 February 2005 The antimicrobial activities of secretions, blood, leukocytes, and lymphatic tissues were recognized as early as the ���last fifteen years of the nineteenth century���1, and between 1920 and 1950 many antimicrobial com- pounds that were isolated from these secretions were shown to be selective for Gram-positive and Gram- negative bacteria1. The list of compounds included a bacteriolytic substance in nasal mucous (which was later named lysozyme2), basic antimicrobial proteins and basic linear tissue polypeptides.Although some of the larger basic proteins were thought to be histone fractions and protamine, the identity of the small tissue polypeptides was unknown. Despite this, the descrip- tions of their characteristics, activities and modes of action were accurate:������antimicrobial basic proteins and polypeptides combine with cell nucleoproteins or other negatively charged surface constituents of bacteria or viruses, thus disrupting important cell function. The union of the basic substances with negatively charged cell surfaces is believed to occur through electrostatic bonding���1. The association of the presence of these antimicrobial substances in normal tissues and fluids with natural resistance to microorganisms was clearly made.They were described as being inducible on expo- sure to infecting microorganisms, to kill or slow the growth of invading microorganisms and to aid allied mechanisms of natural and adaptive immunity. Thus the field of antimicrobial peptide research was born. Shortly afterwards, antimicrobial substances were purified from phagocytic granule extracts by Hirsch3 and correlated with the presence of low-molecular-mass cationic compounds in granule mixtures4���6, including bactericidal/permeability-increasing protein7. The field expanded further when Hans Boman, Michael Zasloff and Robert Lehrer independently isolated and purified insect cecropins,amphibian magainins and mammalian defensins,respectively8���10.Now,more than 880 different antimicrobial peptides have been identified or predicted from nucleic acid sequences (see Anti-infective peptides in the Online links box). These include antimicrobial peptides that are produced in many tissues and cell types of a variety of invertebrate,plant and animal species11���15, certain cytokines and chemokines16���18, selected neuro- peptides and peptide hormones19,20, and fragments of ANTIMICROBIAL PEPTIDES: PORE FORMERS OR METABOLIC INHIBITORS IN BACTERIA? Kim A. Brogden Abstract | Antimicrobial peptides are an abundant and diverse group of molecules that are produced by many tissues and cell types in a variety of invertebrate, plant and animal species. Their amino acid composition, amphipathicity, cationic charge and size allow them to attach to and insert into membrane bilayers to form pores by ���barrel-stave���, ���carpet��� or ���toroidal-pore��� mechanisms. Although these models are helpful for defining mechanisms of antimicrobial peptide activity, their relevance to how peptides damage and kill microorganisms still need to be clarified. Recently, there has been speculation that transmembrane pore formation is not the only mechanism of microbial killing. In fact several observations suggest that translocated peptides can alter cytoplasmic membrane septum formation, inhibit cell-wall synthesis, inhibit nucleic-acid synthesis, inhibit protein synthesis or inhibit enzymatic activity. In this review the different models of antimicrobial-peptide-induced pore formation and cell killing are presented.

NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | MARCH 2005 | 239 R E V I E W S combined with similar studies of the same peptides with microorganisms, have helped to identify the parameters that are required for optimal peptide activity. In this review, the different models of antimi- crobial peptide activity are presented with a discussion on the relevance of the mechanisms to antimicrobial- peptide-induced killing of microorganisms. Anti- microbial peptides can also inactivate nucleic acids and cytoplasmic proteins, and evidence of this as a mechanism for antimicrobial-peptide-induced killing of microorganisms is included. Antimicrobial peptide diversity Antimicrobial peptides are a unique and diverse group of molecules (BOXES 1,2), which are divided into sub- groups on the basis of their amino acid composition and structure15,26���28. The NMR solution structures of selected peptides of these subgroups are shown in FIG. 1. One subgroup contains anionic antimicrobial peptides. Among these are small (721.6���823.8 Da) peptides present in surfactant extracts, bronchoalveolar lavage fluid and airway epithelial cells29���31. They are produced in mM concentrations, require zinc as a cofactor for antimicrobial activity and are active against both Gram- positive and Gram-negative bacteria. They are similar to the charge-neutralizing pro-peptides of larger zymogens, which also have antimicrobial activity when synthesized alone32. A second subgroup contains ~290 cationic peptides, which are short (contain 40 amino acid residues), lack cysteine residues and sometimes have a hinge or ���kink���in the middle26,33 (see Anti-infective peptides in the Online links box). In aqueous solutions many of these peptides are disordered, but in the presence of trifluoroethanol, sodium dodecyl sulphate (SDS) micelles, phospholipid vesicles and liposomes, or Lipid A, all or part of the molecule is converted to an ��-helix26. A good example is LL-37. In water, it exhibits a circular dichroism (CD) spectrum that is consistent with a disordered struc- ture34. However, in 15 mM HCO3���, SO42��� or CF3CO2���, the peptide adopts a helical structure. As has been observed for buforin II, its congeners and LL-37, the extent of ��-helicity correlates with the antibacterial activity against both Gram-positive and Gram-negative bacteria ��� increased ��-helical content correlates with stronger antimicrobial activities35. A third subgroup contains ~44 cationic peptides that are rich in certain amino acids36 (see Anti-infective peptides in the Online links box). This group includes the bactenecins and PR-39, which are rich in proline (33���49%) and arginine (13���33%) residues prophenin, which is rich in proline (57%) and phenylalanine (19%) residues and indolicidin, which is rich in tryptophan residues26,36. These peptides lack cysteine residues and are linear,although some can form extended coils. A fourth subgroup of anionic and cationic peptides have ~380 members, contain cysteine residues and form disulphide bonds and stable ��-sheets (see Anti- infective peptides in the Online links box). This sub- group includes protegrin from porcine leukocytes (which comprises 16 amino acid residues, including larger proteins21���23. In fact, the list of host-derived antimicrobial molecules is increasing so rapidly that one has to question the biological relevance and likely roles in innate immunity, particularly some antimicrobial fragments of larger proteins. Antimicrobial peptides are recognized as a possible source of pharmaceuticals for the treatment of anti- biotic-resistant bacterial infections or septic shock24,25. Studies to assess the mechanisms of natural peptide and peptide congener activity in model membrane systems, Box 1 | Classes of antimicrobial peptides Anionic peptides ��� Maximin H5 from amphibians146. ��� Small anionic peptides rich in glutamic and aspartic acids from sheep,cattle and humans30. ��� Dermcidin from humans147. Linear cationic ��-helical peptides ��� Cecropins (A),andropin,moricin,ceratotoxin and melittin from insects. ��� Cecropin P1 from Ascaris nematodes148. ��� Magainin (2),dermaseptin,bombinin,brevinin-1,esculentins and buforin II from amphibians. ��� Pleurocidin from skin mucous secretions of the winter flounder. ��� Seminalplasmin,BMAP,SMAP (SMAP29,ovispirin),PMAP from cattle,sheep and pigs. ��� CAP18 from rabbits. ��� LL37 from humans. Cationic peptides enriched for specific amino acids ��� Proline-containing peptides include abaecin from honeybees28. ��� Proline- and arginine-containing peptides include apidaecins from honeybees28 drosocin from Drosophila28 pyrrhocoricin from the European sap-sucking bug36 bactenecins from cattle (Bac7),sheep,and goats149 and PR-39 from pigs73,150. ��� Proline- and phenylalanine-containing peptides include prophenin from pigs150. ��� Glycine-containing peptides include hymenoptaecin from honeybees28. ��� Glycine- and proline-containing peptides include coleoptericin and holotricin from beetles28. ��� Tryptophan-containing peptides include indolicidin from cattle151. ��� Small histidine-rich salivary polypeptides,including the histatins from man and some higher primates116. Anionic and cationic peptides that contain cysteine and form disulphide bonds ��� Peptides with 1 disulphide bond include brevinins152. ��� Peptides with 2 disulphide bonds include protegrin from pigs and tachyplesins from horseshoe crabs153. ��� Peptides with 3 disulphide bonds include ��-defensins from humans (HNP-1,HNP-2, cryptidins),rabbits (NP-1) and rats154 ��-defensins from humans (HBD1,DEFB118), cattle,mice,rats,pigs,goats and poultry12 and rhesus ��-defensin (RTD-1) from the rhesus monkey40. ��� Insect defensins (defensin A)95. ��� SPAG11/isoform HE2C,an atypical anionic ��-defensin42. ��� Peptides with 3 disulphide bonds include drosomycin in fruit flies155 and plant antifungal defensins155. Anionic and cationic peptide fragments of larger proteins ��� Lactoferricin from lactoferrin. ��� Casocidin I from human casein. ��� Antimicrobial domains from bovine ��-lactalbumin,human haemoglobin,lysozyme and ovalbumin.