Antimicrobial peptides (AMPs): Ancient compounds that represent novel weapons in the fight against bacteria

Biochemical Pharmacology 133 (2017) 117–138

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Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Review

Antimicrobial peptides (AMPs): Ancient compounds that represent novel weapons in the fight against bacteria J.M. Ageitos a,⇑, A. Sánchez-Pérez b, P. Calo-Mata c, T.G. Villa a a

Department of Microbiology, Faculty of Pharmacy, University of Santiago de Compostela, Spain Faculty of Veterinary Science and Bosch Institute, University of Sydney, NSW 2006, Australia c Department of Food Science and Technology, Faculty of Veterinary, University of Santiago de Compostela, Spain b

a r t i c l e

i n f o

Article history: Received 11 July 2016 Accepted 19 September 2016 Available online 20 September 2016 Keywords: Antimicrobial peptides Defensins Cathelicidins Gram positive Gram negative

a b s t r a c t Antimicrobial peptides (AMPs) are short peptidic molecules produced by most living creatures. They help unicellular organisms to successfully compete for nutrients with other organisms sharing their biological niche, while AMPs form part of the immune system of multicellular creatures. Thus, these molecules represent biological weapons that have evolved over millions of years as a result of an escalating arms race for survival among living organisms. All AMPs share common features, such as a small size, with cationic and hydrophobic sequences within a linear or cyclic structure. AMPs can inhibit or kill bacteria at micromolar concentrations, often by non-specific mechanisms; hence the appearance of resistance to these antimicrobials is rare. Moreover, AMPs can kill antibiotic-resistant bacteria, including insidious microbes such as Acinetobacter baumannii and the methicillin-resistant Staphylococcus aureus. This review gives a detailed insight into a selection of the most prominent and interesting AMPs with antibacterial activity. In the near future AMPs, due to their properties and despite their ancient origin, should represent a novel alternative to antibiotics in the struggle to control pathogenic microorganisms and maintain the current human life expectancy. Ó 2016 Elsevier Inc. All rights reserved.

Contents 1. 2. 3.

4. 5. 6.

7. 8.

9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AMPs in fungi and plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Fungal AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. AMPs produced by plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AMPs produced by Porifera, Cnidarian and Mollusca . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AMPs produced by Arthropoda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fish AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Fish cathelicidins and b-Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Fish hepcidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Fish piscidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AMPs produced by amphibians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AMPs produced by reptiles and birds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Reptilian and avian cathelicidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Reptilian and avian b-Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AMPs produced by mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Mammalian cathelicidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Mammalian defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1. Mammalian a-Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2. Mammalian b-Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding author. E-mail address: [email protected] (J.M. Ageitos). http://dx.doi.org/10.1016/j.bcp.2016.09.018 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.

118 119 122 122 124 124 125 125 125 126 126 126 128 128 128 129 129 129 129 130

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9.2.3. h-Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. AMPs from platelets, liver and eccrine sweat glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Synthetic AMPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1. Synthetic hybrid AMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2. Synthetic analogs of natural AMPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3. Synthetic AMPs not based in natural sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Determination of the inhibitory and bactericidal activity of AMPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Antimicrobial peptides (AMPs, also known as host defense peptides or HDPs) are small polycationic peptides [7–100 amino acids (aa)] that form part of the innate immune response shared by all classes of life. For instance, the granules of polymorphonuclear neutrophils (PMNs) and the natural killer cells produce a variety of AMPs with activity against viruses, fungi, bacteria, protozoa, and even transformed or cancer cells. These compounds are currently being tested as alternatives to classic antibiotic therapies or, at least, as complementary to antibiotics to treat infectious diseases. There are currently more than 3000 experimentally reported AMPs, including both synthetically synthesized and compounds produced by living organisms. These vast numbers of compounds are listed in specialized AMP databases such as APD3 [1], CAMPR3 [2] and LAMP [3]. The importance of AMP research is emphasized by more than 330,000 articles published in this field, with an average of 12,000 articles per year in the last five years. As mentioned above, all AMPs, regardless of their biological origin, share common features; these include a small size and either a

130 130 131 131 131 132 132 132 132

linear or cyclic structure. The linear structure encompasses amphipathic a-helices, while the cyclic structure contains one or more disulfide bridges forming a b-sheet [4]. The classical action mechanism of AMPs involves cell membrane damage [5] (Fig. 1). Although cationic AMPs can electrostatically interact with the surface of the negatively charged cell membrane [6], some AMPs disrupt cell membranes by specific interaction with membrane compounds, while other compounds target intracellular molecules (Fig. 1). AMPs usually exhibit broad-spectrum activity against parasitic microorganisms and, since these compounds do not interact with specific receptors, their microbial targets rarely develop resistant phenotypes. However, resistance to some AMPs has been documented. Two such examples are: a) the resistance to dermcidin developed by Staphylococcus aureus, by induction of a specific protease, and b) the decreased sensitivity to cationic AMPs exhibited by some Gram
bacteria, achieved by modifying their lipopolysaccharide through a specific sensor (PhoP/PhoQ) system [7]. These mechanisms, however, only result in a moderate effect on the minimal inhibitory concentration (MIC) required for the AMPs to be effective [6].

Fig. 1. Mechanisms of action of antimicrobial peptides. This figure was compiled from a variety of sources [24,61,166,197,200,289].

J.M. Ageitos et al. / Biochemical Pharmacology 133 (2017) 117–138

119

In addition to antimicrobial abilities, some AMPs display unexpected actions, due to their putative role as regulators of specific cellular functions. The cathelicidins are a good example of AMPs with additional activities. These compounds are enzymatically processed, through the action of proteinase 3, to produce cathelin and the mature LL-37 peptide. Although both molecules are bioactive, cathelin also acts as a proteinase inhibitor, thus participating in a variety of biochemical processes [13]. Another well documented AMP is the liver-produced hepcidin whose primary role appears to be hormonal, serving as a negative regulator of iron homeostasis by binding to ferroportin molecules on the surface of enterocytes and macrophages [14]. The regulatory role of AMPs could depend, at least in some cases, on additional internal regulators that are specific for each bacterial group. In bacteria such as Listeria monocytogenes and S. aureus, the AMPs could be active in one bacterial group but not in the other [15], thus apparently contradicting the tennet of AMPs having an antimicrobial action with a ‘‘broad biological activity”. The so called ‘‘Superbugs”, or microorganisms displaying multi-resistance against antibiotics, have unfortunately become common in healthcare settings (including the lethal methicillin-resistant S. aureus (MRSA), the vancomycin-resistant enterococci (VRE), the multidrug-resistant Pseudomonas aeruginosa, and the all-resistant Mycobacterium tuberculosis) that they produce thousands of global annual deaths. Based on the intensive research and the results obtained so far, AMPs will represent a novel alternative to antibiotics in the control of human pathogens in the near future. 2. Bacterial AMPs

Fig. 2. Secondary structure of Gramicin D, Micasin, a-1-Plurothionin, Aurelin, Tachyplesin I, and Thanatin, antimicrobial peptides produced by bacteria, fungi, plants, and invertebrates. The molecular models were obtained from the Protein Data Bank (PDB; http://www.rcsb.org/pdb) and the molecular graphics were performed with the UCSF Chimera package (http://www.cgl.ucsf.edu/chimera). Hydrophobic residues are depicted in yellow and hydrophilic amino aids in blue.

The sensitivity of cells to AMPs is not only directly related to the physicochemical properties of the lipids found on the target cell membranes, but also depends on the composition and secondary structure of the AMPs. Unfortunately, due to our inability to predict protein structure based on the primary amino acid sequence, we have yet to develop a practical application for AMPs in general therapies, which limits a rational pharmacological approach to dose-response assessment. Current and future advances in this field involve computer-assisted design of antimicrobial peptides and, although this represents a gargantuan task, given the number of amino acids that exist in nature and their possible combinations (ca. 5  10111 according to Cherkasov and co-workers [8]), the dream of designing and using antimicrobial peptides against antibiotic-resistant bacterial pathogens should soon become a reality. We are currently aware that AMPs containing D-amino acids are less sensitive to protease activity [9], meaning that Damino acids reduce microbial resistance and increase the biological effect of AMPs [10]. For instance, Tyrothricin was the first ever clinically used AMP (clinical trials carried out in 1939 [11]) and this compound spans a mixture of linear and cyclic D-amino acids. It is very encouraging that, after continual clinical use for over 60 years, no microbial resistance to tyrothricin has yet been described [12].

Tyrothricin, the first peptidic antibiotic clinically used in humans (1939 [11]) is a mixture of cyclic (Tyrocidins 70–80%) and linear (Gramicidin D 20–25%) AMPs produced by Bacillus brevis. Gramicidin D (Fig. 2), the first AMP isolated [16], is a mixture of 3 compounds, gramicidin isoforms A, B, and C (Table 1), present in a 7:1:2 ratio, respectively [17]. Gramicidins are synthesized nonribosomally by a multienzyme complex [18]. These AMPs contain acids, which allow the peptides to adopt a b-helical structure and interact with bacterial membranes. Gramicidin kills bacteria by increasing the permeability of their cell membranes to monovalent cations, thus destroying the intracellular ion gradient [17]. These AMPs are active against Gram+ bacteria (with the exception of Bacillus) and some Gram
organisms (such as Neisseria sp.) [19]. Gramicidin S (Table 1) is a cyclic biosynthetic derivative of Gramicidin, with a cyclic b-hairpin [20], that contains ornithine (Orn) [cyclo(VOrnLDFP)2]. This AMP is active (MIC 3– 11 lM) against both Gram+ and Gram
bacteria [21], although its high hemolytic activity limits its clinical applications [22]. Gramicidin S not only disrupts the cellular membrane, but it is also a potent inhibitor of the bacterial cytochrome bd-type quinol oxidase [22]. Tyrocidines (Table 1) constitute a cyclic AMP family of compounds, produced by B. brevis, which are the major constituent of Tyrothricin [12]. Tyrocidines include more than 28 compounds [23], they have a conserved sequence cyclo(VOrnLDFPX1DX2NQYVX3L) and are related to Gramicidin S. The more clinically relevant tyrocidines contain Trp1,2/Phe1,2 or Lys3/Orn3 in the X position above. Apart from Tyrocidines A, B and C, there are additional analogs such as tryptocidine and phenycidine. These AMPs act on the cellular membrane, but they also have intercellular targets, indeed, Tyrocidines can inactivate the bacterial enzyme glucose dehydrogenase [11]. Tyrocidines are active against both Gram+ and Gram
bacteria [24,25]. Gageotetrins (A–C) are non-ribosomally synthesized lipoAMPs produced by a marine Bacillus subtilis isolate. The C-terminal lipid present in Gageotetrin A is 3-hydroxy-8,10-dime thyldodecanoic acid, while both Gageotetrin B and C contain a D-amino

120

Table 1 Antimicrobial peptides (AMP) produced by bacteria1, fungi2 and plants3. Organism

B. brevis1

B. subtilis

1

L. lactis1 E. coli

1

P. polymyxa1 A. otae2 C. cinerea2

P. nigrella2 R. pusillus2 T. longibrachiatum2 T. saturnisporum2 T. viride2 A. hippocastanum3 C. parviflora3 C. ternatea3 D. merckii3 O. affinis3 P. longipes3 S. tuberosum3 T. aestivum3

1

Gramicidin A Gramicidin B Gramicidin C Gramicidin S Tyrocidines Bacitracin A Gageotetrin A Gageotetrin B Gageotetrin C Nisin A Microcin J25 Microcin C7 Microcin B17 Polymyxin B Polymyxin E Micasin Copsin

Plectasin Sillucin Trichogin GA IV Tricholongin BI Saturnisporin SA II Alamethicin Ah-AMP1 Circulin A Circulin B Ct-AMP1 Dm-AMP1 Kalata B1 Cyclopsychotride A Snakin-1 a1Purothionin Puroindoline A

Sequence

CHOVGADLADVVDVWDLWDLWLW NHCH2CH2OH CHOVGADLADVVDVWDLFLDWLW NHCH2CH2OH CHOVGADLADVVDVWDLYLDWLW NHCH2CH2OH cyclo(VOrnLDFP)2 cyclo(VOrnLDFPX1DX2NQYVX3L); X (W1,2/F1,2; K3/Orn3) 1-(N-((2-(1-amino-2-methylbutyl)-4,5-dihydro -4-thiazolyl)carbonyl)LDEI-cyclo(KDOrnIDFHDDN) LE-C14H27O3 LLLE-C14H27O3 LLLE-C14H27O3 IDhbcyclo(AIDhaLA)cyclo(AbuPGA)Kcyclo(AbuGAL MGA)NMKcyclo(AbuAAbuAHA)SIHVDhaK VGIGTPIFSYGGGAGHVPEYF MRTGNAD-1-[(3-aminopropyl)(50 -adenosyl)phosphono]amide VGIGGGGGGGGGGSCGGQGGGCGGCSNGCSGGNGGSGGSGSHI (S)-6-Methyloctanoyl-DabTDab-cyclo(DabDabDFLDabDabT) (S)-6-Methyloctanoyl-BTB-cyclo(BBDLLBBT) APATNNAAVDAAADATPAVEKRGFGCPFNENECHAHCLSIGRKFGFCAGPLRATCTCGKQ ATTVPGCFAECIDKAAVAVNCAAGDIDCLQASSQFATIVSECVATSD CTALSPGSASDADSINKTFNILSGLGFIDEADAFSAADVPEERDLTGLGRVLPVEK RQNCPTRRGLCVTSGLTACRNHCRSCHRGDVGCVRCSNAQCTGFLGTTCTCINPCPRC GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY ACLPNSCVSKGCCCGBSGYWCRQCGIKYTC nOct-AiBGLAiBGGLAiBGI-Lol AcAibGFAibAibQAibAibAibSLAibPVAibAibQQLol AcAibAAibAAibAQAibLGAibPVAibIvaQQPhl AcAibPAibAAAQAibVAibGLAibPVAibAibEQPhl LCNERPSQTWSGNCGNTAHCDKQCQDWEKASHGACHKRENHWKCFCYFNC GIPCGESCVWIPCISAALGCSCKNKVCYRN VIPCGESCVFIPCISTLLGCSCKNKVCYRN NLCERASLTWTGNCGNTGHCDTQCRNWESAKHGACHKRGNWKCFCYFDC ELCEKASKTWSGNCGNTGHCDNQCKSWEGAAHGACHVRNGKHMCFCYFNC GLPVCGETCVGGTCNTPGCTCSWPVCTRN SIPCGESCVFIPCTVTALLGCSCKSKVCYKN GSNFCDSKCKLRCSKAGLADRCLKYCGICCEECKCVPSGTYGNKHECPCYRDKKNSKGKSKCP KSCCRSTLGRNCYNLCRARGAQKLCAGVCRCKISSGLSCPKGFPK DVAGGGGAQQCPVETKLNSCRNYLLDRCSTMKDFPVTWRWWKWWKGGCQELL GECCSRLGQMPPQCRCNIIQGSIQGDLGGIFGFQRDRASKVIQEAKNLPPRCNQGPPCNIPGTIGY

Activity

Ref.

G+

G


+ + + + + +

+ + + + +

+ + + +

+ + + +

+ +

+ + + + + + + + + + + + + + + +

+ + + + + +

[17]

[20] [11] [34] [26]

[37] [43] [45] [48] [27] [29] [56] [59]

[58] [55] [52] [53] [54] [51] [66] [72] +

+ + + +

[66] [66] [72] [72] [67] [60] [63]

Bacteria; 2fungi; 3Plants; G+: Gram positive bacteria; G
: Gram negative bacteria. Orn: ornithine; Dhb: didehydroaminobutyric acid; Dha: didehydroalanine; Abu: animobutidic acid; Dab: diaminobutyric acid. Ac: Acetylated. AiB:

a-aminoisobutyric acid. Phl: phenylalaninol. nOct: n-octanoyl; Lol: Leucinol. Iva: Isovaline.

J.M. Ageitos et al. / Biochemical Pharmacology 133 (2017) 117–138

B. subtilis1

AMP name

Table 2 Antimicrobial peptides (AMP) produced by Porifera1, Cnidaria2, Mollusca3, Arthropoda4 and Fish5. Organism

AMP name

D. kiiensis1

T. tridentatus3 L. polyphemus3 A. mellifera4

C. capitata4

D. melanogaster4 H. cecropia4 P. maculiventri4 P. apterus4 C. semilaevis5 G. morhua5 M. glutinosa5 M. saxatilis5 x M. chrysops5 O. melastigmus5 O. mykiss5

P. americanus5 2

3

4

CHODAFDPDtLtLDWRDCys(O3H)TNMeGlnDLNTSar DAb-BrPhePtLbMeIleDWRDCTcyclo(NMeGlnDVPD)

KRRACADLRGKTFCRLFKSYCDKKGIRGRÑMRDKCSYSCGCRNH2 AACSDRAHGHICESFKSFCKDSGRNGVKLRANCKKTCGLC GFGCPNDYPCHRHCKSIPGRXGGYCGGXHRLRCTCYR GCASRCKAKCAGRRCKGWASASFRGRCYCKCFRC SCASRCKGHCRARRCGYYVSVLYRGRCYCKCLRC TCGSLCKAHCTFRKCGYFMSVLYHGRCYCRCLLC HPHVCTSYYCSKFCGTAGCTRYGCRNLHRGKLCFCLHCSR KWCFRVCYRGICYRRCRNH2 RRWCFRVCYRGFCYRKCRNH2 RRWCFRVCYKGFCYRKCRNH2 YVPLPNVPQPGRRPFPTFPGQGPFNPKIKWPQGY GNNRPVYIPQPRPPHPRI GNNRPVYIPQPRPPHPRL GNNRPIYIPQPRPPHPRL QERGSIVIQGTKEGKSRPSLDIDYKQRVYDKNGMTGDAYGGLNIRPGQPSRQHAGFEFGKEYKNGFIKGQSEVQRGPGGRLSPYFGINGGFRF GIGAVLKVLTTGLPALISWIKRKRQQ SIGSALKKALPVAKKIGKIALPIAKAALP SIGSAFKKALPVAKKIGKAALPIAKAALP SLGGVISGAKKVAKVAIPIGKAVLPVVAKLVG SIGTAVKKAVPIAKKVGKVAIPIAKAVLSVVGQLVG GKPRPYSPRPT(O)SHPRPIRV KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAKNH2 GSKKPVPIIYCNRRTGKCQRM VDKGSYLPRPT(O)PPRPIYNRN LPLDQVQETEGVGMVRGAGMSDTPAAANEETSVDQWITPYHARVKR SRSGRGSGKGGRGGSRGSSGSRGSKGPSGSRGSSGSRGSKGSRGGRSGRGSTIAGNGNRNNGGTRTA WSCPTLSGVCRKVCLPTEMFFGPLGCGKEFQCCVSHFF GFFKKABrTrpRKVKHAGRRVLDTAKGVGRHYVNNBrTrpLNRYR FFHHIFRGIVHVGKTIHRLVTG FIHHIFRGIVHAGRSIGRFLTG MGSSHHHHHHSSGLVPRGSHMIPVNGVTELEEAASNDTPVAARHEMSMQSWMMPNHIREKRQSHLSMCSVCCNCCK-NYKGCGFCCRF RRSKVRICSRGKNCVSRPGVGSIIGRPGGGSLIGRP RICSRGKNCVSRPGVGSIIGRPGGGSLIGRPGGGSV RRGKDSGGPKMGRKNSKGGWRGRPGSGSRPGFGSGI GWGSFFKKAAHVGKHVGKAALTHYL

Activity

Ref.

G+

G


+ + + + + + +

+

+ + + +

+ + + + + + + + +

+ + + + +

+

+

[75] [78] [81] [79] [83]

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

[86] [84] [99] [96]

[100] [95] [1101]

[103] [89] [93] [102] [113] [120] [125] [119] [129]

J.M. Ageitos et al. / Biochemical Pharmacology 133 (2017) 117–138

Discodermin A Polydiscamide-A Damicornin Aurelin Defensin A Mytilin-A Mytilin-B Mytilin-G1 Myticin-B Tachyplesin I Polyphemusin I Polyphemusin II Abaecin Apidaecin IA Apidaecin IB Apidaecin II Hymenoptaecin Melittin Ceratotoxin A Ceratotoxin B Ceratotoxin C Ceratotoxin D Drosocin Cecropin A Thanatin Pyrrhocoricin CsHEP codCath Cod b-defensin HFIAP-1 Piscidin 1 Piscidin 3 Pro-Omhep1 rtCATH_1(R146-P181) rtCATH_1(R151-V186) rtCATH_2(R143-I178) Pleurocidin

P. damicornis1 A. aurita2 Mytilus sp.3

1

Sequence

[126] [122] [123] [122] [127]

5

Porifera; Cnidaria; Mollusca; Arthropoda; Pisces; G+: Gram+ bacteria; G
: Gram
bacteria. tL: tert-leucine; Cys(O3H): cystenoic acid; Sar: sarcosine. b-BrPhe: b-hydroxy-pbromophenylalanine; bMeIle: b-methylisoleucine; NMeGln: N-methyl glutamine. NH2: Amidated C-terminus. (O): O-glycosylated; BrTrp: Bromotryptophan.

121

122

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3-hydroxy-11-methyltridecanoic acid (Table 1). These short linear AMPs, containing 2–4 amino acids, are highly active (MIC 0.02– 0.06 lM) against S. aureus, B. subtilis, Salmonella enterica subsp. enterica serovar Typhimurium, and P. aeruginosa [26]. Polymyxin B (Table 1) is a cyclic AMP produced by Paenibacillus polymyxa. It contains diaminobutyric acid (Dab) and D-phenylalanine, responsible for the planar ring structure displayed by this AMP [27]. There are 6 different isoforms of Polymyxin B (B1, B1-I, B2, B3, and B6) that differ in the initial fatty acid and in one of the amino acids forming the planar ring [28]. Polymyxin B is active against a broad range of Gram
bacteria, it acts as a surfactant, permeabilizing the bacterial cell membrane. Polymyxin E (Colistin, Table 1) is a cyclic AMP, similar to Polymyxin B, produced by P. polymyxa var. colistinus [29]. Colistin has been used in Japan and Europe since the 1950s, and in United States from the 1960s, but it was discontinued in 1980 due to its high nephrotoxicity [30]. However, it is currently part of the lastline antibiotic arsenal against multi-drug resistant Gram
bacteria [31]. In fact, Polymyxin B and Colistin are among the few remaining antibiotics for the treatment of multi-drug resistant P. aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae infections [30,31]. The mechanism of action of Colistin involves two steps; an initial electrostatic interaction between the AMP and the lipopolysaccharide membrane (Fig. 1), followed by the displacement of the Mg2+ and Ca2+ ions that stabilize the cellular membrane. This increases the membrane permeability and results in cell death [30]. Bacitracin (Table 1) is an AMP, discovered in 1943, produced by B. subtilis and Bacillus licheniformis [32]. Bacitracin is only active in the presence of a divalent metal ion, such as Zn2+, Mn2+, Co2+, Ni2+, and Cu2+ [33]. Bacitracin is a complex mixture of cyclic polypeptides, of which Bacitracin A is the most active [34]. The mechanism of action of Bacitracin involves the inhibition of the biosynthesis of the bacterial peptidoglycan layer by binding to the undecaprenyl pyrophosphate intermediate [33]. Nisin is the most prominent member of the lantibiotic family, a group of ribosomically synthetized polycyclic AMPs characterized by the presence of uncommon amino acids, such as animobutidic acid (Abu), didehydroalanine (Dha) and didehydroaminobutyric acid (Dhb) [35]. Nisin is produced by Lactococcus lactis and contains one lanthionine (A-S-A) and two (b-methyl) lanthionine (Abu-S-A) rings [36]. There are two naturally produced forms of Nisin, A (Table 1) and Z, which only differ in one amino acid. The antibiotic effect of Nisin was first described by Rogers in 1928 [37], and this compound constitutes the only ‘‘antibiotic” approved as a food preservative by the United States Food and Drug Administration (FDA) [38]. Nisin has a broad spectrum of action against several Gram+ (including S. aureus, Streptococcus pyogenes, Clostridium botulinum, and L. monocytogenes) and Gram
(Neisseria spp., Klebsiella spp., Aeromonas spp., Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, Yersinia enterocolitica, and Pseudomonas fluorescens) pathogenic bacteria [39]. Nisin exerts its activity by forming pores on the cell membrane and inhibiting peptidoglycan biosynthesis (Fig. 1); it specifically targets lipid II, the membrane-bound peptidoglycan precursor [40]. Microcins are a family of plasmid-encoded AMPs secreted by Enterobacteria [41], giving the bacteria a competition advantage in their biological niche within the intestinal microbiota [42]. One of these compounds, Microcin J25 (MccJ25, 21 aa, Table 1), is produced by E. coli and can inhibit the growth of a range of Enterobacteriaceae (E. coli, Salmonella sp. and Shigella sp.) at nanomolar concentrations (e.g. E. coli AB1133, MIC 0.009 lM; S. enterica Serotype Newport, MIC 0.005 lM) [43]. Microcin J25 cellular uptake, in E. coli, requires the presence of the outer membrane receptor FhuA, as well as specific inner membrane proteins (TonB, ExbD, ExbB, and SbmAhas). This AMP has a dual mode of action; it can both inhibit the bacterial RNA polymerase

and act on the respiratory chain, inducing the production of reactive oxygen species (ROS) [44]. The short AMP Microcin C7 (MccC7, Microcin C and Microcin C51, 7 aa, Table 1) is produced by E. coli and has a posttranslational modification in the C-terminus (adenosine monophosphate). Microcin C7 enters bacterial cells through membrane transporters (such as YejABEF) [45] and it is intracellularly processed by proteases. This process releases Asp-NH-adenosine monophosphate, which binds aspartyl-tRNA synthetase and inhibits protein synthesis [46,47]. Microcin B17 (MccB17, 43 aa, Table 1) is produced by E. coli strains carrying pMccB17 or related plasmids, and targets DNA gyrase (a prokaryotic type II DNA topoisomerase) causing the cleavage of double-stranded DNA [48]. This results in fragmentation of the bacterial chromosome, caused by a massive DNA degradation following the induction of the SOS response [48,49]. 3. AMPs in fungi and plants 3.1. Fungal AMPs Peptaibols peptides are a big family of AMP compounds of fungal origin characterized by the presence of non-proteinogenic amino acids, such as a-aminoisobutyric acid (AiB). The Nterminal residues of peptaibols are usually acetylated (Ac) and they display an amino alcohol at the C-terminus of the molecule [such as phenylalaninol (Phl) or leucinol (Lol)] [50]. Alamethicin (Table 1) is the most widely studied peptaibol, produced by Trichoderma viride [51]. This compound has a broad spectrum of action, acting on bacteria, fungus, animal and insect cells [50]. This AMP is known to exert its action on Gram+ bacteria, such as Enterococcus faecalis, Staphylococcus haemolyticus, Streptococcus viridans, and S. aureus [51]. Alamethicin self-associates into hexameric barrel–stave transmembrane helices, and permeabilizes membranes by forming channels through them [5]. Trichogin GA IV (Table 1) is a related lipopeptaibol, isolated from Trichoderma longibrachiatum, which spans a lipophilic acyl chain at the N-terminus [n-octanoyl (nOct)], and AiB residue, and a C-terminal Lol. This lipopeptaibol is mainly effective against S. aureus (MIC 7.5–15 lM) [52] and is resistant to protease activity. Tricholongins B (I and II) (Table 1) are highly hydrophobic peptaibols (19 aa), isolated from T. longibrachiatum and active against Gram+ bacteria [53]. Saturnisporins SA (II and IV) (Table 1) are peptaibols (20 aa) produced by Trichoderma saturnisporum and active against S. aureus [54]. Sillucin is an AMP (30 aa, Table 1) with 4 disulfide bonds, produced by Rhizomucor pusillus (synonym: Mucor pusillus), and active against Gram+ bacteria at the level of RNA metabolism [55]. Micasin (Fig. 2, Table 1) is a defensing-like AMP encoded by Arthroderma otae (anamorph: Microsporum canis). Micasin is lethal to Bacillus megaterium at concentrations as low as 0.05 lM, and it is also effective against a broad range of Gram+ and Gram
bacteria (ie. P. aeruginosa at 0.94 lM). Micasin does not produce significant membrane permeabilization; it is in fact believed to interfere with intracellular protein folding [56]. Plectasin (40 aa, Table 1) was the first defensin isolated from fungi (it is homologous to mussel and insect defensins), it was described by Mygind and coworkers in the Ebony cup (Pseudoplectania nigrella) [57]. This AMP is effective against Gram+ bacteria, especially S. pyogenes (MIC 0.015 lM), Corynebacterium jeikeium (MIC 0.2 lM), Corynebacterium diphtheriae (MIC 0.5 lM), and MRSA (MIC 0.9 lM) [58]. Plectasin targets the bacterial cell wall precursor Lipid II [57]. Copsin (184 aa, Table 1) is another fungal defensin (isolated from Coprinopsis cinerea) with the same antibacterial mechanism of action. It is very effective against L. monocytogenes (MIC 0.04–0.08 lM), Micrococcus luteus (MIC 0.1 lM), B. subtilis (MIC 0.2 lM), and Enterococcus faecium (MIC 0.3–0.7 lM) [59].

Table 3 Amphibian, 2Reptilian and 3Avian antimicrobial peptides (AMPs).

1

AMP name

Sequence

A. loloensi1 B. gargarizans1

Cathelicidin AL Buforin I Buforin II Maximin 3 Bombinin Bombinin H2 Fallaxin Lf-CATH1 Dermaseptin B2 Dermatoxin B1 Plasticin B1 Phylloxin B1 Dermaseptin S1 Dermaseptin S5 Phylloseptin S1 Cathelicidin PY Ranateurin 1 Ranalexin Esculentin 1 Palustrin 3a Palustrin 3b Magainin I Magainin II CPF-St5 XPF-St8 Pseudin 2 Cathelicidin BF TEWP Crotamine TBD-1 OH-CATH Omwaprin Pelovaterin dCATH AvBD1 AvBD2 AvBD7 chCATH-B1 CHP1 CHP2 Fowlicidin 1

RRSRRGRGGGRRGGSGGRGGRGGGGRSGAGSSIAGVGSRGGGGGRHYA AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY TRSSRAGLQFPVGRVHRLLRK GIGGKILSGLKTALKGAAKELASTYLHNH2 GIGALSAKGALKGLAKGLAEHFANNH2 IIGPVLGLVGSALGGLLKKINH2 GVVDILKGAAKDIAGHLASKVMNKLNH2 PPCRGIFCRRVGSSSAIARPGKTLSTFITV GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAVNH2 SLGSFLKGVGTTLASVGKVVSDQFGKLLQAGQGNH2 GLVTSLIKGAGKLLGGLFGSVTGGQSNH2 GWMSKIASGIGTFLSGMQQNH2 ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ GLWSKIKTAGKSVAKAAAKAAVKAVTNAV FLSLIPHIVSGVASIAKHFNH2 RKCNFLCKLKEKLRTVITSHIDKVLRPQG SMLSVLKNLGKVGLGFVACKINKQC FLGGLIKIVPAMICAVTKKC GIFSKLGRKKIKNLLISGLKNVGKEVGMDVVRTGIDIAGCKIKGEC GIFPKIIGKGIKTGIVNGIKSLVKGVGMKVFKAGLNNIGNTGCNEDEC GIFPKIIGKGIKTGIVNGIKSLVKGVGMKVFKAGLSNIGNTGCNEDEC GIGKFLHSAGKFGKAFVGEIMKS GIGKFLHSAKKFGKAFVGEIMNS GVFGLLAKAALKGASKLIPHLLPSRQQ GFMSKVANFAKKFAKGGVNAIMNQK GLNALKKVFQGIHEAIKLINNHVQ KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF QKKCPGRCTLKCGKHERPTLPYNCGKYICCVPVKVK YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG YDLSKNCRLRGGICYIGKCPRRFFRSGSCSRGNVCCLRFG KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPF KDRPKKPGLCPPRPQKPCVKECKNDDSCPGQQKCCNYGCKDECRDPIFVG DDTPSSRCGSGGWGPCLPIVDLLCIVHVTVGCSGGFGCCRIG KRFWQLVPLAIKIYRAWKRR GRKSDCFRKSGFCAFLKCPSLTLISGKCSRFYLCCKRIW LFCKGGSCHFGGCPSHLIKVGSCFGFRSCCKWPWNA QPFIPRPIDTCRLRNGICFPGICRRPYYWIGTCNNGIGSCCARGWRS PITYLDAILAAVRLLNQRISGPCILRLREAQPRPGWVGTLQRRREVSFLVEDGPCPPGVDCRSCEPGALQHCVGTVSIEQQPTAELRCRPLRPQ GRKSDCFRKSGFCAFLKCPSLTLISGKCSRFYLCCKRIR GRKSDCFRKNGFCAFLKCPYLTLISGLCSFHLC RVKRVWPLVIRTVIAGYNLYRAIKKK

B. maxima1 B. variegata1 L. fallax1 L. fragilis1 P. bicolor1

P. sauvagii1

P. yunnanensis1 R. catesbeiana1 R. esculenta1 R. palustris1 X. laevis1 1

X. tropicalis

P. paradoxa1 B. fasciatus2 C. caretta2 C. d. terrificus2 E. orbicularis2 O. hannah2 O.microlepidotus2 P. sinensis2 A.platyrhynchos3 G. gallus3

Activity

Ref.

G+

G


+ + + + + +

+ + + + + + + + +

+ + + + + + + + + + +

+ + + + + + + + +

+ + + + + + + + + + + + + + + + + + + +

+ + + + + + + + +

+ + + + + + + + +

[147] [145] [141] [136] [139] [163] [149] [152] [157] [156] [160] [152] [153] [162] [148] [173] [175] [151] [164] [165] [165] [170] [170] [172] [180] [188] [186] [185] [179] [181] [187] [184] [192] [192] [193] [183] [191] [191] [182]

J.M. Ageitos et al. / Biochemical Pharmacology 133 (2017) 117–138

Organism

1

Amphibian; 2Reptilian; 3Avian; G+: Gram positive bacteria; G
: Gram negative bacteria. NH2: amidated C-terminus.

123

124

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Snakins mechanisms of action are unknown, but their activities are affected by divalent cations. Cyclotides are a family of AMPs with a head-to-tail cyclic backbone found in plants, bacteria and animals [68]. These amphipathic peptides have separate clusters of hydrophobic and hydrophilic residues, a conserved cyclic cysteine knot (CCK), and three conserved disulfide bonds [69]. Cyclotides provide resistance against insect infections [70], and carry out their antimicrobial activity by forming pores in bacterial and fungal membranes (Fig. 1) [70,71]. Several members of this family display antibacterial activity; for instance, Kalata B1 from Oldenlandia affinis and Circulins A and B from Chassalia parviflora (Table 1) can inhibit S. aureus at low concentrations (MIC 0.3, 0.2 and 8.5 lM, respectively). Kalata B1 and Circulin A are relatively ineffective against Gram
bacteria, while Circulin B can inhibit Gram
bacteria, such as E. coli (MIC 0.4 lM), Klebsiella oxytoca (MIC 8.2 lM), and Proteus vulgaris (MIC 6.8 lM) [72]. Cyclopsychotride A (Table 1), from Psychotria longipes [73], has an antimicrobial spectrum of activity similar to Circulin A (S. aureus, MIC 0.2 lM) [72].

4. AMPs produced by Porifera, Cnidarian and Mollusca

Fig. 3. Secondary structure of some antimicrobial peptides from vertebrates. The molecular models were obtained from the Protein Data Bank (PDB; http://www. rcsb.org/pdb) and the molecular graphics were performed with the UCSF Chimera package (http://www.cgl.ucsf.edu/chimera). Hydrophobic residues are depicted in yellow and hydrophilic amino acids are in blue.

3.2. AMPs produced by plants Plants produce several AMPs as part of their defense response, with Purothionins and plant defensins as the most relevant. Purothionins (a1, a2 and b; a-1-Purothionin is depicted in Fig. 2, Table 1) are cationic AMPs, spanning 45 amino acids and present on the endosperm of wheat (Triticum aestivum) and other cereal species [60]. These AMPs are toxic to mammalian cells and effective against several pathogenic bacteria, such as gamma proteobacteria (Xanthomonas phaseoli, Xanthomonas campestris, Erwinia amylovora, etc.), as well as Gram
(such as Ralstonia solanacearum) and Gram + organisms (such as Rhodococcus fascians, Curtobacterium flaccumfaciens, Clavibacter michiganensis, L. monocytogenes, Listeria innocua, and Listeria ivanovii) [60–62]. Puroindolines [isoforms A (Table 1) and B] are cationic AMPs isolated from wheat grain endosperm and containing a tryptophan-rich domain [63]. Puroindolines can inhibit the growth of some Gram+ (such as S. aureus, M. luteus, and Bacillus cereus) and Gram
(Klebsiella sp.) bacteria [64]. Plant defensins (c-thionins) are cationic peptides between 45 and 54 amino acids long [65], with mainly antifungal activity, as opposed to other defensins. However, some c-thionins (such as CtAMP1 from Clitoria ternatea, Ah-AMP1 from Aesculus hippocastanum, and Dm-AMP1 from Dahlia merckii (Table 1)) can inhibit the growth of B. subtilis (MIC 3, 17 and 27 lM, respectively) [66]. Snakins (1 and 2) are AMP-related plant defensins that display a broader action spectrum, as they can act on Gram+ and Gram
bacteria. Snakin-1 (Table 1), an AMP isolated from potato plants (Solanum tuberosum), is active (MIC 1 lM) against L. monocytogenes, L. innocua, L. ivanovii [62], and the plant pathogen C. michiganensis (MIC < 10 lM) [67].

AMPs are part of the humoral defense of invertebrates and they are usually produced as a response to pathogen infections. The sponges of the genera Discodermia produce a family of AMPs termed Discodermins (A–H) that are active against fungi and bacteria, by causing cell membrane permeabilization [74]. Discodermin A (Table 2) is a non-ribosomal AMP, isolated from Discodermia kiiensis [75,76], that contains unusual residues [tert-leucine (tL), cystenoic acid (Cys(O3H), and sarcosine (Sar)] and D-amino acids. Discodermin A inhibits the growth of B. subtilis (MIC 2 lM), S. aureus, E. coli, P. aeruginosa, Proteus mirabilis (MIC 1 lM), and Morganella morganii (MIC 8 lM) [75,77]. Discodermia sp. also produces Polydiscamide-A (Table 2), a partially cycled Depsi-AMP, capable of inhibiting B. subtilis with a MIC of 2 lM [78]. Aurelin (40 aa, Table 2, Fig. 2) is an AMP isolated from the jellyfish Aurelia aurita, active against B. megaterium (with a MIC of 10 lM), L. monocytogenes (MIC 5 lM) and E. coli (MIC 2 lM) [79,80]. Aurelin has 3 cysteine bonds and is 40% homologous to the K+ channels-blocking toxins from sea aeromones [80]. Scleractinian corals (e.g. Pocillopora damicornis) produce Damicornin (Table 2), an AMP similar to Aurelin [81] active against some Gram+ bacteria (S. aureus, MIC 5 lM; M. luteus, 1.25 lM; Corynebacterium stationis, MIC 10 lM). Damicornin has a limited activity against Gram
bacteria; indeed the coral pathogen Vibrio coralliilyticus can repress Damicornin expression in the coral [81]. Several Cysteine-rich AMPs have been isolated from marine mollusks; in particular 2 defensins, 5 Mytilins, 2 Myticins, and a Mytimycin were isolated from the mussel (Mytilus sp.) [82,83]. With the exception of Myticilyn, which only displays activity against fungi, the other AMPs have antibacterial activity. Mytilus defensins (Table 2) are structurally similar to the defensins produced by Arthropoda. Defensins and myticins are mainly active against Gram+ bacteria [83]. Different mytilin isoforms (Table 2) can display different activities; isoforms A, B, C, and D are active against both Gram
and Gram+ bacteria, while mytilin G1 (Table 2) is only active against Gram+ bacteria [83]. Tachyplesins are a family of AMPs isolated from horseshoe crabs (Tachypleus tridentatus and Limulus polyphemus) hemocytes [84]. Tachyplesin I (Table 2, Fig. 2) is a 17 amino acid long AMP with a positive charge and a structure spanning two antiparallel b-sheets stabilized by two disulfide bonds between four cysteine residues [85]. Tachyplesin I (Table 2) is active against Gram+ and Gram
bacteria, yeasts and virus [86], and it specifically binds to the DNA minor groove [87]. Tachyplesin I can inhibit sequence-specific protein binding

J.M. Ageitos et al. / Biochemical Pharmacology 133 (2017) 117–138

to DNA, such as the assembly of transcription factors [88]. Polyphemusins (I and II, 18 aa, Table 2) are AMPs produced by L. polyphemus which have two disulfide bonds (Cys 4-Cys17 and Cys8-Cys13) and form an anti-parallel b-structures. Polyphemusin I is twice as potent as Polyphemusin II, and both are active against Gram+ and Gram
bacteria [84].

5. AMPs produced by Arthropoda Cecropins (A, B and D) (Cecropin A, Table 2) are positively charged linear AMPs, cecropins A and B were originally isolated by Boman’s group from the hemolymph of the giant silk moth (Hyalophora cecropia) [89,90]. Their mechanism of action is based on the formation of membrane channels and results in cell lysis (Fig. 1). Gram
bacteria are more sensitive to these AMPs, for instance Cecropin B is 40 times more active against E. coli (MIC 1.7–3.3 lM) than against S. aureus [6,90]. Thanatin (Table 2, Fig. 2), an AMP produced by the hemipteran Podisus maculiventri [91,92], is highly active (MIC < 1.2 lM) against Gram
bacteria (S. enterica subsp. enterica serovar Typhimurium, E. coli, and K. pneumoniae); but it is less active (MIC < 5 lM) against Gram+ bacteria (Aerococcus viridans, M. luteus, B. megaterium, and B. subtilis) [93]. Thanatin stops respiration in bacteria, but does not permeabilize their inner cellular membranes [93]. Melittin (Table 2) is an amphipathic a-helical AMP isolated from the venom of the European honeybee (Apis mellifera) [94]. This AMP is highly active (MIC < 1–8 lM) against Gram
(E. coli, P. aeruginosa, S. enterica subsp. enterica serovar Typhimurium, Enterobacter cloacae, and K. pneumoniae) and Gram+ bacteria (B. megaterium, B. subtilis L. monocytogenes, S. aureus, and Staphylococcus epidermidis), including several antibiotic-resistant strains of E. coli, P. aeruginosa, S. aureus, and S. enterica subsp. enterica serovar Typhimurium [95]. Apidaecins (Ia, Ib and II) (Table 2) are a family of short AMPs (18 aa) produced by A. mellifera after bacterial infection [96]. These compounds are heat-stable and non-helical, and highly active against Gram
bacteria. Their mechanism of action is not based on the formation of pores [97]. Apidaecins enter inside the cell by a permease/ transporter-mediated peptide uptake [98] and they target the heat shock protein DnaK and the chaperonin GroEL (Fig. 1). Abaecin (Table 2) is another major A. mellifera AMP with antimicrobial activity [99]. This inducible, proline-rich, a-helical AMP is produced after bacteria infect the bee, and it is active against several Gram
bacteria associated with plants. Abaecin acts synergically with Hymenoptaecin (Table 2), another infection-inducible AMP rich in proline produced by A. mellifera, highly active (MIC 0.5–1 lM) against B. megaterium, E. coli and X. campestris [100]. Hymenoptaecin can form pores in both Gram+ and Gram
bacterial membranes (Fig. 1) and, once inside the cell, Abaecin interacts with the chaperone protein DnaK [101]. Similarly to Apiadecins and Abaecin, Pyrrhocoricin (from Pyrrhocoris apterus, Table 2) [102] and Drosocin (from Drosophila melanogaster) [103] target the internal proteins DnaK and GroEL [98]. The chaperone DnaK and the chaperonin GroEL recognize aberrantly folded proteins/peptides [104], and the AMPs cause the accumulation of misfolded proteins [104]. The threonine residue of Pyrrhocoricin is O-glycosylated with either a monosaccharide [a-n-acetylgalactosamine (a-GalNAc)] or a disaccharide (b-galactopyranosyl-a-GalNAc) sugar moiety. Pyrrhocoricin inhibits the ATPase actions of the chaperone, thus preventing the chaperone-assisted protein folding [105]. This AMP can inhibit the growth of Gram
(E. cloacae, E. coli and P. aeruginosa) and some Gram+ (such as B. megaterium and M. luteus) bacteria [102]. Drosocin (Table 2) is a proline-rich AMP that contains an O-glycosylated threonine residue [103]. This O-glycosylation essentially provides an extended/rigid conformation to this AMP [106], while the sugar moiety affects the activity of Drosocin [107], with

125

O-glycosylated Drosocin more active than its non-glycosylated counterparts. As mentioned above, Drosocin binds to the bacterial protein DnaK, but unlike Pyrrhocoricin, this AMP does not affect DnaK ATPase activity [105]. Drosocin is mainly effective against Gram
bacteria (e.g. E. coli, S. enterica subsp. enterica serovar Typhimurium and S. enterica subsp. enterica serovar Typhi), but its glycosylated and non-glycosylated analogs have different patterns of activity [106–108]. Ceratotoxins (A–D) (Table 2) are a family of sex-specific amphiphilic AMPs expressed in the female reproductive accessory glands of Ceratitis capitata (Mediterranean fruit fly) [109,110]. Unlike other insect AMPs, their expression is induced by sexual maturity, not by bacterial infection [111]. Ceratotoxins share homology with the caerulein precursor factor (CPF), an AMP produced by Xenopus laevis [109]. Ceratotoxins are hemolytic and can inhibit the growth of Gram
(e.g. E. coli, P. aeruginosa, S. enterica subsp. enterica serovar Typhimurium) bacteria and B. subtilis [110,112]. Ceratotoxins lyse bacterial cells after increasing the permeability of their inner and outer cell membranes [112].

6. Fish AMPs As is the case for other vertebrates, fish also produce AMPs as part of their immune system, and these fish compounds are known as cathelicidins, b-Defensins, hepcidins and piscidins. For a detailed view of fish AMPs, please see the excellent review article by Masso-Silva and Gill [113].

6.1. Fish cathelicidins and b-Defensins Cathelicidins (CATH) are short cathionic AMPs with a conserved cathelin domain (cathelin is an inhibitor of the cysteine proteinase cathepsin L) that are stored in the secretory granules of neutrophils and macrophages and released after leukocyte activation; they are widely found in vertebrates (such as fish, reptiles, birds, and mammals) [113–116]. Cathelicidins are characterized by the presence of a conserved cathelin domain, but they differ in length, amino acid sequence and structure; they and can adopt several conformations such as a-helical, elongated or b-hairpin structures [117]. The first fish Chatelicidin described was found in the intestine of the Atlantic hagfish (Myxine glutinosa), and named hagfish intestinal antimicrobial peptide (HFIAPs 1–3) [118]. HFIAP-1 (37 aa, Table 2) is a potent AMP with a broad-spectrum of action (Gram+, MIC 0.1– 3.5 lM; Gram
, MIC 0.4–7.0 lM) [119], and contains brominated Tryptophan (BrTrp7 and BrTrp32) residues [118]. HFIAP-3 (30 aa, BrTrp2 and BrTrp7) has an activity and spectrum similar to HFIAP-1, whereas HFIAP-2 has the same sequence as HFIAP-1 but the only brominated Tryptophan residue is at position 7 [118,119]. Another fish AMP is the glycine-rich cathelicidin from the Atlantic cod (Gadus morhua), codCath (67 aa, Table 2). This AMP has lytic activity against Gram
bacteria (MIC 5–10 lM) and B. megaterium (MIC 5 lM), and is salt-sensitive [120]. CodCath has low cytotoxicity for fish cells (not observed at concentrations up to 40 lM) [120] and it is expressed in liver, spleen and kidney as a response to bacterial infection [121]. Two cathelicidins, rtCATH_1 and rtCATH_2, are produced by the rainbow trout (Oncorhynchus mykiss) and the Atlantic salmon (Salmo salar). The peptides rtCATH_1(R146-P181) and rtCATH_2(R143-I178) (Table 2) are highly active against the fish pathogen Lactococcus garvieae (MIC 0.1–8 lM) and several Gram
bacteria (MIC 0.5–10 lM) [122]. Another rtCATH fragment, rtCATH_1 (R151-V186) (Table 2) is active (MIC 2 lM) against the fish pathogens Vibrio anguillarum and L. garvieae [123]. Several cathelicidin genes have also been described in other fish species [124].

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In addition, several b-Defensins genes have been found in fish, but all the b-Defensins experimentally tested so far have displayed a low antimicrobial efficacy [113], with the exception of cod bDefensin (Table 2), which is highly active against the fish pathogen Planococcus citreus, with a MIC of 0.4–0.8 lM [125]. 6.2. Fish hepcidins Hepcidins are iron-regulating hormones with antimicrobial properties that were initially described in humans. Fish hepcidins are cysteine-rich AMPs containing hairpin-shaped b-sheets with four disulfide bridges, which have been identified in 37 different fish species [113]. Hepcidins belong to two different families, HAMP1 and HAMP2; the first family is an ortholog of mammalian hepcidin, while the second supports innate immunity [126]. Fish Hepcidins display low in vitro activity, with the exception of CsHEP (Table 2) from the half-smooth tongue sole (Cynoglossus semilaevis), with a MIC of 2.92 lM for Edwardsiella tarda and V. anguillarum, and the prohepcidin Pro-Omhep1 (Table 2) from the Indian medaka (Oryzias melastigmus), with a MIC of 1.5–3 lM for S. aureus and Aeromonas hydrophila [126]. 6.3. Fish piscidins Piscidins are a family of fish-specific linear amphipathic AMPs belonging to the cecropin family. They are characterized by adopting an a-helix structure similar to that of magainins and cecropins [113]. Pleurocidin (25 aa, Table 2) was isolated from the mucus of the winter flounder (Pleuronectes americanus) [127], and constitutes the first characterized Pisicidin. It is active against several Gram+ (such as E. coli, MIC 2.2–3.3 lM, and Flavobacterium hydatis, MIC 2.2–4.4 lM) and Gram
(B. subtilis, MIC 1.1–2.2 lM) bacteria in a salt-independent manner. Pleurocidin can permeabilize bacterial membranes and also inhibit macromolecular synthesis in E. coli [128]. Piscidin 1–3 (22 aa, Table 2) were isolated from a hybrid striped bass (Morone saxatilis  Morone chrysops) and are expressed in mast cells, blood, skin, gills, and the gut. Piscidin 1 is effective (MIC 3.1 lM) against Gram+ and Gram
bacterial fish pathogens, including several antibiotic-resistant strains. Piscidin 2 has the same sequence and activity as Piscidin 1, except for the fact that the lysine at position 18 is substituted by an arginine. Piscidin 1 and 3 exert both a potent inhibitory and bactericidal effect on A. hydrophila, with a MIC of 0.8 and 1.6 lM, respectively; in addition, their minimal bactericide concentration (MBC) value is the same as their MIC. Piscidin 1 is twice as potent as Piscidin 3, and both AMPs are stronger hemolytic agents than Magainin 2, although they are less potent than Melittin [129]. 7. AMPs produced by amphibians The amphibian skin currently constitutes a good source of AMPs [130], produced in their dermatous granular glands [131], with more than 500 different AMPs described in the Amphibia animal class [132]. The term Bombinins describes a family of AMPs initially described in the skin secretions of toads belonging to the genus Bombina [133]. Bombinins are highly structurally homologous hemolytic glycine-rich AMPs, with an aminated residue as their C-terminal amino acid. The AMP Bombinin (Table 3) was isolated from the yellow-bellied toad (Bombina variegata) [130,134,135] and can inhibit the growth, but not kill, 84% of the tested strains of S. aureus and 72% of the E. coli strains, at a MIC ranging from 3 to 14 lM [136]. B. variegata [136] and Bombina orientalis [137] produce other AMPs, known as Bombinin-like peptides (BLPs). B. variegata also produces another family of hydrophobic AMPs, named Bombinins H (H1–H5) (Bombinin H2

is depicted in Fig. 3, Table 3), with peptides H3, H4 and H5 containing D-allo-isoleucine (DaIle) [138]. Bombinins H are described as active (at MIC < 5 lM) against S. aureus and E. coli; while Bombinin H2 is also active (MIC 4 lM) against antibiotic-resistant strains of E. faecium, Stenotrophomonas maltophilia and A. baumannii [139]. A related toad species, the Chinese red belly toad (Bombina maxima) produces several families of AMPs in its skin secretions, known as Maximins H (H1–H4) (homologous to Bombinins H) [140,141], Maximins (1–9) [141,142] and Maximins S (S1–S5) [143]. Maximin 3 (Table 3) has a potent antimicrobial activity, with a MIC of 0.3 lM, against B. megaterium, Shigella dysenteriae and E. coli, as well as against S. aureus and K. pneumoniae, with a MIC of 1.1 lM [141]. The number of B. maxima AMPs described is continuously increasing, with more than 79 different AMPs currently known [144]. Buforin I and II (Table 3) are AMPs isolated from Bufo gargarizans stomach [145], these cationic peptides have antibacterial activity (MIC 1–2 lM) against Gram+ (B. subtilis, S. aureus, S. mutants, Streptococcus pneumoniae, and Pseudomonas putida) and Gram
(E. coli, S. enterica subsp. enterica serovar Typhimurium and Serratia sp.) bacteria. Buforin II is more active than Buforin I, but both AMPs share a common domain containing the histone H2A, a highly basic protein rich in arginine and lysine. Buforin II enters the cell without significant disruption of the cell membrane, and mainly targets DNA [145,146]. Although Cathelicidins are widely found in nature [113–116], they had not been described in amphibians until recently. Cathelicidin-AL (isolated from Amolops loloensi; Table 3) was the first Cathelicin found in amphibians [147], it is active (MIC 6.5 lM) agaisnt S. aureus and P. aureginosa. Other Chathelicins are currently known, produced by other frogs, such as Cathelicidin-PY (Paa yunnanensis, Table 3) [148], Lf-CATH1 and 2 (Limnonectes fragilis, Table 3) [149] and Cathelicidin-RC1 (Rana catesbeiana, Table 3) [150]. Those AMPs are active agaisnt Gram+ and Gram
bacteria, Cathelicidin-PY is effective against E. coli (MIC 4.7 lM), Lf-CATH1 against S. aureus (MIC 1.3 lM) and Cathelicidin-RC1 against S. dysenteriae and K. oxytoca (MIC 1.4 lM). Esculentins [1 (a and b) and 2 (a and b)] are a family of AMPs with a C-terminal disulfide bridge, initially isolated from the edible frog Rana esculenta [151]. Esculentins have a broad antimicrobial activity, and are higly active (MIC < 1 lM) against Gram+ and Gram
bacteria. Dermaseptins (S1–S9) are cationic and amphipathic AMPs with 24–34-residues, isolated from the skin of the tree frog Phyllomedusa sauvagii [131,152]. They are active against bacteria, yeast, protozoa, and filamentous fungi; they inhibit protein synthesis [146] and induce cell apoptosis [104]. Dermaseptins are synthesized as high-molecular-mass precursors that undergo proteolytic maturation to generate the active AMPs [152]; although they share common structural properties, their action spectrum is specific. While Dermaseptins S1 (Table 3) to S4 are highly active against the amphibian pathogen Aeromonas caviae (MIC 0.5–1 lM), Dermaseptin S5 (Table 3) has a remarkable activity against S. aureus (MIC 2 lM) [153]. Dermaseptins are also found in other South American frogs, such as Phyllomedusa bicolor, Phyllomedusa hypochondrialis, Phyllomedusa distincta, Phyllomedusa oreades, Phyllomedusa tarsius, Phyllomedusa trinitatis, Pachymedusa dacnicolor, Agalichnis annae, Agalichnis callidryas, Agalichnis litodryas, and Hylomantis lemur [154,155]. Adenoregulin (Dermaseptin B2, Table 3) was isolated from the giant leaf frog P. bicolor, this AMP has an a-helix structure and contains D-amino acids [152]; it belongs to the Dermaseptin B family (B1–B8) (24–34 aa) [156]. Dermaseptin B2 is the more active among these compounds, inhibiting the growth of the tested microorganisms with a MIC in the lM range. Dermatoxin B1 (32 aa, Table 3) [157] is a glycinerich ortholog of the dermaseptin AMP family of compounds; it was isolated from P. bicolor and displays sequence homology to the xenopsin precursor factor (XPF) of X. laevis. This AMP mainly

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J.M. Ageitos et al. / Biochemical Pharmacology 133 (2017) 117–138 Table 4 Mammalian and synthetic antimicrobial peptides (AMPs). Organism

AMP name

Sequence

B. bubalis B. taurus

buCATHL4C Bactenecin Indolicidin LAP TAP GNCP-1 GNCP-2 eCATH-1 Dermcidin HBD-1 HBD-2 HBD-3 HBD-4 HD-5 HD-6 Hepcidin 20 HNP-1 LEAP-1 LEAP-2 LL-37 TC-1 TC-2 RTD-1 RTD-2 RTD-3 Cryptdin-1 PG1 AMP72 AMP126 AMP2041 BP100 C16-KGGDK CAMEL0 Dhvar1 Dhvar2 Dhvar4 Dhvar5 FL9 GS14K4 P-Der Pexiganan RW-BP100 RN7-IN6 V Peptide 4 WLBU2 WMR-NH2

RIRFPWPWRWPWWRRVRG RLcycic(CRIVVIRVC)R ILPWKWPWWPWRRNH2 GFTQGVRNSQSCRRNKGICVPIRCPGSMRQIGTCLGAQVKCCRRK NPVSCVRNKGICVPIRCPGSMKQIGTCVGRAVKCCRKK RRCICTTRTCRFPYRRLGTCIFQNRVYTFCC RCICTTRTCRFPYRRLGTCLFQNRVYTFCC KRFGRLAKSFLRMRILLPRRKILLAS SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK GIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK FELDRICGYGTARCRKKCRSQEYRIGRCPNTYACCLRKWDESLLNRTKP ATCYCRTGRCATRESLSGVCEISGRLYRLCCR AFTCHCRRSCYSTEYSYGTCTVMGINHRFCCL ICIFCCGCCHRSKCGMCCKT ACYCRIPACIAGERRYGTCIYQGRLWAFCC DTHFPICIFCCGCCHRSKCGMCCKT MTPFWRGVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES AELRCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIVQKKLAGDES NLAKGKEESLDSDLYAELRCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVEVIATLKDGRKICLDPDAPRIKKIVQKKLAGDES cyclic(GFCRCLCRRGVCRCICTR) cyclic(GVCRCLCRRGVCRCLCRR) cyclic(GFCRCICTRGFCRCICTR) LRDLVCYCRTRGCKRRERMNGTCRKGHLMYTLCCR RGGRLCYCRRRFCVCVGR KGCALVKVRGLTLKVCK KWCRKWQWRGVKFIKCV HKCAKIKWRGVHVKYCA KKLFKKILKYLNH2 C16H31O2-KGGDK KWKLFKKIGAVLKVLNH2 KRLFKELKFSLRKY KRLFKELLFSLRKY KRLFKKLLFSLRKY LLLFLLKKRKKRKY GVVDILKGLAKDIAGHLASKVMNKLNH2 cyclo(VKLDKVDYPLKVKLDYP) ALWKTMLKKAAHVGKHVGKAALTHYLNH2 GIGKFLKKAKKFGKAFVKILKKNH2 RRLFRRILRWLNH2 FLGGLIKWWPWRRNH2 Ac-cyclo(CVKVQVKVGSGVKVQVKVC)NH2 RVVRVVRRWVRRVRRVWRRVVRVVRRWVRRVRRWVRRVVRVVRRWVRR WGIRRILKYGKRSNH2

C. porcellus E. asinus H. sapiens

M. mulatta

M. musculus S. scrofa Synthetic

Activity G+

G


+ + + + + + + + +

+ + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + +

+ + + + + + + +

+ + + + + + + +

Ref.

[206] [214] [205] [243] [241] [227] [227] [219] [277] [250] [254] [251] [262] [231] [231] [275] [201] [274] [276] [200] [273] [273] [267] [268] [268] [236] [211] [300] [300] [300] [284] [301] [283] [292] [292] [293] [293] [294] [297] [128] [289] [286] [288] [298] [299] [295]

G+: Gram positive bacteria; G
: Gram negative bacteria. NH2: amidated C-terminus.

acts on Acholeplasma laidlawii and B. megaterium, and has low activity against Gram
bacteria. Plasticins (23–29 aa) are Dermaseptin-related peptides described in P. bicolor and other frogs belonging to the Phyllomedusinae subfamily [154,156,158]. These flexible AMPs adopt various structures when interacting with cell membranes, and it was suggested that their activity not only relates to membrane disruption [158,159]. For instance, the cationic Plasticin-B1 (Table 3) [156] binds to cell membranes forming pores on them (Fig. 1), and it is active against Gram
(P. aeruginosa, S. enterica and S. enterica subsp. enterica serovar Typhimurium; MIC 3.1 lM) and Gram+ (B. megaterium, MIC 3.2 lM; S. aureus and S. pneumoniae; MIC 6.3 lM) bacteria [155,158]. Phylloxin B1 (19 aa, Table 3) is another AMP, isolated from P. bicolor and, similarly to Dermatoxin, with sequence homology to XPF [160]. It inhibits not only the growth (MIC  10 lM) of A. laidlawii and B. megaterium, but also of Corynebacterium glutamicum (MIC 1.6 lM), M. luteus (MIC 3 lM) and E. coli (MIC 10 lM). Interestingly, Phylloxin inhibits the growth, but does not kill the bacterial strains [160] tested. Phylloseptins are a family of AMPs isolated from South American tree frogs (P. sauvagii, P. bicolor, Phyllomedusa hypochondrialis, P. oreades and Hylomantis lemur)

[155,161,162] that are 20 amino acids long and are amidated at the C-terminal region. Phylloseptin S1 (Table 3) displays a broad spectrum against Gram+ (E. faecalis, S. aureus and S. pyogenes) and Gram
(A. baumannii, E. coli and P. aeruginosa) bacteria, with a MIC in the range of 3–8 lM [161,162]. Phylloseptins are believed to produce cell membrane permeabilization using the carpet-like mechanism (Fig. 1), creating leakage of the intracellular cell content and resulting in cell death [162]. Fallaxin (Ocellatin-F1; Table 3) is a 25 amino acids long AMP, isolated from the frog Leptodactylus fallax [163], active against Gram
bacteria (E. coli, MIC 40 lM; E. cloacae, MIC 20 lM; P. aeruginosa, MIC 80 lM; and K. pneumoniae, MIC 80 lM). Palustrins [1 (a to d), 2 (a to c) and 3 (a and B)] were isolated from the North American pickerel frog (Rana palustris) [164]. They include Palustrin-3a and Palustrin-3b, the two most active compounds, with 48 amino acids and a Cterminal cyclic hexapeptide region (Table 3); they display a MIC of 1 lM for E. coli, but are inactive against Gram+ bacteria. Magainins (1 and 2, Table 3) are a family of a-helical AMP ionophores, isolated from the skin of X. laevis (African clawed frog) [165], which disrupt transmembrane ion concentration gradients. Magainins 1 and 2 are active against Gram+ (S. epidermidis MIC

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4 lM) and Gram
(E. coli, MIC 2 lM; K. pneumoniae, MIC 4 lM; P. putida, MIC 4 lM) [165]. Magainins have little or not well-defined secondary structure in aqueous solutions at physiological pH, adopting the functional a-helical structure after binding to the cell membrane phospholipid bilayer [166]. Several Magainin-related AMPs have been isolated from other Xenopus species, such as a) Magainin-M1 (E. coli, MIC 12.5 lM) and Magainin-MW1 (E. coli, MIC 50 lM) [167], isolated from X. muelleri; b) Magainin-AM2 (E. coli, MIC 50 lM) from X. amieti [168]; c) Magainin-F3, isolated from X. fraseri and X. andrei [169]; d) and Magainin-St1, obtained from X. tropicalis [170]. X. tropicalis was found to produce 12 highly active AMPs, such as the caerulein precursor factor (CPF) and the xenopsin precursor factor (XPF). Additional AMPs include CPF-St5 (Table 3) (which has a MIC of 1 lM for S. aureus or E. coli, and a MIC 4–8 lM for P. aeruginosa) and XPF-St8 (Table 3), with a MIC 1 lM for E. coli, and 4 lM for P. aeruginosa MIC [170]. X. laevis also produces XPFs, but with a lower activity than those from X. tropicalis [171]. Pseudins (1–4) are AMPs isolated from the skin of the frog Pseudis paradoxa, with Pseudin-2 as the more abundant and active family member [172]. Pseudin-2 (Table 3) exerts its activity by interacting with RNA, which results in protein synthesis inhibition [95]. This AMP enters into the cell by forming pores on the cell membrane (Fig. 1), and specifically binds to RNA. It is active (MIC 2–8 lM) against Gram+ (B. subtilis, B. megaterium, L. monocytogenes, and S. aureus) and Gram
(E. coli, P. aeruginosa, S. enterica subsp. enterica serovar Typhimurium, E. cloacae, and K. pneumoniae) bacteria [95]. Ranatuerins (1–9) are a family of AMPs isolated from the American bullfrog Rana catesbeiana. Ranatuerin-1 (Table 3) is the more potent of these compounds (although it lacks hemolytic activity at the concentrations tested), which is active against E. coli (MIC 2 lM), Streptococcus agalactiae (MIC 5 lM) and S. aureus (MIC 50 lM) [173,174]. Ranalexin (Table 3) [175], another AMP isolated from R. catesbeiana, has a heptapeptide ring in the C-terminal region that is stabilized by a single intramolecular disulfide bond. This AMP is highly active (MIC 0.7–2.6 lM) against several bacteria, such as Methicillin-resistant S. aureus (MRSA), Methicillinresistant coagulase-negative staphylococci (MR-CoNS) and S. pneumoniae or E. coli [176]. 8. AMPs produced by reptiles and birds As it is the case with other vertebrates, the more prominent AMPs present in reptiles and birds [114,115] belong to the cathelicidin and defensin families. Based on genomic analyses, thus far 50 cathelicidins and 34 b-Defensins have been predicted in reptiles, and 44 putative cathelicidins and 714 putative b-Defensins described in 53 avian genomes [114,177]. However, so far only a fraction of those AMPs have been studied and tested. As mentioned above, Cathelicidins are short cationic AMPs stored in the secretory granules of neutrophils and macrophages and released after leukocyte activation. The b-Defensins represent another major group of AMPs present in reptiles [114] and birds [115], they are cationic AMPs with a triple-stranded b-sheet structure connected with a region forming a loop of b-hairpin turn and stabilized by three cysteine bridges. Reptiles and birds only express type b-Defensins, whereas mammals additionally produce a-Defensins and hDefensins. The homology found among the Defensin families originating from the different living things indicates that they all have evolved from a common ancestral gene [178]. 8.1. Reptilian and avian cathelicidins Cathelicidins have been identified from several elapid snakes, such as Ophiophagus hannah and Bungarus fasciatus, and some of

them have even been cloned and chemically-synthetized. The cathelicidin produced by the king cobra (O. hannah), OH-CATH, is 34 amino acids long (Table 3) and is active on Gram
bacteria, with a remarkable antimicrobial activity against E. cloacae (MIC 1 lM), Enterobacter aerogenes and P. aeruginosa (MIC 2 lM) [179]. Cathelicidin-BF (Table 3), a 30 amino acids long AMP produced by the banded krait (Bungarus fasciatus), was reported to be highly active against a broad spectrum of microorganism, in particular B. cereus (MIC 0.6–2.6 lM), S. aureus (MIC 1.3 lM), S. enterica subsp. enterica serovar Typhimurium (MIC 0.3 lM), E. coli (MIC 0.2– 0.6 lM), and P. aeruginosa (MIC 0.3–5.4 lM) [180]. The 50 amino acids long AMP Omwaprin (Table 3) is produced by the Inland taipan (Oxyuranus microlepidotus) and is relatively active against the Gram+ bacteria B. megaterium and Staphylococcus warneri [181]. Fowlicidins (1–3; Fowlicidin 1 sequence is depicted in Table 3) are chicken cathelicidins with antimicrobial (MIC 0.4–2 lM) activity against Gram+ and Gram
bacteria, including the multidrugresistant S. enterica subsp. enterica serovar Typhimurium DT104 and MRSA [182]. Chickens also produced chCATH-B1 (Table 3), an AMP preferentially expressed in the bursa of Fabricius, which is highly active against P. aeruginosa (MIC 0.63 lM), S. aureus (MIC 1.25 lM) and E. coli (MIC 2.5 lM) [183]. Another avian cathelicidin, dCATH (Table 3), was recently chemically synthetized based on the published CDNA sequence from duck (Anas platyrhynchos) [184]. This AMP can inhibit the grown of Gram+ and Gram
bacteria, with a geometric mean MIC value of 4 lM. 8.2. Reptilian and avian b-Defensins Despite the fact that putative b-Defensin genes were predicted from their genomic sequences, the first reptilian b-Defensin was only recently identified. Stegemmann and collaborators [185] described in 2009 the isolation of turtle b-Defensin-1 (TBD-1, 40 aa, Table 3) from leukocytes of the European pond turtle (Emys orbicularis). TBD-1 is highly active, under low-salt concentrations, against E. coli and L. monocytogenes (MIC 0.6 lM), but a higher concentration of the compound (MIC 5.6 lM) is required against MRSA. Several defensin-like AMPs were previously isolated, but their cysteine bond pattern usually differs from that of the canonical b-Defensins (Cys1–Cys5, Cys2–Cys4 and Cys3–Cys6). Crotamine (42 aa, Table 3) is a toxin found in the venom of the South American rattlesnake (Crotalus durissus terrificus). This compound is homologous to b-Defensins, as it shares with them characteristics such as the presence of 3 disulfide bonds (Cys4–Cys36; Cys11– Cys30 and Cys18–Cys37). Crotamine is not only toxic to eukaryotic cells, but is also effective against Gram+ (S. aureus) and Gram
(E. coli) bacteria, and its activity is similar to that of human bDefensin 2 (hBD-2) [186]. Pelovaterin (42 aa, Table 3) is produced by the Chinese soft-shelled turtle (Pelodiscus sinensis) and contains 6 cysteines; but it differs from the canonical b-Defensins as only two of these amino acids form a cysteine bond (Cys8 and Cys38). This AMP exhibits a half maximal effective concentration (EC50) of 0.1 lM for both P. aeruginosa and P. vulgaris [187]. The Turtle egg-white protein TEWP (Table 3) is a 36 amino acids long Defensine-like AMP, isolated from the Red sea turtle (Caretta caretta), with 6 cysteines forming 3 disulfide bonds (Cys4–Cys30; Cys8–Cys29 and Cys12–Cys24). However, TEWP does not share structural homology with canonical b-Defensins. This compound is more effective against S. enterica subsp. enterica serovar Typhimurium [half of inhibitory concentration (IC50) 2.8 lM] and E. coli (IC50 3.3 lM) than against S. aureus (IC50 5.1 lM) [188]. Avian b-Defensins can be classified according to their heterophil (avian granulocytes) or non-heterophil origin [189]. The chicken heterophilic AMPs CHP1 and CHP2 (Table 3) were isolated after the induction of inflammation [190]. Those AMPs display the same

J.M. Ageitos et al. / Biochemical Pharmacology 133 (2017) 117–138

disulfide pattern as mammalian b-Defensins, as well as their antimicrobial activity against Gram+ and Gram
bacteria, in particular against Campylobacter jejuni (CHP1, MIC 0.4 lM; CHP2 MIC 1.1 lM) and S. enterica subsp. enterica serovar Typhimurium (CHP1, MIC 0.9 lM; CHP2, MIC 2.1 lM) [191]. Gallinacins 1 and 2 (Gal 1 and Gal 2) [192], currently known as avian b-Defensins 1 and 2 (AvBD1 and AvBD2) (AvBD2 is depicted in Fig. 3), were obtained from the same origin and using the same technology. They were purified from bone marrow, together with AvBD7 [193], AvBD1, AvBD2, and AvBD7 (Table 3), and display a very strong antimicrobiotical activity against Gram+ and Gram
bacteria. Indeed, AvBD1 and AvBD7 display a similar activity (MIC under 0.33 lM) for both bacterial types, and in particular against S. aureus (with a MIC of 0.08 and 0.11 lM, respectively) [193]. AvBD2, although less active, is more effective against Gram+ (MIC < 0.47 lM) than Gram
bacteria (MIC < 6.05 lM) [193]. Gallimacin-3 (AvBD3) [194] and Spheniscins [Sphe-1 (AvBD103a) and Sphe-2 (AvBD103b)] [195] were obtained from the king penguin (Aptenodytes patagonicus) and represent b-Defensins found in epithelial cells. Other examples of avian b-Defensins described are the turkey heterophil AMPs [THP1 (AvBD1), THP2 (AvBD2) and THP3 (25 aa)] [190], Ostricacins from ostrich (OSP-1 to -4) [196] and mallard duck b-Defensins (AvBD2 and AvBD9) [197]. Despite their varied origin, the avian b-Defensins share considerable homology, therefore, it has been proposed to homogenize the nomenclature of the 14 types of avian b-Defensins [197,198] currently known. 9. AMPs produced by mammals The mammalian innate immune response includes the production of antimicrobial compounds such as inorganic substances (i.e. hydrogen peroxide or nitric oxide) and antimicrobial proteins (i.e. lysozyme, azurocidin, cathepsin G, phospholipase A2 or lactoferrin), including AMPs [199]. The major mammalian AMPs belong to Cathelicidin (Section 9.1) and Defensin (Section 9.2) families, but there are AMPs, such as Platelet antimicrobial proteins (PMPs), Hepcidins and Dermcidin (Section 9.3) that do not belong to those two families. 9.1. Mammalian cathelicidins Cathelicidins are a family (with more than 30 members) of cationic AMPs widely found in mammals (human, cattle, horses, pigs, sheep, goats, chickens, rabbits). The cationic AMP LL-37 (37 aa, also known as hCAP-18, FALL-39 or CAMP, Table 4) (Fig. 3) is the only Cathelicidin found in humans, rhesus monkeys, mice, rats, and guinea pigs [116,200]. LL-37 is active against Gram+ and Gram
bacteria [200], in particular against L. monocytogenes (MIC 0.3 lM), E. coli (MIC 0.1 lM), E. faecium (MIC 0.1 lM), and S. enterica subsp. enterica serovar Typhimurium (MIC 0.4 lM) [201,202]. LL-37 is an amphipathic AMP with disordered structure while in solution, but it acquires an a-helical structure when interacting with cell membranes. LL-37 is believed to act by forming a pore on the cell membrane or by inhibiting cell wall biogenesis (Fig. 1) [203]. LL-37 has been proposed as an aid in the treatment of infected wounds [203], due to its ability to prevent bacterial biofilm formation at concentrations below its MIC [204]. Indolicidin (Cathelicidin-4, Table 4) is a short (13 aa) tryptophan-rich cationic AMP isolated from the cytoplasmic granules of bovine neutrophils [205]. This compound has a broad spectrum of action and is active against both Gram+ (B. cereus, MIC 0.8 lM; S. aureus, MIC 3 lM) and Gram
(E. coli, MIC 0.8 lM) bacteria [206]. Indolicidin acts by displacing divalent cations from their binding sites on the surface of the cell membrane, causing cell permeabilization [205].

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However, this AMP also has the ability to interact with the cell membrane and on the DNA inside the cell [205,207]. Indolicidin not only forms pores in the membrane [205], but can also inhibit DNA processing enzymes (Fig. 1) [207,208]; this eventually leads to bacterial filamentation, as the DNA synthesis is inhibited [209]. This DNA interaction involves the cationic residues of Indolicidin, two terminal arginines and an internal lysine residue [208]. Seven different Cathelicidin-4 varieties (buCATHL4 A–G) have been recently identified in water buffalo (Bubalus bubalis), of which the arginine-rich buCATHL4C (18 aa, Table 4) displays high activity against S. aureus (MIC 0.2–0.4 lM) and B. cereus (MIC 0.8 lM), although it is less effective against E. coli (MIC 12.5 lM) [206]. Protegrins are a family (PG1–PG5) of small cationic cysteine-rich Cathelicidins (16–18 aa) isolated from porcine leukocytes [210]. Their secondary structure is characterized by an amphipathic bsheet comprising two antiparallel strands stabilized by two intermolecular disulfide bridges [92]. Protegrin-1 (PG1, Table 4) is active against E. coli, P. aeruginosa, E. faecalis, and S. aureus (including MRSA strains) [211]. Protegrin-1 can act synergically with other mammalian AMPs, such as Indolicidin and Bactenecins [212]. Bactenecins are short cyclic, arginine-rich, Cathelicidins present in bovine, ovine and caprine neutrophil granules [213]. These compounds are more effective against Gram
(E. coli, P. aeruginosa, S. enterica subsp. enterica serovar Typhimurium, Burkholderia pseudomallei, and K. pneumoniae) than Gram+ (S. aureus) bacteria; they are also strongly cytotoxic to rat embryonic neurons, fetal rat astrocytes and human glioblastoma cells [214–216]. These AMPs act by permeabilizing the cell membrane and inhibiting protein and RNA synthesis in bacteria [217]. Scocchi and coworkers were the first to describe equine Cathelicidins (eCATH-1, eCATH-2, eCATH-3) [218]. One of the compounds with the highest antibacterial activity is eCATH-1 (Table 4), capable of inhibiting E. coli (MIC 1 lM), Serratia marcescens (MIC 4 lM), S. enterica (MIC 1.9 lM), P. aeruginosa (MIC 4 lM), K. pneumoniae (MIC 1–1.9 lM), S. epidermidis (MIC 1.9 lM), and B. megaterium (MIC 4 lM) [219]. This AMP has been successfully used in animal models for the treatment of Rhodococcus equi infections (6.4 lM), with no significant toxicity observed [220]. 9.2. Mammalian defensins Defensins are a family of AMPs that form part of the immune system of vertebrates [221]; they have six cysteine residues and are divided into 3 subfamilies, a-Defensins, b-Defensins and hDefensins, according to the alignment of their disulfide bridges [222,223]. 9.2.1. Mammalian a-Defensins a-Defensins (29–35 aa) are cationic AMPs with a three-stranded antiparallel b sheet structure, produced by promyelocytes, the neutrophil precursor cells [224], and in the intestinal Paneth’s cells after bacterial infection [225]. These AMPs play an important role in the non-oxidative killing of microorganism after phagocytosis [201]. a-Defensins antibacterial effect was initially described (1966) in neutrophil granulocytes of guinea pigs (Cavia porcellus) as part of a mixture of ‘‘lysosomal cationic proteins” [226]. Guinea pig neutrophil peptides (GPNP) were subsequently purified by Lehrer and collaborators (1975) as part of the nonoxidative fungicidal mechanisms of mammalian granulocytes [224]. GPNPs were later sequenced and identified as GNCP-1 (31 aa) and GNCP-2 (32 aa) (Table 4) [227], both active against S. aureus (MIC  3 lM) and E. coli (MIC  5 lM). Related a-Defensins have also been described in rabbit (NP-1, NP-2, NP3a, NP-3b, NP-4, and NP-5) [228], rat (RatNP-1, RatNP3 and RatNP4) [229] and hamster (HaNP-1 to 4) [230]. In humans, a-Defensins (HNP1–4) are also primarily found in neutrophils, representing between 5 and 7% of

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their total protein content [231]. HNP-1 (Fig. 3, Table 4) is one of the most studied human a-Defensins; it is active against Gram+ and Gram
bacteria, in particular against S. aureus (MIC 0.6 lM), S. epidermis (MIC 1.5 lM), B. subtilis (MIC 1.9 lM), and E. coli (MIC 0.5 lM) [201]. Despite the fact that HNP-1 is a cationic AMP, the cysteine bridges (Cys1–Cys5, Cys2–Cys4 and Cys3– Cys6) are essential for its activity; in fact, the MIC increases by tenfold when using the same compounds lacking the disulfide bonds [232]. More cationic a-Defensins, such as NP-1 or RatNP-1, are five to ten times more active in vitro than HNP-1 [233]. The microbicide effect of HNP-1 occurs in several steps [234]; the compound initially forms dimers in solution, then the dimers electrostatically interact with the cell membrane, and later the dimers oligomerize to form transmembrane pores 20–25 Å in diameter (Fig. 1). These pores increase the membrane permeability, resulting in leakage of cellular content [234]; in addition HNP-1 has been described to inhibit DNA and protein synthesis [24]. While neutrophil a-Defensins are found in the cytoplasmic pagolysosome, Paneth cell a-Defensins are secreted extracellularly, with a local concentration between 25 and 100 mg/ml at the point of release [235]. Cryptdins were initially isolated from mouse [Cryptdins (1–6)] [236], but they are also found in rat [Cryptdins (1–6), Cryptdin-1 sequence is depicted in Table 4] [237], horse (DEFA1) [238], rhesus macaque (RED-1 to RED-6) [239], and humans (HD-5 and HD-6, Table 4) [231]. Paneth cell a-Defensins are secreted in response to the presence of bacteria or their antigens [225,235] and are active against Gram+ and Gram
bacteria. It has been suggested that the level of these compounds can affect the composition of the enteric microbiome [235]. 9.2.2. Mammalian b-Defensins Mammalian b-Defensins are AMPs closely related to aDefensins, and both have a similar tridimensional structure, but the separation of the cysteine residues and the distribution of the disulfide bridges are different [222,223,231]. The fact that the older vertebrate groups, such as fish, reptiles and birds have b-Defensins but not a-Defensins (Fig. 3) indicates that both a- and b-families have evolved from a common ancestral b-Defensin gene [178]. As is the case for a-Defensins, b-Defensins have also been isolated from neutrophils and other leukocytes; however, the latter are mainly produced by nongranular mucosal epithelial cells in the respiratory, gastrointestinal, and genitourinary tracts [240]. bDefensins are an important part of the innate immune system and have so far been found in every mammalian species studied [199]. Patil and coworkers [240] published a comprehensive study of the complete repertoires of b-Defensins in humans, chimpanzees, mice, rats, and dogs following systemic, genome-wide computational tools analyses. b-Defensins were firstly isolated in 1991 from bovine mucosal epithelial cells [241]. The tracheal antimicrobial peptide (TAP, Table 4) is active against Gram+ (S. aureus, MIC 6–12 lM) and Gram
bacteria (E. coli, MIC 3–6 lM; K. pneumoniae, MIC 3–6 lM; P. aeruginosa, MIC 6–12 lM; and the bovine pathogens Mannheimia haemolytica, MIC 0.8 lM; Histophilus somni, MIC 1.1 lM; and Pasteurella multocida, MIC 0.6 lM) [241,242]. Another related AMP, the lingual antimicrobial peptide (LAP, Table 4) was isolated from a swollen cattle tongue [243], but it is expressed in other tissues [244] and can be present in milk [245]. LAP is active (MIC 3.5–7.1 lM) against E. coli or Nocardia farcinica and, to a lesser extent (MIC 14–28 lM), against S. aureus and P. aeruginosa [243,246]. Bovine neutrophil b-Defensins constitute a family of AMPs (BNBD-1 to 13) active against Gram+ and Gram
bacteria [247]. As opposed to cattle, b-Defensins are mainly produced by the epithelial mucosa in other mammals. Human bDefensins (HBD-1 to 4, Table 4) are also found in epithelial cells. HBD-1 (Fig. 3) was isolated from a blood filtrate, but it is expressed by astrocytes, skin keratinocytes, cornea, mammary gland, and the

reproductive, digestive, urinary, and respiratory tract epithelial cells [231,248,249]. HBD-1 is considered the more important AMP in epithelial defense against infections and it is expressed constitutively, in contrast to HBD-2, -3 and 4, which are inducible [250]. HBD-1 is mainly active against Gram
bacteria [251]. As opposed to HNP-1 [232], HBD-1 is more active against anaerobic Gram+ commensal bacteria (Bifidobacterium and Lactobacillus species) after its disulfide bonds are reduced [252]. This is a quite interesting fact since, although HBD-1 is ubiquitously expressed, it is considered one of the weaker HBD-family compounds against aerobic bacteria [253]. Schroeder and coworkers proposed that HBD-1 and Thioredoxin are constitutively expressed in mammals to provide a mechanism of resistance against opportunist anaerobic pathogenic or commensal bacteria [252]. HBD-2 (41 aa, Table 4) is an AMP homologous to HBD-1 and TAB, which is expressed after inflammation in keratinocytes (in fact, it was originally isolated from psoriasis skin lesions [254]) and other epithelial cells (such as those in lung, gut, trachea, small intestine, urogenital system, pancreas, and adenoid), as well as leukocytes and in bone marrow [201,231,255]. HBD-2 is mainly active against Gram
bacteria (E. coli, LD90 2.3 lM, MIC 14.5 lM; P. aeruginosa, LD90 2.3 lM, MIC 14.5 lM; and E. faecalis, MIC 3.5 lM) [249,256], however, it is also active against some oral Gram+ bacteria, such as S. mutans (MIC 0.6–1.2 lM) and Actinomyces naeslundii (MIC 1–1.6 lM) [257]. HBD-3 (Table 4) [251] is considered the most active member of the human b-Defensins, mainly because it is salt-insensitive [251,258] and can kill bacteria in physiological conditions [259]. This AMP has a broad spectrum of action and can kill a variety of microorganisms, such as S. aureus (MIC 0.6 lM), S. mutans (MIC 0.5–1 lM), A. naeslundii (MIC 0.8–1.4 lM), E. coli (MIC 1 lM), and P. aeruginosa (MIC 1 lM) [257,259]. HBD-3 displays synergistic action with LL-37 [260] and classical antibiotics [261]. HBD-4 (Table 4) is expressed in testis, gastric antrum, by neutrophils, in thyroid gland, lung, and kidney. The activity of this AMP is lower than that of the other HBD members, however, it is highly active against Streptomyces carnosus (MIC 1 lM) and P. aeruginosa (MIC 0.9 lM) [262]. As is the case for HBD-2, HBD-4 analogs with only one disulfide bond displayed a minimum of a twofold increase in antimicrobial activity against E. coli, P. aeruginosa and S. aureus [263]. Although there have been significant advances in the field, according to computational genomics there are still 25 additional HBD not yet identified in humans [264]. 9.2.3. h-Defensins h-Defensins are a family of AMPs found in leukocytes and bone marrow in some non-human primates. They possess a unique macrocyclic backbone [265], and constitute the only known cyclic protein found in animals [266]. h-Defensins are formed by ligation of two truncated a-Defensins (Demidefensins, 9 aa) from an aDefensin-related precursor. The rhesus macaque (Macaca mulatta) produces three h-Defensins (RTD-1 to 3, Table 4) (RTD-1 is structure depicted in Fig. 3), isolated from leukocytes [267,268] with relative cellular abundances of 29:1:2 (RTD-1:RTD-2:RTD-3, respectively) [268], and from bone narrow (RTD-2 and RTD-3) [269]. Those cyclicAMPs are resistant to exoprotease activity and their bactericide activities are both salt- and serum-independent, as opposed to non-circular a-Defensins [225]. Interestingly, the linear analogs of the cyclicAMPs are indeed salt-sensitive [267]. RTDs are highly active against S. aureus (RTD-1 and RTD-3, MIC 1 lM; RTD-2, MIC 0.5 lM), E. coli (RTD-1 and RTD-3, MIC 0.5 lM; RTD-2, MIC 1.9 lM) and L. monocytogenes (RTD-1, MIC 1.2 lM) [267,268]. 9.3. AMPs from platelets, liver and eccrine sweat glands Platelets, although less studied than other blood cells (neutrophils, eosinophils and macrophages), have granules containing

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cationic proteins and peptides with antimicrobial activity [270]. Platelet microbicidal proteins (PMPs) are thermostable AMPs active against Gram+ and Gram
bacteria [271]. One such example is the thrombin-induced PMPs (tPMP-1) that include the human Thrombocidins TC-1 (68 aa) and TC-2 (83 aa) (Table 4), which are very effective against B. subtilis (MBC 0.4 lM and 0.7 lM, respectively) [272]. Rabbit (PMP1 to 5, and tPMP1 and 2) [273], cattle and swine [271] PMPs display significant antibacterial activity against E. coli, B. subtilis and S. aureus. Hepcidins (20–25 aa) are cysteine-rich peptides initially isolated from humans; they are found in plasma [274] and urine [275], but they are produced in the liver. Human Hepcidin 20 (Table 4) has four disulfide bridges (Cys2–Cys18, Cys5–Cys17, Cys6–Cys14 and Cys8–Cys9) that form a b-sheet structure, and displays a moderate antimicrobial activity against E. coli, with a MIC 30 lM [275]. Hepticin 25 (aka Liver expressed peptide-1; LEAP-1, Table 4) also displays a moderate activity against M. luteus (MIC 1.8 lM) [274]. Hepticins main role is iron-regulation in blood [14]. Another LEAP, LEAP-2 (40 aa, Table 4), is expressed after bacterial invasion of epithelial cells; this AMP is active against several Gram+ bacteria, especially B. megaterium (MIC 12.5 lM) [276]. Dermcidin (47 aa, Table 4) is an anionic AMP constitutively expressed in sweat glands, but it is also present in neutrophil granules and other human and primate cell types [7,277,278]. This AMP is proteolytically processed from an inactive precursor and has a broad-range activity in a salt- and pH-independent manner. The MIC of Dermcidin for E. coli, E. faecalis and S. aureus is 0.2 lM, while its concentration in sweat was found to be 0.2–2.1 lM. These results indicate that Dermcidin plays a role in the regulation of human skin microbiota [278]. 10. Synthetic AMPs In general terms, cationic peptides are difficult to produce by recombinant expression, since they are toxic to cells. This is why most of the AMPs reported above are produced by chemical synthesis; however, some AMPs, such as Cathelicidins and Defensins, can be expressed in bacteria [279] as part of fusion proteins, tandem multimers, or in inclusion bodies [58,122,123,125,126,149, 181,234,238,239,251,258]. This section will provide a summary of AMPs that are not only not naturally produced by organisms, but there are not even encoded in their genomes. Classical synthetic modifications of AMPs include amidation of their carboxyl end, acetylation of their amino-terminus, modification of the amino acid sequence, and the use of unnatural or D-amino acids [9,24]. 10.1. Synthetic hybrid AMPs At the end of the eighties and beginning of the nineties, Boman and Merrifield reported pioneer studies on the synthesis of Cecropin A and Melittin A hybrids [9,280–282]. Since that time, synthetic hybrid peptides based on natural AMPs have been created to increase the activity of the compounds, to change the targets of the AMPs, or to decrease their cytotoxicity. CAMEL0 (15 aa, Table 4) is a Cecropin A (1–7)-Melittin A (2–9) hybrid, that constitutes one of several natural AMP analogs synthesized to target anaerobic bacteria, with MIC ranging from 0.6 to 4.6 lM. It was found that the analogs containing D-amino acids were more stable than their L-amino acid-containing counterparts [283]. BP100 (11 aa, Table 4) is another synthetic Cecropin A-Melittin hybrid AMP, produced by combinatorial chemistry [284], that can to kill Gram
bacteria (MIC 0.7–8 lM) and has moderate cytotoxicity [285,286]. This AMP makes the cell membrane permeable and produces membrane depolarization [287]. Several BP100 analogs have been synthetized with the aim of increasing the activity and the

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spectra of action of this compound; two such examples are RBP100 and RW-BP100 [286], where lysines were substituted by arginines. This modification resulted in the analogs acting on both Gram+ and Gram
bacteria. One of the analogs, RW-BP100 (Table 4), displays a significantly improved activity on Gram+ (MIC 0.3–1 lM) and Gram
bacteria (MIC 0.1–2.5 lM), although it also causes greater cytotoxicity [286]. Several synthetic hybrid AMPs of Indolicidin and Ranalexin were recently reported [288]. Among them RN7-IN6 (13 aa, Table 4) displays the highest antibacterial activity (S. pneumoniae, MIC 4.6–9.1 lM; E. coli, MIC 4.6 lM; S. aureus and MRSA, MIC 4.6 lM); it is approximately 4 times more potent than Indolicidin and 8-fold stronger than Ranalexin. P-Der (26 aa, Table 4) is an amidated hybrid of pleurocidin and dermaseptin that can inhibit E. coli macromolecular synthesis at sub-lethal concentrations (MIC 0.7 lM). P-Der does not produce membrane permeation, even at concentrations 5 times above its MIC, but it can inhibit DNA and RNA synthesis within 20 and 5 min after treatment, respectively. This AMP enters into the cell by translocation, using a flip-flop mechanism (Fig. 1) [128] different from that proposed for the parental AMP dermaseptin, which produces membrane depolarization following the ‘‘carpet-like” model (Fig. 1) [166]. Additionally, pleurocidin requires a concentration 10-fold above its MIC to produce inhibition of RNA synthesis [128]. 10.2. Synthetic analogs of natural AMPs Pexiganan (MSI-78, 22 aa, Table 4) is a synthetic analog of Magainin II with a potent and broad spectrum of action; it kills bacteria by forming toroidal pores in their cell membranes (Fig. 1) [289]. This AMP was found to be active in 3108 clinical bacterial isolates (2692 aerobes and 416 anaerobes), with a MIC ranging from 0.8 to 6.5 lM in 87% of the tested bacterial isolates. These clinical isolates included Gram+ and Gram
bacteria resistant to several antibiotics, such as oxacillin, cefazolin, cefoxitin, imipenem, ofloxacin, ciprofloxacin, gentamicin, and clindamycin. In addition, bacterial susceptibility to Pexiganan appeared to be the same for either antibiotic-sensitive or resistant bacterial strains, and no resistance to the compound was detected. The clinical pathogen A. baumannii is one of the bacteria with the highest sensitivity to this AMP, with a MIC ranging from 0.4 to 3.2 lM, while E. faecalis and S. sanguis are the more resistant to it, requiring a MIC above 6.5 lM [290]. Pexiganan is one of the most studied AMPs and has undergone clinical trials, however, in 1999 FDA initially denied its approval for infected diabetic foot ulcers treatment after phase III [289]. Additional trials with placebo arm were reported in 2008, results showed that Pexiganan cream might be an effective alternative to oral antibiotic therapy in treating diabetic patient, reducing the risk of selecting antimicrobialresistant bacteria [291]. Histatins (1, 3 and 5) are cationic histidine-rich AMPs expressed in the salivary glands of humans and other primates [201]. Natural Histatins have little antibacterial activity, acting mainly as antifungals. Dhvar1 and Dhvar2 (Table 4) are two multi-site substituted analogs of Histatin 5 with a broad spectrum of action, which can inhibit the growth of oral pathogens such as Prevotella intermedia, S. mutans and S. sanguis [292]. Dhvar4, Dhvar5 (Table 4) and their dimmers are also Histatin 5 analogs that are active, both in vivo and in vitro, against MRSA. Dimeric Dhvar4 and Dhvar5 have an in vitro lethal concentration 50 (LC50, concentration required to reduce viable counts by 50% compared to incubations without AMP) of 0.4 lM against MRSA [293]. FL9 (an analog of Fallaxin, Table 4) [294] can also affect MRSA (MIC 24.7–49.4 lM) [295]; it binds to DNA and induces the SOS response. Interestingly, FL9 mechanism of action depends on the compound concentration; at concentrations below its MIC it mainly targets DNA, while at

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higher concentrations it forms pores and disrupts the cellular membrane resulting in cell death (Fig. 1) [295]. WMR-NH2 (13 aa, Table 4) is an analog of the fish AMP Myximidin but it displays stronger activity against a variety of microorganisms (E. coli, MIC 2 lM; P. aeruginosa, MIC 2 lM; S. enterica subsp. enterica serovar Typhimurium, MIC 1.2 lM; K. pneumonia, MIC 3 lM; and S. aureus, MIC 2.2 lM). Cantisani et al. [296] analyzed key residues in Myximidin by alanine scanning, and were able to identify and modify critical amino acids to design novel peptides with increased activity. Lee and Hodges [297] tested 29 synthetic analogs based on Gramicidin S [cyclo(VOrnLDFP)2] and found the peptide GS14K4 (Table 4) to be the most effective. This AMP has low hemolytic activity and can inhibit the growth of several Gram+ (S. epidermis, MIC 1.2 lM; E. faecalis, MIC 0.9 lM; Corynebacterium xerosis, MIC 1 lM) and Gram
(E. coli, MIC 1.8–3.7 lM; S. enterica subsp. enterica serovar Typhimurium, MIC 2.4–6.5 lM; P. aeruginosa, MIC 1.8– 14.8 lM) bacteria [297]. 10.3. Synthetic AMPs not based in natural sequences Synthetic cationic amphipathic AMPs, such as V-Peptides (V peptide 4 sequence is depicted in Table 4), were designed to improve the AMP attachment to lipopolysaccharide and Lipid A, present in Gram
bacterial membranes, and have a b-sheet structure stabilized by a disulfide bridge (Cys1–Cys19). These synthetic peptides display a potent activity (MIC 0.004–0.692 lM) against the Gram
bacteria. V peptides are 208 fold more active than the polymyxin B used as control [298]. Deslouches and collaborators designed cationic amphipathic [containing the (Arginine)2(Valine)2 motive] AMPs with an a-helix structure and studied the effect of AMP length, as well as amino acid substitutions, on their activity (Tryptophan). The authors found that, while the activity of the compounds did not increase when AMPs longer than 24 residues were employed, tryptophan substitution made the compounds less sensitive to high-salt concentration. Of the peptides tested, WLBU2 (3 Tryptophan substitutions, 24 aa, Table 4) had the lower MIC (0.3 lM) for P. aeruginosa and S. aureus, and this was independent from the salt concentration or the bacterial strain analyzed [299]. Romani and coauthors described the activity of novel in silico-developed antimicrobial compounds against a panel of bacterial pathogens, using ad hoc screening software. The peptides span 17 amino acids forming a two-stranded antiparallel bsheet, stabilized by three to six interstrand hydrogen bonds and one disulfide bond. AMP72, AMP126 and AMP2041 (Table 4) are effective against Gram+ (MBC 0.94–20.65 lM) and Gram
(MBC 0.17–10.12 lM) bacteria, with low hemolytic and cytotoxic activities [300]. Highly active ultra-short antimicrobial lipopeptides have been produced using solid-phase peptide synthesis. These AMPs consist of four L and D amino acids with the sequence KXXK (where X represents either L, A, G, K, or E), with a lipophilic amino acid at the Nterminus [301]. C16-KGGDK (Table 4) is the most potent of the assayed lipo-AMPs, with MIC values, against both Gram+ (S. aureus, MIC 6.25 lM) and Gram
(E. coli, MIC 3 lM; P. aeruginosa, MIC 6.25 lM; A. baumannii, MIC 12.5 lM) bacteria, in the lM range [301]. 11. Determination of the inhibitory and bactericidal activity of AMPs This review summarizes the published inhibitory and/or bactericidal concentrations for different AMPs, but one must be aware that different approaches were employed for determining those values; hence a direct comparison between those figures is very problematic. Most of the studies consider the MIC as the AMP

concentration at which no bacterial growth is detected. MIC determination is usually achieved by serial microdilution assays in multiwell plates, analyzing multiple conditions and many bacterial strains in a reproducible manner. Nevertheless, the MIC value is influenced by the initial concentration of microorganisms (inoculum) and the assay cannot discriminate between bactericidal and bacteriostatic properties. Minimal bactericidal concentration (MBC) or minimal lethal concentration (MLC) are usually determined by subculturing the bacteria used in the MIC assay on liquid media or agar plates without the AMP, and are defined at the concentration that reduces the viability of the initial bacterial inoculum by P99%. The agar dilution method allows directly determining the MBC/MLC value, but the amount of AMP is significantly higher than that used by other approaches. Parameters such as LC50 (lethal concentration 50, the AMP concentration required to reduce viable bacterial counts by 50%, as compared to bacterial counts obtained without AMP) or the half maximal effective concentration (EC50) are also determined by linear regression studies at different AMP concentrations. Lethal concentrations are often calculated by measuring inhibition zone diameters, where different concentrations of AMP are tested in disks or wells. Other methods to determine bacterial viability include alamarBlue dye [182,220,221], LIVE/DEADÒ BacLightTM [220], and acridine orange accumulation [280]. The inoculum used in the studies is usually between 104 and 107 colony-forming units (CFU)/ml, but the methodology varies significantly between different studies, and those differences are not necessarily reported. In addition, a wide variety of bacterial strains are used to test the antibacterial activity of AMPs. Usually collection type bacterial specimens are used, but in occasion these are replaced by clinical or environmental microbial isolates that are relevant for the organisms producing the AMPs. Another difference between publications is the pH and salt concentrations used in the culture media, even when growing the same microorganism. In order to standardize the quantitative susceptibility testing data between laboratories, some authors follow either the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS, http://clsi.org/) [58,122,161, 163,174,176,184,202,211,219,283,290] or the Hancock Laboratory Methods (Department of Microbiology and Immunology, University of British Columbia, British Columbia, Canada. ‘‘http://www.cmdr. ubc.ca/bobh/methods.htm”) [129,294,295]. Acknowledgment This work was supported by the Spanish Ministry of Economy and Competitiveness, under grant AGL2013-48244-R. References [1] G. Wang, X. Li, Z. Wang, APD3: the antimicrobial peptide database as a tool for research and education, Nucleic Acids Res. 44 (2015) D1087–D1093, http:// dx.doi.org/10.1093/nar/gkv1278. [2] F.H. Waghu, R.S. Barai, P. Gurung, S. Idicula-Thomas, CAMP R3: a database on sequences, structures and signatures of antimicrobial peptides, Nucleic Acids Res. 44 (2015) gkv1051, http://dx.doi.org/10.1093/nar/gkv1051. [3] X. Zhao, H. Wu, H. Lu, G. Li, Q. Huang, LAMP: a database linking antimicrobial peptides, PLoS One 8 (2013) e66557, http://dx.doi.org/10.1371/journal. pone.0066557. [4] H.G. Boman, Peptide antibiotics and their role in innate immunity, Annu. Rev. Immunol. 13 (1995) 61–92, http://dx.doi.org/10.1146/annurev. iy.13.040195.000425. [5] R.M. Epand, H.J. Vogel, Diversity of antimicrobial peptides and their mechanisms of action, Biochim. Biophys. Acta 1462 (1999) 11–28, http://dx. doi.org/10.1016/S0005-2736(99)00198-4. [6] R.E. Hancock, Cationic peptides: effectors in innate immunity and novel antimicrobials, Lancet Infect. Dis. 1 (2001) 156–164, http://dx.doi.org/ 10.1016/S1473-3099(01)00092-5. [7] Y. Lai, A.E. Villaruz, M. Li, D.J. Cha, D.E. Sturdevant, M. Otto, The human anionic antimicrobial peptide dermcidin induces proteolytic defence mechanisms in staphylococci, Mol. Microbiol. 63 (2007) 497–506, http://dx. doi.org/10.1111/j.1365-2958.2006.05540.x.

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