CMLS, Cell. Mol. Life Sci. 60 (2003) 2651–2668 1420-682X/03/122651-18 DOI 10.1007/s00018-003-3106-8 CMLS Cellular and Molecular Life Sciences © Birkhäuser Verlag, Basel, 2003 Review Antimicrobial and cytolytic peptides of venomous arthropods L. Kuhn-Nentwig Zoological Institute, University of Bern, Baltzerstrasse 6, 3012 Bern (Switzerland), Fax: +41 31 631 48 88, e-mail: [email protected],URL: http://www.zoology.unibe.ch/ecol/ Received 17 March 2003; received after revision 11 June 2003; accepted 17 June 2003 Abstract. As a response to invading microorganisms, the interact together, resulting in extremely rapid immobi- innate immune system of arthropods has evolved a com- lization and/or killing of prey or aggressors. This review plex arrangement of constitutive and inducible antimicro- provides an overview of antimicrobial peptides identified bial peptides that immediately destroy a large variety of in the hemolymph of venomous arthropods, and espe- pathogens. At the same time, venomous arthropods have cially of cytolytic peptides in their venom. For these pep- developed an additional offensive system in their venom tides a dual role is proposed: acting as antimicrobials as glands to subdue their prey items. In this complex venom well as increasing the potency of the venom by influenc- system, several enzymes, low-molecular-mass com- ing excitable cells. pounds, neurotoxins, antimicrobial and cytolytic peptides Key words. Antimicrobial peptides; cytolytic peptides; venom; arthropods; synergism. Introduction membrane of bacteria and parasitic protozoans. The un- structured antimicrobial peptides are electrically at- As strategies in antagonistic relationships, prokaryotic tracted to negatively charged groups of the cell surface, and eukaryotic organisms have developed hundreds of where they adopt an a-helical conformation and accumu- different cytolytic peptides and proteins during their evo- late on the membrane. This can result in the formation of lution in different phyla of the plant [1] and animal king- transient pores, membrane perturbation and cell lysis [8]. dom [2]. Many cytolytic peptides are specific antimicro- Antimicrobial peptides in vertebrates and invertebrates bially acting peptides that are part of the innate immune can either be induced by microbial infection and inflam- system of invertebrates and vertebrates. They serve as mation or produced constitutively. In vertebrates, biogen- primary defense weapons against invading prokaryotic esis occurs in circulating phagocytes, destroying intracel- and eukaryotic microorganisms, a fact which is well doc- lularly phagocytosed pathogens by defensins and like- umented by a constantly increasing number of publica- wise in different tissues and epithelia from where they are tions and reviews [3–6]. actively secreted into the lumen, as has been shown for Gene-encoded antimicrobially acting peptides show great enteric a-defensins [9]. In some crustaceans [10, 11] and variety in amino acid sequence, structure and target chelicerates, these peptides are stored constitutively in specificity. Many of them are cationic, amphipathic pep- hemocytes and released into the hemolymph by exocyto- tides with molecular masses lower than 10 kDa and show sis after microbial injury as an ancient form of host de- a higher specificity to prokaryotic than to eukaryotic cells fense [12, 13]. Insects exhibit a further mode of action in [7]. The main site of antimicrobial activity is the plasma response to microbial challenge. Depending on the im- munogenic character of the microbial invaders, the tran- scription of different antimicrobial peptides, mainly in * Corresponding author. the fat body, is induced by two different pathways. The 2652 L. Kuhn-Nentwig Antimicrobial peptides of arthropods Imd pathway controls the immune response to Gram-neg- Antimicrobial peptides in the hemolymph of ative bacteria, and the Toll pathway is activated after in- venomous arthropods fection with fungi and Gram-positive bacteria [5]. After induction, a rapid release of diverse antimicrobial pep- Antimicrobial peptides isolated from the hemolymph of tides into the hemolymph follows within hours [14]. Fur- actively venomous arthropods have quite different struc- ther constitutive expressions of these peptides are also re- tures from the cytolytic peptides identified in their ven- ported in hemocytes, salivary glands [15] and in the oms. According to their structure, antimicrobial peptides midgut of Anopheles gambiae [16, 17]. can be arranged into four families: a-helical, linear pep- Additionally, other cytolytic peptides are used facilita- tides devoid of cysteines, the proline-rich and glycine- tively for the improvement of living conditions and access rich peptides, and the cysteine-rich, open-ended cyclic to nutrition. Bacteria that produce cytolytic peptides peptides such as insect defensins [14]. seem to have a growth advantage over their natural com- In the hemolymph of the scorpion Leiurus quinquestria- petitors for the same growth substrate and in the same tus (Buthidae) [26] a 4-kDa scorpion defensin was iden- ecological niche [18, 19]. This bactericidal activity can be tified with a high sequence homology to an insect de- specifically targeted at bacteria of the same group [20] or fensin from the dragonfly Aeschna cyanea [27]. The scor- may occur as a more unspecific cytolytic activity against pion defensin is a cationic 39-residue peptide with a prokaryotic and eukaryotic cell membranes, as in the case CSab fold and three intramolecular disulfide bridges, a of gramicidin S [21]. Cytolytic peptides from the para- common structural motif also present in scorpion toxins sitic protozoans Entamoeba histolytica and Entamoeba [28]. A further defensin from the hemolymph of the scor- dispar (amoebapores and disparpores) primarily destroy pion Androctonus australis, also belonging to the family the phagocytosed bacteria as food. The cytolytic activity Buthidae, showed 95% identity with the Leiurus de- toward eukaryotic cells that some of them exert seems to fensin, both acting mainly on Gram-positive bacteria be a mere side effect [22, 23]. [29]. These high sequence similarities suggest a common On the other hand, arthropods such as scorpions, spiders ancient peptide structure for scorpion neurotoxins and in- and stinging hymenopterans have developed venom sect defensins [30]. From the same hemolymph, two fur- glands that mainly produce three different groups of com- ther antimicrobially acting peptides were characterized: ponents, based on their molecular mass. The first group buthinin, with three disulfide bridges, active against both consists of proteins, including several enzymes such as Gram-positive and Gram-negative bacteria, and androc- hyaluronidases, phospholipases and sphingomyelinases. tonin, with two disulfide bridges. Androctonin is bacteri- The second group is represented by a peptide fraction cidal in the micromolar range to both Gram-positive and with molecular masses around and lower than 10 kDa, Gram-negative bacteria. The peptide additionally exhibits containing several neurotoxic and cytolytic compounds. a fungicidal activity and is nonhemolytic up to a concen- A third group is composed of low-molecular-mass sub- tration of 150 mM [31]. Its sequence shows a close simi- stances such as ions, free amino acids, biogenic amines, larity to antimicrobial tachyplesins and polyphemusins neurotransmitters, acylpolyamines, heterocyclic com- isolated from the hemolymph of the horseshoe crabs pounds and alkaloids [24, 25]. The cooperation of all Tachypleus tridentatus and Limulus polyphemus, belong- these mentioned components in the venom enables these ing to the old marine arthropod group Xiphosura [32, 33]. arthropods to paralyze and/or to kill other arthropods in As assumed for cytolytic peptides from the venom of terms of foraging and defense. As a reproduction strategy, scorpions, antimicrobial peptides in the hemolymph also parasitic wasps also paralyze other arthropods and lay an seem to be constitutively present, because their concen- egg inside or outside of the prey, which serves as a nutri- tration does not increase after bacterial injury [26, 29]. tion source for their offspring. This review will focus on Antimicrobial peptides from the hemolymph of spiders antimicrobial and cytolytic peptides from the hemolymph are currently known from one unchallenged mygalo- and venom glands of poisonous arthropods identified morph species, Acanthoscurria gomesiana (Theraphosi- over the last 40 years. Common to all peptides is their am- dae), from which gomesin had been characterized [34]. phipathic structure and their ability to disturb membrane This peptide contains four cysteines and is active against structures. In context with the fact that all external world- different Gram-negative bacteria (0.4–6.25 mM), Gram- facing epithelia, ducts and glands dispose of growth-in- positive bacteria (0.2–12.5 mM) and fungi (0.4–25 mM). hibiting substances against potential invaders, a possible Gomesin exhibits hemolytic activity of 16% at 1 mM, dual role, especially for the peptides identified in the which does not increase up to concentrations of 100 mM. venom, will be discussed. Interestingly, the viability of Leishmania amazonensis promastigotes was reduced to 50% in the presence of 2.5 mM gomesin. Structural and sequence alignment of gomesin with protegrin from porcine leukocytes [35] and androctonin revealed several similarities in their amino CMLS, Cell. Mol. Life Sci. Vol. 60, 2003 Review