Antimicrobial Peptides Isolation from Anemonia Sulcata (Cnidaria)

ISJ 11: 182-191, 2014 ISSN 1824-307X RESEARCH REPORT First evidence of antimicrobial activity of neurotoxin 2 from Anemonia sulcata (Cnidaria) MR Trapani1,2, MG Parisi1, M Toubiana2, L Coquet3, T Jouenne3, P Roch2, M Cammarata1 1Marine Immunobiology Laboratory, Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo, Italy 2Ecologie des Systèmes Marins Côtiers, CNRS-Université Montpellier 2, place E. Bataillon, Montpellier, France 3Plateforme de Protéomique PISSARO, CNRS-Université de Rouen, Place E. Blondel, Mont-Saint-Aignan, France Accepted June 6, 2014 Abstract We investigated the antibacterial activity of Anemonia sulcata (Cnidaria, Anthozoa) tentacle and body acidic extracts. Biochemical purification consisted of first step on solid phase Sep-Pak C8 column followed by several HPLC runs on C18 column using different conditions. Anti-Micrococcus lysodeikticus activity has been detected in 40 % acetonitrile fractions. The resulting purified molecule from tentacles had a molecular mass determined by MALDI-TOF mass spectrum of 4946,299 Da and has been completely sequenced. Its aa sequence revealed identity with the Neurotoxin 2 (ATX-II), a Na+ channel blocking toxins. Consequently, ATX-II appeared to display a dual role as toxin and as antibacterial. Key Words: antimicrobial peptide; Anemonia sulcata; ATX II; neurotoxin; Micrococcus lysodeikticus Introduction The sea anemone Anemonia sulcata is a 2002). There is evidence that antimicrobial peptides widespread Mediterranean species. Sea anemones are widespread in invertebrates (Chisholm and are generally poisonous animals that spend most of Smith, 1992), especially in tissues such as the gut their lives in a sessile form. Capturing activities and and respiratory organs in marine invertebrates, defense mechanisms are strongly associated with subjected to a first exposure to pathogens. In spite toxin production (Ruppert and Barnes, 1994). The of variations in structure and size, most of neurotoxin ATX II is a type 1 sodium channel toxin antimicrobial peptides are amphiphilic, displaying and consists of 47 amino acid residues, linked by both hydrophilic and hydrophobic surfaces (Tincu et three disulfide bridges. ATX II specifically binds to al., 2004). AMP are exciting candidates as new the sodium channel (site 3), thus delaying its antibacterial agents due to their broad antimicrobial inactivation during signal transduction; has a strong spectra, highly selective toxicities, and the difficulty effect on crustaceans and insects and a weaker for bacteria to develop resistance to these peptides effect on mammals. These toxins specific for sodium (Boman, 1998; Lehrer and Ganz, 1999; Hancock et channels have been thoroughly investigated also al., 2000). Therefore, AMPs from marine organisms because they constitute a major fraction of the represent a largely unexploited resource that can venom (Moran et al., 2009). afford design of new antibiotics with broad-spectrum Antimicrobial peptides are important defense antimicrobial activity (Ovchinnikova et al., 2006). It is molecules in marine invertebrates covering a broad- plausible that certain functional molecular motifs, spectrum of bacteria and fungi (Boman, 2003; Bulet such as amphipathic α-helices and β-sheets, would et al., 2004). They are defined as molecules of less have featured predominantly in the arsenal than 10 kDa characterized by immediate and rapid employed by our distant eukaryotic ancestors. Over response to invading microorganisms (Bartlett et al., time, these would have diversified through mutation ___________________________________________________________________________ and selection pressure to produce the great variety of AMPs and other molecules (Smith et al., 2010). Corresponding author: Several AMPs have been isolated from phylum Matteo Cammarata of Cnidaria. Have been reported the purification of a Marine Immunobiology Laboratory Department of Biological, Chemical 40-residue AMP from the mesoglea of a scyphozoid Pharmaceutical Science and Technology jellyfish, Aurelia aurita (Ovchinnikova et al., 2006). University of Palermo, Palermo, Italy The peptide was named aurelin and exhibited E-mail: [email protected] activity against gram-positive and gram-negative 182 Fig. 1 Tentacles and body acid extracts of Anemonia sulcata were loaded onto solid phase column (Sep-Pak C8). The resulting 10, 40 and 80 % acetonitrile fractions (10 μl) were tested for the antibacterial activity toward Micrococcus lysodeikticus by filling wells performed in Petri dishes containing trypticase soy agar (A). The 40 % active fraction have been also assayed at different dilution (B). bacteria. Furthermore, a detailed biochemical Antibacterial assays characterization of the antimicrobial activity in Hydra Solid phase elution fractions have been freeze- revealed the presence of conserved AMPs such as dried and suspended in 50 μl sterile ultra pure water Hydramacin-1 and novel taxon-specific AMPs such (UPW). Antibacterial activity has been tested against as Arminin 1a and Periculin-1 which have no M. lysodeikticus by filling wells performed in Petri counterpart in the transcriptomes of any other dishes containing 10 ml of trypticase soy agar (TSA) organisms (Bosch et al., 2009). It could be possible and 3 ml of 0.5 McFarland turbidity. Each well to obtain, from this molecule, synthetic peptides with received 10 μl of sample and Petri dishes were significant antibacterial activity and relatively low incubated overnight at 37 °C. Diameter of clear ring cytotoxicity to human erythrocytes, as in the case of around the wells paralleled the antibacterial activity. stycholysin I and II (Tejuca et al., 1996), 2 pore- Liquid growth inhibition assay has been used to forming cytolysins from the sea anemone check for antibacterial activity in all HPLC fractions. Stychodactyla helianthus. Briefly, fractions were freeze-dried and suspended in In this paper we reported i) the presence of 50 μl UPW. Twenty μl aliquots were added to 20 μl of anti-Micrococcus lysodeikticus activity in acidic a suspension of M. lysodeikticus (5x106 bacteria/ml) extracts from tentacles and body of A. sulcata; ii) in Poor-Broth nutrient medium (1 % Bactotrypton, 0.5 the biochemical purification of the active peptide, % NaCl, w/v) in 96 wells microtiter plates. Bacterial and iii) its complete aa sequence revealing identity growth was measured at A600 with a microplate with Neurotoxin 2 (ATX II). reader Tecan (Infinite 200 M) during 16 h at 37°C. Minimal inhibitory concentration (MIC) was obtained Material and Methods by testing serial doubling dilutions of selected purified peptide in liquid growth inhibition assay as Animals and tissues sampling above and corresponded to the lowest concentration Anemonia sulcata (Anthozoa) adults have been that caused 100 % growth inhibition. Minimal collected from Termini Imerese (Palermo, Italy) and bactericidal concentration (MBC) was determined by maintained in oxygenated sea water at 18 °C. Acid plating the contents of the first 5 wells with no visible extractions were performed from tentacles and body. growth of bacteria onto TSA Petri dishes. The lowest Briefly, tentacle were separated from the animal concentration that prevented any colony formation body with a forceps and both suspended in Tris unit (CFU) as observed after 18 h incubation at 37 buffered solution (TBS, 150 mM NaCl, 10 mM Tris- °C corresponded to the MBC. HCl, pH 7.4). After homogenization in ice bath with Ultra-Turrax for 5 min, 10 % acetic acid was carefully Solid phase extraction and reversed phase HPLC added. The 2 samples have been sonicated purification (Branson Model B15, Danbury, CT) 3 times for 30 Tentacles and body acid extracts were loaded sec each, then centrifuged at 21,000xg, for 20 min at onto Sep-Pak C8 Vac cartridges (Waters 4 °C. Protein content of both supernatants was Associates) equilibrated with acidified water (0.05 % determined using the NanoDrop ND-1000 TFA in UPW). After washing with acidified water, spectrophotometer. three successive elutions were performed 183 Fig. 2 Tentacles and body acid extracts of Anemonia sulcata were loaded onto solid phase column (Sep-Pak C8). Minimum inhibition concentration of the 40 % active acetonitrile fraction was assayed toward Micrococcus lysodeikticus obtained after incubation in the microplate reader TECAN for 16 h at 37 °C. The data obtained show an antibacterial activity at concentrations up to 0.244 mg/ml. successively with 50 ml of 10, 40 and 80 % Mass spectrometry acetonitrile in acidified water. Fractions were freeze- The molecular mass of the active peak content dried and reconstituted with UPW. As the bulk of the was determined by matrix-assisted laser antimicrobial activity was detected in the 40 % desorption/ionization time-of-flight mass fraction, only this fraction was submitted to reversed spectrometry with an Ultraflex TOF/TOF (Bruker phase HPLC on a silica column C18 Interchrom Daltonics). The 2,5-dihydroxybenzoic acid (DHB) UP5ODB-25QS (250x4.6 mm). Elution of the 50 μl matrix was prepared in Acetonitrile/Ultrapure sample was performed with a linear gradient of 0 - Water/TFA (20/80/0.1 %) for a final concentration of 60 % acetonitrile in acidified water over 60 min at a 5 mg/mL. One µL of sample dissolved in 0.1 % TFA flow rate of 0.5 ml/min. Fractions corresponding to was mixed with 1 μL of the matrix solution on the absorbance peaks detected at both 280 and 225 nm steel target and dry in ambient air. The calibration were collected

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