Antimicrobial Peptides in Spider Venoms - Springer Link

Antimicrobial Peptides in Spider Venoms

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Daniel M. Santos, Pablo. V. Reis, and Adriano M. C. Pimenta

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antimicrobial Peptides as Candidates for Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Structural Features of AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible Modes of Action of AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AMPs from Spiders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Linear Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclic Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Antimicrobial Peptides in Spider Venoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potential Commercial Use of AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Historically, toxinologists have regarded venom studies focusing on lethality and other apparent toxic effects using mammals and insects as models. Nevertheless, with the development of sensitive and accessible analytical techniques, novel structures, especially peptides lacking observable effects in mammal and insect systems, have been increasingly noticed. Among such novel structures and activities are the antimicrobial peptides (AMPs). In this chapter, we review the current literature dealing with AMPs from spider venoms since their first appearance back in 1998, when a peptide was isolated from the venom of a species of wolf spider. It is also worth mentioning that the description of such peptides is constantly expanding, along with the information gathered regarding their structure and functional relationships over the last decade.

D.M. Santos • P.V. Reis • A.M.C. Pimenta (*) Departamento de Bioquı´mica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil e-mail: [email protected]; [email protected]; [email protected]; [email protected] # Springer Science+Business Media Dordrecht 2016 P. Gopalakrishnakone et al. (eds.), Spider Venoms, Toxinology, DOI 10.1007/978-94-007-6389-0_19

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Introduction Venoms from spiders are a complex mixture of molecules with enormous diversity. The chemicals they contain, such as small polypeptides, acylpolyamines, free acids, biogenic amines, glucose, free amino acids, inorganic salts, and ions, may have pharmacological actions, as neurotransmitters, modulators, and/or blockers of ion channels and pore formers in plasma membranes. In addition, high molecular weight molecules, including enzymes and other proteinaceous neurotoxins, can be found in these venoms (Escoubas et al. 2000; Estrada et al. 2007). Among the peptide constituents of spider venoms, a group that has emerged onto the scientific field consists of the antimicrobial peptides. The first report of antimicrobial activity in the venom of a spider was made by Xu et al. in 1989. These authors reported a peptide from the venom of the spider Lycosa singoriensis that was active against Escherichia coli. Since then, other antibacterial peptides have been found in the venoms of these arthropods (Kuhn-Nentwig 2003, 2009; Remijsen et al. 2006; Pukala et al. 2007; Shlyapnikov et al. 2008; Cerovsky et al. 2008). This is not a surprise, as there is a close structural similarity between some toxins and a specific class of antimicrobial peptides, the defensins (Dimarcq et al. 1998).

Antimicrobial Peptides as Candidates for Antibiotics Interest in the use of antimicrobial peptides (AMP) has led to the development of a new generation of peptide antibiotics, and has increased in recent years owing to the rise of microorganisms that are resistant to conventional antibiotics. Some remarkable features make possible treatment with AMPs an attractive alternative proposition: the direct action against microorganisms, the possible combination with conventional antimicrobials to promote an additive or synergistic effect, immunomodulatory action, and the effect on endotoxin neutralization that prevents associated complications (Miranda et al. 2009). The AMPs have generally accelerated pharmacokinetics, showing a high rate of excretion and rapid clearance from the circulation, with subsequent penetration into extravascular tissues, thereby facilitating accumulation at sites of infection. By radioactive labeling with technetium-99 m, the biodistribution of AMPs and the diagnosis of sites of infection have been analyzed in real time in vivo scintigraphy (Welling et al. 2001). Different AMPs have shown a broad spectrum of activity, being effective against Gram-positive and Gram-negative bacteria (Brogden 2005), fungi, protozoa, and viral envelopes (Daffre et al. 2001; Bulet et al. 2004), and have been studied as potential molecules to combat tumor cells (Mader and Hoskin 2006). These activities indicate AMPs as a future alternative to conventional antibiotics, especially in the treatment of infections produced by microorganisms resistant to existing antibiotics (Table 1; Ferre et al. 2009).

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Table 1 Spider antimicrobial peptides against Gram-positive and Gram-negative bacteria, fungi, and insect and mammal activities Peptide CIT 1a CIT 1b CIT1c CIT 1d CIT1e CIT 1f CIT 1g CIT 1h Cupiennin 1a

Biological assay Bac + Bac + + NT NT NT NT NT NT NT NT NT NT NT NT NT NT + +

Fung NT NT NT NT NT NT NT NT NT

Pro NT NT NT NT NT NT NT NT +

Inse + NT NT NT NT NT NT NT +

Mam + NT NT NT NT NT NT NT +

GsMTx4 Juruin Lycocitin 1 Lycocitin 2 Lycocitin 3 Lycotoxin I Lycotoxin II Ltc1 Ltc2a

+ + NT NT NT NT NT + +

+ + NT NT NT + + + +

NT + NT NT NT + + + +

NT NT NT NT NT NT NT NT NT

NT NT NT NT NT + + NT NT

NT + NT NT NT + + + +

Ltc3a Ltc3b Ltc4a Ltc4b Ltc5 LtTx-1a LtTx-1b LtTx-2a LtTx-2b LtTx-2c LyeTx I Oh-defensin Oxt 4a Oxyopinin1 Oxyopinin2a Oxyopinin2b Oxyopinin2c Oxyopinin2d PcFK1 PcFK2

+ + + + + NT NT NT NT + + + + + + + + + + +

+ + + + + NT NT NT NT + + + + + + + + + + +

+ + + + + NT NT NT NT NT + + NT NT NT NT NT NT + +

NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT + +

NT NT NT NT NT NT NT NT NT + NT NT NT NT NT NT NT NT NT NT

+ + + + + NT NT NT NT NT + NT NT + + + + + + +

References (Vassilevski et al. 2008) (Vassilevski et al. 2008) (Vassilevski et al. 2008) (Vassilevski et al. 2008) (Vassilevski et al. 2008) (Vassilevski et al. 2008) (Vassilevski et al. 2008) (Vassilevski et al. 2008) (Kuhn-Nentwig et al. 2002) (Kuhn-Nentwig et al. 2011) (Jung et al. 2006) (Ayroza et al. 2012) (Budnik et al. 2004) (Budnik et al. 2004) (Budnik et al. 2004) (Yan and Adams 1998) (Yan and Adams 1998) (Kozlov et al. 2006)) (Kozlov et al. 2006) (Vorontsova et al. 2011) (Kozlov et al. 2006) (Kozlov et al. 2006) (Kozlov et al. 2006) (Kozlov et al. 2006) (Kozlov et al. 2006) (Kuzmenkov et al. 2013) (Kuzmenkov et al. 2013) (Kuzmenkov et al. 2013) (Kuzmenkov et al. 2013) (Kuzmenkov et al. 2013) (Santos et al. 2010) (Zhao et al. 2011) (Dubovskii et al. 2011) (Corzo et al. 2002) (Corzo et al. 2002) (Corzo et al. 2002) (Corzo et al. 2002) (Corzo et al. 2002) (Choi et al. 2004) (Choi et al. 2004)

Bac+ Gram-positive bacteria, Bac Gram-negative bacteria, Pro protozoa, Inse insects, Mam mammal; + active, NT not tested ISSN International Centre: The ISSN Register. 2010 (updated 2010 Feb 19; cited 7 April 2010). http:// www.issn.org

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In addition to their direct action on microorganisms, AMPs possess immunomodulatory function, as observed for example, in the regulation of inflammatory responses in vertebrates (Hancock and Sahl 2006). Some AMPs can activate “Tolllike receptors” (TLR), and may also regulate the production of proinflammatory cytokines and chemokines (Vora et al. 2004). Other actions include the stimulation of the recruitment and proliferation of T cells, macrophages, neutrophils, and eosinophils, thus stimulating phagocytosis and the release of prostaglandin (Niyonsaba et al. 2001). AMPs can also act on the differentiation of dendritic cells and stimulate angiogenesis (Bowdish et al. 2005).

General Structural Features of AMPs Regarding the chemical properties, most AMPs are cationic and amphipathic molecules (Brogden 2005), spanning from 10 to 80 amino acid residues, often with around 50 % hydrophobic residues and positively charged hydrophilic residues at physiological pH. For many AMPs, the cationic region of the molecule seems to be responsible for its attraction for the anionic membranes of bacteria and, finally, the arrangement and composition of AMPs allow the formation of structures that cause the death of the microorganism by cell permeabilization (Ferre et al. 2009; Brogden 2005). The amino acid sequences and secondary structure of AMPs are important for establishing their amphipathic characteristics, which cause larger or smaller interactions with different membrane types. Their primary sequences are able to structure these peptides in α-helix, folded (hairpin), and other random coiled structures, depending on which medium the molecule is in, determining areas that interact with surfaces or enter the hydrophobic interior of the membrane microorganisms (Jenssen et al. 2006; Epand et al. 2010; Nguyen et al. 2010). Antimicrobial peptides are usually grouped into five main classes, according to their amino acid composition and conformation (Brogden 2005): 1. Peptides with linear amphipathic helices, such as magainin (Zasloff 1987). 2. β-sheets stabilized by disulfide bonds, such as defensin A, isolated from the mosquito larvae Phormia terraenovae (Dimarcq et al. 1998; Schmidt et al. 2011). 3. With the predominance of certain amino acid residues in their sequences, such as histatin present in human saliva, with a high content of histidine (Den Hertog et al. 2004). 4. Peptides with clip structures (loop) owing to the presence of disulfide bonds, as gomesin isolated from the hemolymph of the Brazilian spider Acanthoscurria gomesiana (Silva et al. 2000). 5. Extended as indolizidine peptides isolated from bovine neutrophils (Falla et al. 1996).

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Studies in the literature report the antimicrobial activity of anionic peptides rich in aspartate and glutamate, such as maximin H5 peptide isolated from frog Bombina maxima, which seems to be specific against Staphylococcus aureus (Lai et al. 2002). Also, another peptide rich in D and E, isolated dermcidin anionic peptide (DCD) was isolated from human sweat glands, has a wide spectrum of action (Schittek et al. 2001). However, the mechanism of action of these anionic peptides is still unclear. Another class of peptides, known as cryptides, is derived from protein hydrolysis, such as those originating from the cleavage of lactoferrin, lactoferricin (Neto 2006), casein casocidin 1 (Somkuti et al. 2010) or hemoglobin Hb 33–61 (Ivanov et al. 1997).

Possible Modes of Action of AMPs One hypothesis about the mechanism of action of AMPs involves the ability of these molecules to cause a collapse of the membrane owing to the formation of pores, by interaction with lipids of the cell surface of microorganisms, leading to bacterial cell death. As a result, the leakage of ions and metabolites, membrane depolarization, disruption of the process of respiration, and the synthesis of biopolymers could lead to cell death (Jenssen et al. 2006; Daffre et al. 2001). It is also possible that some peptides may enter the cell without forming pores, reaching the cytosolic environment and interfering in the synthesis of biomolecules or other processes that are crucial for microorganism survival or metabolism. According to pore-forming theory, cationic peptides are attracted to the exterior surface of the bacterial membrane owing to the electrostatic attraction of negatively charged molecules such as phospholipids, lipopolysaccharides (LPS) of Gram-negative, and teichoic acid of Gram-positive bacteria, which are located asymmetrically in the architecture of the cell membrane of the microorganisms. Furthermore, the positively charged amino acid residues that constitute AMPs could also interact with membrane lipids through specific receptors, activating different mechanisms that may lead to cell death (Barbosa Pelegrini et al. 2011). Intrinsic and extrinsic parameters are reported to be a threshold for determining the concentration of the antimicrobial peptide on the surface of the membrane of the microorganism to cause cell death. The intrinsic parameters, i.e., the characteristics of the peptide itself, include the ability of the peptides to oligomerize and stay bound to the membrane, while the extrinsic parameters are determined by lipid composition and membrane fluidity, together with the structure of the polar head of the lipid. These factors influence the membrane potential, which is critical in defining the peptide concentration required to cause lysis/cell death (Yeaman and Yount 2003). There are three main mechanisms of action proposed for AMPs on the membrane:

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(a) Barrel-shaped pores: After reaching a certain concentration in the medium, peptides can interact with themselves laterally, in an oligomerization state, and then with the membrane, forming a channel that passes throughout the membrane from one side to the other. Therefore, the oligomerized peptides form a ring-shaped drum, opening a pore in the membrane (Jenssen et al. 2006). (b) Toroidal pores: The toroidal pore is a transmembrane structure formed of peptides that fall into the bilayer, forming a complex of peptides and lipids alternating between their chains (Jenssen et al. 2006; Barbosa Pelegrini et al. 2011). (c) Carpet model This mechanism is characterized initially by the binding of the peptide in its monomeric or oligomeric form onto the surface of the cell membrane by electrostatic attraction. This mode of action is similar to carpet formation and, hence, the carpet causes displacement of the lipid, altering membrane fluidity, and reducing its cellular barrier properties (Yeaman and Yount 2003; Barbosa Pelegrini et al. 2011).

The cytoplasmic membranes of bacteria and multicellular organisms have different lipid compositions. The cytoplasmic membrane of the bacteria E. coli contains 70–80 % of neutral lipids whose heads consist of phosphatidylethanolamine (PE), 20–25 % with the head consisting of phosphatidylglycerol (PG), which has a negative charge, and other lipids that can be found in a small percentage (Dowhan 1997). In eukaryotic cells, the extracellular face of the cytoplasmic membrane is composed of predominantly lipids whose head contains phosphatidylcholine (PC), while the inner face also has negatively charged lipids (phosphatidylserine: PS) (Mateo et al. 2006). The difference in the net charges presented by the extracellular layers of the cytoplasmic membranes of bacteria or eukaryotic cells is regarded as the primary key to the selectivity of antimicrobial peptides (Zasloff 2002; Yeaman and Yount 2003; Mateo et al. 2006). In multicellular organisms, the hydrophobicity of the peptide is regarded as the main factor responsible for the alignment of the AMPs in the lipid bilayers (Zasloff 2002). In the case of bacteria, the peptide approach toward the bilayer applies the additional factor of the electrostatic interactions in addition to the hydrophobic interactions (Zasloff 2002). The extracellular face of the cytoplasmic membrane of many cancer cells has a slightly negative charge compared with the normal load cells, as they are enriched with phosphatidylserine lipids. This charge difference may play an important role in the action of AMPs in cancer cells (Ohsaki et al. 1992; Hoskin and Ramamoorthy 2008) and may shed a light on the reason why AMPs have been increasingly reported to be antitumoral peptides.

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AMPs from Spiders Antimicrobial peptides are produced by many species and have great importance in the defense processes (Brogden 2005). Their biotechnological relevance reaches several areas, such as the pharmaceutical and food industry. Currently, the pharmaceutical industry urgently needs to discover new classes of antibiotics, as only three have been developed over the last 40 years. In addition to interest in the ability that some peptides have to kill multiple-resistant bacteria, there is also the possibility of using them in combination with known commercial antibiotics, to obtain a synergistic effect and increased potency and selectivity (Kuhn-Nentwig et al. 2002). The interest of the food industry lies in the fact that the peptides inhibit the growth of certain bacteria, therefore allowing the production of more healthy and natural products. With the advent of techniques such as mass spectrometry and the chemical synthesis of peptides, there is an increase in the description and characterization of novel antimicrobial peptides in animal venoms, enabling the search for new modes of action and applications. There are, in general, two main groups of AMPs from spiders: the linear and the cyclic peptides. Some examples are mentioned below.

Linear Peptides Latarcins The latarcins are AMPs isolated from the venom of the spider Lachesana tarabaevi (Zodariidae) (Kozlov et al. 2006). They comprise seven new, structurally unrelated groups of membrane-active molecules (Ltc 1, Ltc 2a/2b, Ltc 3a/3b, Ltc 4a/4b, Ltc 5, Ltc 6b/6c, and Ltc 7). These peptides show lytic effects on different organisms, such as Gram-positive and Gram-negative bacteria, rabbit erythrocytes, and yeasts (Kuhn-Nentwig et al. 2011). Latarcins show low levels of similarity with other AMPs from spiders; they have positively charged and hydrophobic residue repetitions, exhibiting an amphipathic structure. The distribution of lysine residues observed in latarcin sequences is similar to that found in lycocitins, lycotoxins, and oxyopinins (Table 2) (KuhnNentwig et al. 1994, 2011). In addition to antimicrobial activity, latarcins caused larval paralysis, and it is possible that this may enhance the overall paralyzing and toxic effect of the crude venom (Kuhn-Nentwig 2003). The NMR analysis of Ltc 2a forms an alpha-helical structure when inserted in micelles or liposomes (Fig. 1a) (Kuhn-Nentwig 2003). The Ltc 2a shows a hairpinlike structure in micelles that consist of two helical regions connected with a non-ordered region. This hinge between the helical regions is essential for the antimicrobial activity (Idiong et al. 2011) of the peptide. Ltc 2a has been suggested to exert its action via the carpet-like mechanism (Kozlov et al. 2006).

Peptide Sequence Oxyopinin1 -FRGLAKLLKIGLKSFARVLKKVLPKAAKAGKALAKSMADENAIRQQNQ——————————————————————————————————————— Oxt4a —GIRCPKSWKCKAFKQRVLKRLLAMLRQHAF————————————————————————————————————————————————————————— Ltc1 –SMWSGMWRRKLKKLRNALKKKLKGE————————————————————————————————————————————————————————————— Ltc3a ——SWKSMAKKLKEYMEKLKQRA—————————————————————————————————————————————————————————————————— Ltc3b ——SWASMAKKLKEYMEKLKQRA————————————————————————————————————————————————————————————————— Ltc4a GLKDKFKSMGEKLKQYIQTWKAKF———————————————————————————————————————————————————————————————— Ltc4b SLKDKVKSMGEKLKQY-QTWKAKF——————————————————————————————————————————————————————————————— Lycocitin1 —GKLQAFLAKMKEIAAQTL———————————————————————————————————————————————————————————————————— Lycocitin2 —GRLQAFLAKMKEIAAQTL————————————————————————————————————————————————————————————————————— Oxyopinin2a —GKFSVFGKILRSIAKVFKGVGKVRKQFKTASDLDKNQ————————————————————————————————————————————————— Oxyopinin2d —GKFSVFSKILRSIAKVFKGVGKVRKQFKTASDLDKNQ—————————————————————————————————————————————————— Oxyopinin2b —GKFSGFAKILKSIAKFFKGVGKVRKQFKEASDLDKNQ—————————————————————————————————————————————————— Oxyopinin2c —GKLSGISKVLRAIAKFFKGVGKARKQFKEASDLDKNQ—————————————————————————————————————————————————— CIT1a –GFFGNTWKKIKGKADKIMLKKAVKIMVKKEGISKEEAQAKVDAMSKKQIRLYLLKYYGKKALQKASEKL CIT1e –GFFGNTWKKIKGKSDKIMLKKAVKIMVKKEGISKEEAQAKVDAMSKKQIRLYLLKYYGKKALQKASEKL CIT1b –GFFGNTWKKIKGKADKIMLKKAVKLMVKKEGISKEEAQAKVDAMSKKQIRLYLLKYYGKKALQKASEKL CIT1d –GFFGNTWKKIKGKADKIMLKKAVKIMVKKEGITKEEAQAKVDAMSKKQIRLYLLKYYGKKALQKASEKL CIT1f –GFFGNTWKKIKGKADKIMLKKAVKIMVKKEGISKEEAQAKVDAMSKKQIRLYLLKHYGKKALQKASEKL CIT1c –GFFGNTWKKIKGKADKIMLKKAVKIMVKKEGISKEEAQAKVDAMSKKQIRLYVLKYYGKKALQKASEKL CIT1g –GFFGNTWKKIKGKADKIMLKKAVKIMVKKEGITKEEAQAKVDAMSKKQIRLYVLKHYGKKALQKASEKL CIT1H –GFFGNAWKKIKGKAEKFFRKKAAKIIAKKEGITKEEAEAKVDTMSKKQIKVYLLKHYGKKALQKASEKL Ltc2a –GLFGKLIKKFGRKAISYAVKKARGKH————————————————————————————————————————————————————————————— LycotoxinI —IWLTALKFLGKHAAKHLAKQQLSKL—————————————————————————————————————————————————————————————— LyeTxI —IWLTALKFLGKNLGKHLAKQQLAKL—————————————————————————————————————————————————————————————— Cupiennin1a —GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME———————————————————————————————————————————————————— Lycocitin3 -KIKWFKTMKSLAKFLAKEQMKKHLGE—————————————————————————————————————————————————————————————— LycotoxinII -KIKWFKTMKSIAKFIAKEQMKKHLGGE———————————————————————————————————————————————————————————— Ltc5 —GFFGKMKEYFKKFGASFKRRFANLKKRL———————————————————————————————————————————————————————————— Bold: positively charged residues.

Table 2 Amino acid sequence of aliphatic antimicrobial peptides (AMPs) derived from spider venoms Structure α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix α- Helix

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Lycotoxins Lycotoxins I and II, from the venom of the spider Hogna carolinensis, were the first toxins isolated from spider venom and were characterized as cationic peptides with a hydrophobic moiety capable of forming alpha-helix in solution (Yan and Adams 1998). Lycotoxins I and II were active against Gram-positive and Gram-negative bacteria and fungi (Candida glabrata and Candida albicans). They were able to cause hemolysis of rabbit erythrocytes and dissipate voltage gradients across muscle membrane. Based on the observed activities mentioned above, it was suggested by the authors that lycotoxins might play a dual role in spider–prey interaction, acting both in the prey capture strategy as well as to protect the spider from potentially infectious organisms arising from prey ingestion (Yan and Adams 1998). From the venom of the spider Lycosa singorienis three new peptides (lycocitin 1, 2, and 3) were characterized later. Lycocitin 3 shows high homology with lycotoxin 2. Both lycocitin 1 and 2 inhibit the growth of Gram-positive and Gram-negative bacteria and fungi (Candida albicans) (Budnik et al. 2004). LyeTx I was purified from the venom of the Brazilian spider Lycosa erythrognatha (Santos et al. 2010). It shows high similarity to lycotoxin I, being active against Gram-positive and Gram-negative and fungi (Candida krusei and Cryptococcus neoformans). Also, it was able to cause hemolysis in rabbit cells and did not show any toxicity against insects (Santos et al. 2009). The secondary structure of LyeTx I was determined by NMR. The C-terminus region of the peptide reveals an amphipathic alpha helix that may support the peptide–membrane interaction and the N-terminus region of LyeTx I can be essential for anchoring the peptide in the phospholipid bilayer (Fig. 1a; Santos et al. 2010). Cupiennins Cupiennins are toxins from the venom of the spider Cupiennius salei, which is able to destroy a great variety of cell types, including bacteria and eukaryotic cells such as myoblasts, and various blood and cancer cells (Kuhn-Nentwig et al. 2002; Pukala et al. 2007). The most frequently investigated cytolytic peptide, cupiennin 1a, is characterized by a helix–hinge–helix structure (Pukala et al. 2007), comparable with the structure of latarcin 2a (Kozlov et al. 2006). Cupiennin 1a assumes alpha-helical conformation in the presence of negatively charged membranes (Fig. 1c). Analysis of cupiennin 1a (Cu1a) in different phospholipid bilayers by solid-state nuclear magnetic resonance (NMR) suggests that the formation of the toroidal pore is the main mechanism of the antimicrobial effect (Pukala et al. 2007). Cu1a exhibits a nonlytic activity at low concentrations (within a submicromolar range); the peptide can combine with complexation with the regulatory protein Ca2+ calmodulin and inhibits the formation of nitric oxide by neuronal nitric oxide synthase (Pukala et al. 2007). This inhibition, together with the formation of pores, can play an important role in the interruption of the vital functions of the prey.

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Fig. 1 NMR structure of antimicrobial peptides. (a) Latarcin 1a (PDB. 2G9P). (b) LyeTx I . (c) Cupiennin 1a (2 K38). (d) Oxyopinin 4a (PDB: 2L3I)

A remarkable aspect of L-Cu1a is its strong synergistic effect on the neurotoxic activity of different neurotoxins. Most likely, L-Cu1a favors the presentation of the neurotoxins to their targets (Wullschleger et al. 2005).

Oxyopinin 1 Five antimicrobial peptides were purified and characterized from the venom of the spider Oxyopes kitabensis (Corzo et al. 2002). Oxyopinin 1 has 48 amino acids and shows similarity to frog peptide dermaseptin and the ant peptide ponericin L2. The Oxyopinins 2a, 2b, 2c, and 2d have 37 amino acids and show high similarity to other oxyopinins 1 and 2 show an alpha-helical structure, activity against E. coli and S. aureus, hemolytic activity, and has a synergic effect with the paralytic neurotoxin oxytoxin against insect larvae (Belokoneva et al. 2004; Nomura and Corzo 2006).

Cyclic Peptides GsMTx-4 A peptide named GsMTx4 was purified from the venom of the tarantula Grammostola spatulata (Jung et al. 2006). Structurally, this peptide is a member of the inhibitory cysteine knot peptide superfamily (Zhu et al. 2003). Three specific disulfide bonds in a 34-amino acid peptide forms a compact structure with two antiparallel beta-strands. GsMTx4 possess an amphipathic structure, having a hydrophobic core and a hydrophilic face that, combined with a net charge of +5, suggests that they might play a role in lipid binding (Suchyna et al. 2004). Antimicrobial assays indicate that GsMTx-4 is more active against Gram-positive than against Gram-negative bacteria.

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In addition to the lytic activity directly in the phospholipid bilayer, the peptide can also affect the activity of mechanosensitive channels in bacteria, but the action mechanism is not yet fully understood (Jung et al. 2006; Hurst et al. 2009).

Oh-Defensin The peptid Oh-defensin was isolated and characterized from the venom of the spider Ornithoctonus hainana, with activity against Gram-positive and Gramnegative bacteria, fungi (Candida albicans), and poor hemolytic activity (Zhao et al. 2011). This peptide chain comprises 52 amino acids, including six cysteines, indicating the formation of three disulfide bonds. The Oh-defensin shows similarity to other arthropod antimicrobial peptides, especially those from wasps and scorpions (defensins). Oxyopinin 4a The oxyopinin 4a is a peptide with 30 amino acids from the venom of the spider Oxyopes takobius. In contrast to other spider AMPs, the oxyopinin 4a has a single disulfide bond (C4–C10), N-terminally located, showing similarity to Rana-box peptides from frogs. NMR analysis indicates that the peptide has a random structure in water, but shows a torpedo-like structure in micelles with notable amphipathic features (Fig. 1d). It shows activity against Gram-positive and Gram-negative bacteria and cytolytic activity against human erythrocytes (Dubovskii et al. 2011). Juruin The peptide juruin was purified from the theraposid spider Avicularia juruensis, with a putative inhibitory cystine knot (ICK) motif. It has 38 amino acids, positive net charge, and shows high similarity to insecticidal toxins from other Chinese theraposid spiders. It shows a potent antifungal activity (MIC spanning from 2.5 to 5 μM for Candida albicans, Candida Krusei, Candida glabrata, Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida guilliermondii), lacking hemolytic activity on human erythrocytes at antimicrobial concentrations (Ayroza et al. 2012; Table 3).

Role of Antimicrobial Peptides in Spider Venoms The exact function of the linear cytolytic peptides found in the venom of spiders is still unclear. It is possible that the antimicrobial activity observed in some of these peptides could be a secondary characteristic (Vassilevski et al. 2008). There are some suggestions regarding the exact function of these peptides, such as the direct toxic effect on prey. This function was proposed for lycotoxins from the venom of the spider Lycosa carolinensis (Lycosidae) (Yan and Adams 1998), for cupiennins from Cupiennius salei (Ctenidae) (Kuhn-Nentwig et al. 2002), and oxyopinins from Oxyopes kitabensis (Oxyopidae) (Corzo et al. 2002). These peptides can also act as a spreading agent, facilitating the passage of neurotoxins through cellular barriers, ensuring their access to target neurons. This cooperation with neurotoxins has also

DCIPTRHECTNNQQN–CCEGHDCKCDYTEIGGAKKE—ICYCKKTLWQKTKDKLSTAGDILKS————

ECIPLYNDCTAFKYNNNCCKDPEKKYQYKCSCIVCKEGKEQCTCQRKETVESMMKCVRFVKKVGEKVIEKV

ECIPLYNDCKEFKYNNNCCKDPEKKYQYKCSCIMCEGGEEQCTCQRKETVENMMKCVRFVKKVVEKV——

ECVPLENDCTKLKYSNPCCKDEKKKYQYKCSCIVDKT–EQCTCQRKETVEKMMKGMKYIKNLGKKI——

RCLPAGKTCVRGPMRVPCCGSCSQNKCT————————————— -MLCKLSMFGAVLGVPACAIDCLPMGKTGGSCEGG———VCGCRKLTFKILWDKKFG

ACGILHDNCVYVPAQNPCCRGLQCRYG—————————————KCLVQVX————————— –FTCAISCDIKVNGKPCKGSGEKKCSG——————————GWSCKFNVCVKV

GCLEFWWKCN–PNDDKCCRPKLKCSKLFK——————————LCNFSF——————————

LtTx-1b

LtTx-2a

LtTx-2b

LtTx-2C

PCFK2 OHdefensin PCFK1 Juruin

GsMTx4

Bold: positively charged residues; Italicized: cysteine residues.

Sequence ECIPTKHDCTNDRKN–CCPGHECKCYNTQIGGSKKE—QCGCKKSLLQKAKNFGGKVITIFKA————

Peptide LtTx-1a

Table 3 Amino acid sequence of cyclic antimicrobial peptides (AMPs) derived from spider venoms Structure α- Helix, β;-Sheet, Turns, SS-bond α- Helix, β;-Sheet, Turns, SS-bond α- Helix, β;-Sheet, Turns, SS-bond α- Helix, β;-Sheet, Turns, SS-bond α- Helix, β;-Sheet, Turns, SS-bond β;-Sheet, Turns, SS-bond α- Helix, β;-Sheet, Turns, SS-bond β;-Sheet, Turns, SS-bond α- Helix, β;-Sheet, Turns, SS-bond α- Helix, β;-Sheet, Turns, SS-bond

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been attributed to cupiennins (Kuhn-Nentwig et al. 2002) and oxyopinins (Corzo et al. 2002). A direct antiseptic role has also been suggested for lycotoxins (Yan and Adams 1998), cupiennins (Kuhn-Nentwig et al. 2002), and latarcins (Kozlov et al. 2006), which disinfect and conserve the paralyzed prey. An additional function of protecting the gland has also been proposed (Kuhn-Nentwig 2003).

Potential Commercial Use of AMPs At first glance, these peptides could be used as alternative therapeutics to organic nonpeptide natural antimicrobials, as they exhibit a broad spectrum of action, have a smaller capacity to induce antibiotic resistance compared with non-peptide antibiotics, and cause the rapid death of microorganisms (Hancock 1997; Hancock and Sahl 2006; Miranda et al. 2009). These peptides may be used alone (monotherapy) or in combination with other antibiotics, providing that there are possible synergistic effects between peptides and other compounds (Gordon et al. 2005). In the last two decades, several peptides with antibiotic action or their derivatives have been widely studied up to clinical testing (relatively few compared with the large number of peptides already discovered) (Fox 2013; Miranda et al. 2009; Pathan et al. 2010). However, until now, few have proven sufficiently effective (Hancock and Sahl 2006): 1. Pexiganan (Genaera, Plymouth Meeting, PA, USA), which is a derivative of magainin 2 (peptide isolated from the skin of Xenopus laevis African frog); currently pexiganan is on phase 3 of clinical trials for the treatment of mild infections of diabetic foot ulcers (ClinicalTrials.gov identifier NCT01594762); 2. Omiganan (Microbiologix Biotech, Vancouver, BC, Canada), which is a derivative of indolicidin (the peptide obtained from bovine neutrophil granules) able to reduce the colonization of microorganisms on catheters (Gordon et al. 2005). Omiganan is in phase 2 of clinical trials (ClinicalTrials.gov identifier: NCT00608959). 3. OP-145, a synthetic peptide derived from LL-37, is in phase 2 of clinical trials for the treatment of chronic bacterial middle-ear infection (Fox 2013). The vast majority of previous studies involve monotherapy and are aimed at topical application (Hancock and Sahl 2006). A limiting factor for systemic administration is the low efficacy at lower doses. In animal studies, certain peptides, such as magainin, were effective only at high doses and usually near-toxic doses (Zasloff 2002). The most frequent use of these compounds has been in the academic field, as knowledge of the chemical, biological, and structural properties of a peptide’s antibiotic action is extremely valuable. Furthermore, the exploitation of structure–activity relationships and understanding of the action mechanisms may lead to novel compounds and analogs with more interesting properties compared with the starting compounds (Kim et al. 2002), and

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to the discovery of other actions, such as the inhibition of enzymes, the promotion of dermal absorption for topical drug delivery, the immobilization of cells with different affinities on solid surfaces, or even incorporation into food packaging (Gregory and Mello 2005; Miltz et al. 2006; Kim et al. 2007).

Conclusion and Future Directions Today, there more than 2,450 natural antimicrobial peptides have been described (Collection of Anti-Microbial Peptides 2015). Nevertheless, there is still great difficulty in turning these AMPs in commercial products. AMPs from spiders and other arthropod venoms can be regarded as new interesting leads, as they show interesting features in terms of structure and activities.

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