Paracentrin 1, a synthetic antimicrobial peptide from the sea-urchin Paracentrotus lividus, interferes with staphylococcal and Pseudomonas aeruginosa biofilm formation
© Schillaci et al.; licensee Springer. 2014
Received: 13 June 2014
Accepted: 5 October 2014
Published: 31 October 2014
The rise of antibiotic-resistance as well as the reduction of investments by pharmaceutical companies in the development of new antibiotics have stimulated the investigation for alternative strategies to conventional antibiotics. Many antimicrobial peptides show a high specificity for prokaryotes and a low toxicity for eukaryotic cells and, due to their mode of action the development of resistance is considered unlikely. We recently characterized an antimicrobial peptide that was called Paracentrin 1 from the 5-kDa peptide fraction from the coelomocyte cytosol of the Paracentrotus lividus. In this study, the chemically synthesized Paracentrin 1, was tested for its antimicrobial and antibiofilm properties against reference strains of Gram positive and Gram negative. The Paracentrin 1 was active against planktonic form of staphylococcal strains (reference and isolates) and Pseudomonas aeruginosa ATCC 15442 at concentrations ranging from 12.5 to 6.2 mg/ml. The Paracentrin 1 was able to inhibit biofilm formation of staphylococcal and Pseudomonas aeruginosa strains at concentrations ranging from 3.1 to 0.75 mg/ml. We consider the tested peptide as a good starting molecule for novel synthetic derivatives with improved pharmaceutical potential.
KeywordsAMP (Antimicrobial peptides) Biofilm Staphylococci Pseudomonas aeruginosa Paracentrotus lividus
Many natural antimicrobial peptides show a high specificity for prokaryotes and a low toxicity for eukaryotic cells, and for their mode of action the development of resistance by pathogenic bacteria is considered unlikely (Hancock and Rozek ). At present, there has been an increase of interest in these molecules as potential new antimicrobials (Bax et al. ; Mor ).
In human medicine, chronic and persistent forms of some infectious diseases depend on the ability of pathogenic bacteria to develop bacterial communities called biofilms. Opportunistic pathogens, such as staphylococcal strains and Pseudomonas aeruginosa show a great ability to produce biofilms that preventing infected wounds to heal, render the treatment extremely challenging. In veterinary medicine, biofilms are believed to be involved in many diseases such as pneumonia, liver abscesses, enteritis, wound infections and mastitis, which is one of the most common diseases in dairy cattle (Clutterbuck et al. ). Staphylococcus aureus, a major pathogen of mastitis has good in vitro antimicrobial susceptibility, but the therapy used to treat the affected animals is often disappointing and results in chronic infections due to the growth of bacteria as biofilms (Melchior et al. ). The biofilm of P. aeruginosa is a severe complicating factor in bovine mastitis, which is often associated with contaminated udder washing water or contaminated intramammary dry-cow preparations (Melchior et al. ).
The treatment of biofilm-associated infections is complicated because microbial biofilms are typically highly resistant to conventional antibiotics (Gilbert et al. ). The discovery of anti-infective agents active not only against planktonic microorganisms but also against biofilms represents an important goal for an effective control of infections (Projan and Youngman ).
The antimicrobial defence system of marine invertebrates is an interesting source of new anti-infective agents (Arizza ). We focused, particularly, on the effector cells of the echinoderm immune system, the coelomocytes. In a recent study, the antimicrobial activity of a 5-kDa peptide fraction from coelomocyte cytosol (5-CC) of the Paracentrotus lividus, the sea-urchin from Mediterranean sea, was demonstrated in relation to a group of important human pathogens. The anti-biofilm activity of 5-CC was shown in S. epidermidis 1457, a clinical strain isolated from an infected central venous catheter, against reference staphylococcal biofilms and against Candida albicans and Candida tropicalis (Schillaci et al. ; Schillaci et al. ). We showed the presence of three principal peptides, in the 5-CC content, whose molecular weights were respectively 1251.7, 2088.1, and 2292.2. These peptides are the (9–19), (12–31), (24–41) fragments of a β-thymosin of P. lividus. We focused particularly on the smallest peptide, that we called paracentrin 1, 11 amino acids in length, because showed the common chemical-physical characteristics of an antimicrobial peptide (Wang and Wang ).
The present study was aimed at evaluating the antibacterial and anti-biofilm activity of a chemically synthesized paracentrin 1 (SP1) against a group of staphylococcal reference strains and isolates and against P. aeruginos a ATCC 15442.
Materials and methods
The SP1 was purchased from CASLO, Lyngby, Denmark utilizing the following sequence EVASFDKSKLK derived from ESI-MS analysis. The peptide was synthesized using Fmoc solid phase technology and the peptide content and purity was determined by high performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis.
Hydrophobic mean value was calculated by using the Liu-Deber hydrophobicity scale ([Liu and Deber 1998]). Secondary structure was evaluated utilizing the algorithm of Wang et al. (), present in antimicrobial peptide calculator web site (http://aps.unmc.edu/AP/prediction/prediction_main.php). The helical wheel projection was performed using the Gautier et al. () algorithm present in the HeliQuest site (http://heliquest.ipmc.cnrs.fr) with a window size of 11 residues.
The staphylococcal reference strains used were: Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, and Staphylococcus epidermidis RP62A, known for its ability to form a biofilm (Schumacher-Perdreau et al. ). Four staphylococcal isolates from the Istituto Zooprofilattico Sperimentale, Sicily (IZS) bacterial collection including strain 657 isolated from a milk sample from an individual sheep affected by mastitis, strain 688 and 700 and strain 702 isolated from bulk milk samples from different sheep flocks. The isolates were selected on blood agar plates and on Mannitol Salt Agar (Difco, Sparks, MD). The colonies were typed by API Staph strip (bio-Mérieux) and tube coagulase test was performed (Canicatti and Roch ).
Pseudomonas aeruginosa ATCC 15442, the reference strain in official tests for antibacterial evaluation in vitro (UNI EN European Standard), was also used in this study.
Minimum inhibitory concentrations (MIC)
MICs were determined by a micro-method described previously (Schillaci et al. ). Briefly, a series of solutions of SP1 was prepared with a range of concentrations from 25 to 0.07 mg/ml(obtained by two-fold serial dilution). The serial dilutions were made in Tryptic Soy Broth (TSB) (Merck) in a 96-wells plate; to each well was added 10 μl of a bacterial suspension from a 24 h culture containing ~1 × 106 CFU/ml. The plate was incubated at 37°C for 24 h; after this time, the MICs were determined by a microplate reader (ELX 800, Bio-Tek Instruments), and defined as the lowest concentration of compound whose O.D., read at 570 nm, was comparable with the negative control wells (broth only, without inoculum).
Evaluation of Biofilm formation
All the bacterial reference strains were tested for their ability to form biofilms. Briefly, bacteria were grown in Tryptic Soy Broth (TSB, Sigma) containing 2% glucose overnight at 37°C in a shaking bath and then diluted 1:200 to a suspension with optical density (OD) of about 0.040 at 570 nm corresponding to ~106 CFU/ml. Polystyrene 24-well tissue culture plates were filled with 1 ml of diluted suspension and incubated for 24-hours at 37°C. Then, the wells were washed three times with 1 ml of sterile phosphate-buffered saline (PBS) and stained with 1 ml of safranin 0.1% v/v for 1 min. The excess stain was removed by placing the plates under running tap water. Plates were dried overnight in inverted position at 37°C. Safranin stained adherent bacteria in each well were re-dissolved to homogeneity in 1 ml of 30% v/v glacial acetic acid, and the OD was read at 492 nm. Each assay was performed in triplicate and repeated at least twice.
Biofilm prevention assay
Each assay was performed in triplicate and assays were repeated at least twice.
Scanning electron microscopy
The effects of SP1 on formation of bacterial biofilm were morphologically evaluated by scanning electron microscopy (SEM). Glass slides in the bottom of a polystyrene 24-well tissue culture plates, were filled with 1 ml of a S. epidermidis RP62A suspension, obtained and diluted as previously seen, and a sub-MIC concentration of 3.1 mg/ml of SP1 was directly added at time zero and incubated at 37°C for 24 hours. After that time, the glass slides were gently washed four times with PBS to remove non-adherent bacteria and fixed with 2.5% glutaraldehyde-2% paraformaldehyde in 0.1 M cacodilate buffer (pH 7.4) for 30 min at 4°C. The bacterial preparation were washed with phosphate saline buffer PBS and post-fixed in osmium tetroxide 1% for 30 min at 4°C, followed by an ethanol dehydration series: 15 minutes in 50:50 ethanol: H2O, 15 minutes in 75:25 ethanol: H2O, 15 minutes in 95:5 ethanol: H2O, and 30 minutes in 100% ethanol than a critical point drying procedure was followed, and the preparations were mounted on aluminium stubs, and gold coated in a sputter coater. Imaging was conducted with a LEO 420 scanning electron microscope as previously reported (Arizza et al. ).
Molecular dynamics simulations
The molecular folding of the peptide in aqueous solution was investigated in silico by molecular dynamics (MD) simulations, following recently reported procedures (Lauria et al. ; Lentini et al. ). In details, a 400 ns of MD simulation was carried out at 300 K, in the explicit water solvent and in the presence of 150 mM Na+ and Cl− counterions, using the Amber99SB-ILDN force field (Lindorff-Larsen et al. ) implemented in the GROMACS 4.6.5 software package (Pronk et al. ).
Molecular Dynamics of SP1
SP1 is an 11-residue-long cationic peptide mainly enriched by residues such as lysine, with a pI of 10.72 and a net charge of +1 at pH 7.0.
Antibacterial activity of SP1
MIC values of SP1 tested against bacterial strains
S. aureus ATCC 25923
S. aureus ATCC 29213
S. aureus ATCC 6538
S. epidermidis RP62A
S. aureus 100
S. aureus 657
S. aureus 700
S. aureus 702
P. aeruginosa ATCC15442
Interference with biofilm formation
The echinoderms are considered a good source for AMPs and a variety of peptides with antimicrobial properties have been isolated from them. Antimicrobial activity has been reported in gonads of the asteroid Marthasterias glacialis (Stabili and Pagliara ), and Paracentrotus lividus contains, in the low molecular weight fraction (<5 kDa) of acid precipitate of their coelomocytes, peptides with antimicrobial activity against staphylococcal biofilms. In addition, we recently reported that immune mediators cells in the echinoderm Holothuria tubulosa is a source of novel AMPs with anti-staphylococcal biofim activity (Schillaci et al. ). Moreover, two cystein-rich AMPs, named centrocyns, have been characterized in the green sea-urchin Strongylocentrotus droebachiensis (Li et al. ).
The synthetic fragment SP1 of β-thymosin extracted from coelomocytes of the P. lividus shares structural characteristics of many antimicrobial peptides: it is a cationic peptide and possesses a significant proportion (~40%) of hydrophobic or non polar residues (Hancock and Lehrer ; Zasloff ). Although the α-helix is the most common secondary structure for the antimicrobial activity of AMPs (Mor and Nicolas ; Skerlavaj et al. ; Storici et al. ; Tossi et al. ) the in silico study suggests that the conformation assumed by SP1 (Figure 4) might be responsible for the observed antimicrobial activity. The helix structure of echinoderm AMPs shares a characteristic amphipathic structure with alternating hydrophobic and polar residues along the primary structure. According to the Shai-Matsuzaki-Huang model, the peptides bind to the membrane surface first and then with their amphipathic structure they enter into the membrane, breaking up the lipid chains and forming transient pore; this process can cause a collapse of the membrane at a critical peptide concentration (Hancock and Chapple ; Huang ; Matsuzaki ; Shai ). The SP1 peptide has a structure very different from other echinoderm AMPs (Schillaci et al. ; Schillaci et al. ). Moreover, such structure, unexpectedly, does not have an amphipathic nature with a hydrophobic face opposite to a hydrophilic one, because the polar charged residues and those hydrophobic are not arranged uniformly. The peptide contains a sequence of four hydrophobic/non polar residues which contribute to constitute a hydrophobic core, flanked at both ends by cationic and polar residues that can solubilize the peptides in aqueous solution providing a binding site for bacterial membranes. This particular conformation is different from other antimicrobial peptides, designed as transmembrane mimetic models and that spontaneously become inserted into the cell membranes (Chan et al. ; [Liu and Deber 1998]; Stark et al. ).
We found that the SP1 showed a broad antimicrobial activity against important pathogens such as S. aureus and P. aeruginosa but it acts at high concentration (12.5 or 6.2 mg/ml) against planktonic forms of these two microorganisms. Such weak activity is comparable to that reported for a different Echinoderm, Holothuria tubulosa (Schillaci et al. ) and to that of some described innate human defence protein like lactoferrin in vitro (de Andrade et al. ). MD results suggest that the observed weak activity could be due to the low stability of SP1, in fact, as showed in RMSD plot (Figure 2), the molecule is stable only in a limited time period and in this period it can present the active conformation of Figure 1. However, SP1 shows an interesting additional antimicrobial effect interfering with biofilm formation in vitro of the above cited pathogens, at lower concentrations than MIC evaluated against the planktonic forms. The prevention of biofilm formation – rather than its elimination – is the best strategy to contrast the growth, as a sessile community, of many pathogens.
We do not know the antimicrobial mechanisms of SP1, but we could speculate as for other cationic non-amphipathic microbial peptides that the positive charges carried by the peptide are essential for the membrane binding through electrostatic interaction between residues with anionic phospholipids. The peptide-membrane interaction could be responsible for membrane aggregation by peptides bridging simultaneously two membranes or for negative curvature of the membrane asymmetry that can form tubes (Lamaziere et al. ). The synthetic SP1 could act like Dermaseptin S9, a non-amphipathic antimicrobial peptides produced by the skin of the South American hylid frog, Phyllomedusa sauvagei, that contains, centrally located, a hydrophobic core that can insert the peptide in interior membrane (Lequin et al. ). Dermaseptin S9 exerts a microbicidal activity by perturbating both the membrane interface and the hydrophobic core of the bacterial membrane (Auvynet et al. ).
In a preliminary evaluation of selective toxicity, it was gratifying to note the general lack of hemolysis of rabbit erythrocytes by SP1, even at peptide concentrations up to 50 mg/ml. Such selectivity of cationic non amphipathic antimicrobial peptides for bacterial membranes may be explicable, in part, by the differences in the compositions of eukaryotic and prokaryotic membranes (Matsuzaki ; Zasloff ). The outer leaflet of mammalian cells is predominantly composed of the zwitterionic phospholipids phosphatidylcholine and sphingomyelin (Verkleij et al. ), along with cholesterol (Turner and Rouser ), while bacterial membranes contain mainly anionic phospholipids and no cholesterol (Brock ). In addition, the outer surfaces of Gram-negative bacteria contain lipopolysaccharides, while those of Gram-positive bacteria contain teichoic acid, which in both cases add to the negative charge of the bacterial surface (Brock ). The cationic nature of native antimicrobial peptides clearly contributes to their preferential recognition by the negatively charged outer surfaces of bacterial membranes (Oren and Shai ; Shai ).
The extensive clinical use of classical antibiotics has led to the growing emergence of many medically relevant resistant strains of pathogens (Patel ). Therefore, the development of a new class of antimicrobials with a different mechanism of action than conventional antibiotics has become critical. The cationic antimicrobial peptides could represent such a new class (Andreu and Rivas ; Hancock ; Sitaram and Nagaraj ). The development of resistance to membrane active peptides whose sole target is the cytoplasmic membrane is not expected because this would require substantial changes in the lipid composition of cell membranes of microorganisms (Hancock ).
The anti-adhesion property showed by SP1 against S. aureus and P. aeruginosa strains is very interesting considering that the two opportunistic pathogens are able to form biofilms in open wounds, such as chronic diabetic foot ulcers, or infected wounds in clinical and veterinary medicine. New antimicrobial agents that are effective against staphylococci and P. aeruginosa to treat infected wounds are needed, and a potential topical application of SP1 could be supposed. Moreover, the two species are also involved in food spoilage and biofilm formation of food transmitted pathogens and an application of SP1 in the food processing is possible and closer, at this stage of our study, than application in clinical or veterinary health.
A chemotherapeutic approach combining conventional antibiotics and novel anti-biofilm agents could be a new strategy for the treatment of biofilm-associated infections like mastitis in veterinary field or the topical treatment of infected wounds in clinical and veterinary setting.
Finally, the tested synthetic peptide is a good starting point to design new synthetic derivatives with modified chemical-physical properties, with the aim to improve their antimicrobial activity against pathogens and their pharmaceutical potential (Brogden and Brogden ; Huang et al. ).
Financial support from Fondi di Ateneo 2006, 2007 Università degli Studi di Palermo (Italy) and Ministero della Salute IZS is gratefully acknowledged.
- Andreu D, Rivas L: Animal antimicrobial peptides: an overview. Biopolymers 1998, 47(6):415-433. 10.1002/(SICI)1097-0282(1998)47:6<415::AID-BIP2>3.0.CO;2-DPubMedGoogle Scholar
- Arizza V: Marine biodiversity as source of new drugs. Ital J Zool 2013, 80(3):317-318. doi:10.1080/11250003.2013.830370Google Scholar
- Arizza V, Parrinello D, Cammarata M, Vazzana M, Vizzini A, Giaramita FT, Parrinello N: A lytic mechanism based on soluble phospholypases A2 (sPLA2) and beta-galactoside specific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractile hemocytes against K562 cell line and mammalian erythrocytes. Fish Shellfish Immun 2011, 30(4–5):1014-1023. doi:10.1016/j.fsi.2011.01.022Google Scholar
- Arizza V, Vazzana M, Schillaci D, Russo D, Giaramita FT, Parrinello N: Gender differences in the immune system activities of sea urchin Paracentrotus lividus . Comp Biochem Physiol A Mol Integr Physiol 2013, 164(3):447-455. doi:10.1016/j.cbpa.2012.11.021PubMedGoogle Scholar
- Auvynet C, El Amri C, Lacombe C, Bruston F, Bourdais J, Nicolas P, Rosenstein Y: Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9. FEBS J 2008, 275(16):4134-4151. doi:10.1111/j.1742-4658.2008.06554.xPubMedGoogle Scholar
- Bax R, Mullan N, Verhoef J: The millennium bugs - the need for and development of new antibacterials. Int J Antimicrob Ag 2000, 16(1):51-59. doi:10.1016/S0924-8579(00)00189-8Google Scholar
- Brock TD: Biology of Microorganisms. Prentice-Hall, Inc, Englewood Cliffs N.J; 1974.Google Scholar
- Brogden NK, Brogden KA: Will new generations of modified antimicrobial peptides improve their potential as pharmaceuticals? Int J Antimicrob Ag 2011, 38(3):217-225. doi:10.1016/j.ijantimicag.2011.05.004Google Scholar
- Canicatti C, Roch P: Studies on Holothuria polii (Echinodermata) Antibacterial Proteins.1. Evidence for and Activity of a Coelomocyte Lysozyme. Experientia 1989, 45(8):756-759. doi:10.1007/Bf01974579Google Scholar
- Chan C, Burrows LL, Deber CM: Helix induction in antimicrobial peptides by alginate in biofilms. J Biol Chem 2004, 279(37):38749-38754. doi:10.1074/jbc.M406044200PubMedGoogle Scholar
- Clutterbuck AL, Woods EJ, Knottenbelt DC, Clegg PD, Cochrane CA, Percival SL: Biofilms and their relevance to veterinary medicine. Vet Microbiol 2007, 121(1–2):1-17. doi:10.1016/j.vetmic.2006.12.029PubMedGoogle Scholar
- de Andrade FB, de Oliveira JC, Yoshie MT, Guimarães BM, Gonçalves RB, Schwarcz WD: Antimicrobial activity and synergism of lactoferrin and lysozyme against cariogenic microorganisms. Braz Dent J 2014, 25: 165-169. 10.1590/0103-6440201302257PubMedGoogle Scholar
- Fischer D, Li Y, Ahlemeyer B, Krieglstein J, Kissel T: In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 2003, 24(7):1121-1131. doi:10.1016/S0142-9612(02)00445-3PubMedGoogle Scholar
- Gautier R, Douguet D, Antonny B, Drin G: HELIQUEST: a web server to screen sequences with specific α-helical properties. Bioinformatics 2008, 24(18):2101-2102. doi:10.1093/bioinformatics/btn392PubMedGoogle Scholar
- Gilbert P, Allison DG, McBain AJ: Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance? J Appl Microbiol 2002, 92: 98s-110s. doi:10.1046/j.1365-2672.92.5s1.5.xPubMedGoogle Scholar
- Hancock REW: Peptide antibiotics. Lancet 1997, 349(9049):418-422. doi:10.1016/S0140-6736(97)80051-7PubMedGoogle Scholar
- Hancock RE: Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001, 1(3):156-164. doi:10.1016/S1473-3099(01)00092-5PubMedGoogle Scholar
- Hancock RE, Chapple DS: Peptide antibiotics. Antimicrob Agents Chemother 1999, 43(6):1317-1323.PubMed CentralPubMedGoogle Scholar
- Hancock RE, Lehrer R: Cationic peptides: a new source of antibiotics. Trends Biotechnol 1998, 16(2):82-88. 10.1016/S0167-7799(97)01156-6PubMedGoogle Scholar
- Hancock RE, Rozek A: Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol Lett 2002, 206(2):143-149. 10.1111/j.1574-6968.2002.tb11000.xPubMedGoogle Scholar
- Huang HW: Action of antimicrobial peptides: Two-state model. Biochemistry 2000, 39(29):8347-8352. doi:10.1021/Bi0009461PubMedGoogle Scholar
- Huang YB, Huang JF, Chen YX: Alpha-helical cationic antimicrobial peptides: relationships of structure and function. Protein Cell 2010, 1(2):143-152. doi:10.1007/s13238-010-0004-3PubMedGoogle Scholar
- Lamaziere A, Burlina F, Wolf C, Chassaing G, Trugnan G, Ayala-Sanmartin J: Non-metabolic membrane tubulation and permeability induced by bioactive peptides. Plos One 2007, 2(2):e201. doi:10.1371/journal.pone.0000201PubMed CentralPubMedGoogle Scholar
- Lauria A, Bonsignore R, Terenzi A, Spinello A, Giannici F, Longo A, Almerico AM, Barone G: Nickel(II), copper(II) and zinc(II) metallo-intercalators: structural details of the DNA-binding by a combined experimental and computational investigation. Dalton Trans 2014, 43: 6108-6119. http://dx.doi.org/10.1039/c3dt53066c 10.1039/c3dt53066cPubMedGoogle Scholar
- Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo Piccionello A, Pibiri I: Towards a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol Pharm 2014, 11: 653-664. http://dx.doi.org/10.1021/mp400230s 10.1021/mp400230sPubMed CentralPubMedGoogle Scholar
- Lequin O, Ladram A, Chabbert L, Bruston F, Convert O, Vanhoye D, Chassaing G, Nicolas P, Amiche M: Dermaseptin S9, an alpha-helical antimicrobial peptide with a hydrophobic core and cationic termini. Biochemistry 2006, 45(2):468-480. doi:10.1021/bi051711iPubMedGoogle Scholar
- Li C, Haug T, Moe MK, Styrvold OB, Stensvåg K: Centrocins: Isolation and characterization of novel dimeric antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis. Dev Comp Immunol 2010, 34: 959-968. doi: 10.1016/j.dci.2010.04.004PubMedGoogle Scholar
- Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO, Shaw DE: Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 2010, 78: 1950-1958. http://dx.doi.org/10.1002/prot.22711PubMed CentralPubMedGoogle Scholar
- Liu L-P, Deber CM: Guidelines for membrane protein engineering derived from de novo designed model peptides. Pept Sci 1998a, 47(1):41-62. doi:10.1002/(SICI)1097-0282(1998)47:1<41::AID-BIP6>3.0.CO;2-XGoogle Scholar
- Liu LP, Deber CM: Guidelines for membrane protein engineering derived from de novo designed model peptides. Biopolymers 1998b, 47(1):41-62. doi:10.1002/(SICI)1097-0282(1998)47:1<41::AID-BIP6>3.0.CO;2-XGoogle Scholar
- Matsuzaki K: Magainins as paradigm for the mode of action of pore forming polypeptides. Biochim Biophys Acta Rev Biomembr 1998, 1376(3):391-400. doi:10.1016/S0304-4157(98)00014-8Google Scholar
- Matsuzaki K: Why and how are peptide–lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim Biophys Acta Rev Biomembr 1999, 1462(1–2):1-10. http://dx.doi.org/10.1016/S0005-2736(99)00197-2Google Scholar
- Melchior MB, Vaarkamp H, Fink-Gremmels J: Biofilms: a role in recurrent mastitis infections? Vet J 2006, 171(3):398-407. doi:10.1016/j.tvjl.2005.01.006PubMedGoogle Scholar
- Melchior MB, van Osch MHJ, Graat RM, van Duijkeren E, Mevius DJ, Nielen M, Gaastra W, Fink-Gremmels J: Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates: Evidence for lack of penicillin-resistance in Agr-type II strains. Vet Microbiol 2009, 137(1–2):83-89. doi:10.1016/j.vetmic.2008.12.004PubMedGoogle Scholar
- Mor A: Peptide-based antibiotics: A potential answer to raging antimicrobial resistance. Drug Develop Res 2000, 50(3–4):440-447. doi:10.1002/1098-2299(200007/08)50:3/4<440::Aid-Ddr27>3.0.Co;2–4Google Scholar
- Mor A, Nicolas P: The Nh2-terminal alpha-helical domain 1–18 of Dermaseptin is responsible for antimicrobial activity. J Biol Chem 1994, 269(3):1934-1939.PubMedGoogle Scholar
- Oren Z, Shai Y: Mode of action of linear amphipathic α-helical antimicrobial peptides. Pept Sci 1998, 47(6):451-463. doi:10.1002/(SICI)1097-0282(1998)47:6<451::AID-BIP4>3.0.CO;2-FGoogle Scholar
- Patel R: Biofilms and antimicrobial resistance. Clin Orthop Relat Res 2005, 437: 41-47. 10.1097/01.blo.0000175714.68624.74PubMedGoogle Scholar
- Projan SJ, Youngman PJ: Antimicrobials: new solutions badly needed. Curr Opin Microbiol 2002, 5(5):463-465. 10.1016/S1369-5274(02)00364-8PubMedGoogle Scholar
- Pronk S, Pall S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Shirts MR, Smith JC, Kasson PM, van der Spoel D, Hess B, Lindahl E: GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 2013, 29: 845-854. http://dx.doi.org/10.1093/bioinformatics/btt055PubMed CentralPubMedGoogle Scholar
- Schillaci D, Petruso S, Sciortino V: 3,4,5,3 `,5 `-Pentabromo-2-(2 `-hydroxybenzoyl)pyrrole: a potential lead compound as anti-Gram-positive and anti-biofilm agent. Int J Antimicrob Ag 2005, 25(4):338-340. doi:10.1016/j.ijantimicag.2004.11.014Google Scholar
- Schillaci D, Arizza V, Parrinello N, Di Stefano V, Fanara S, Muccilli V, Cunsolo V, Haagensen JJ, Molin S: Antimicrobial and antistaphylococcal biofilm activity from the sea urchin Paracentrotus lividus . J Appl Microbiol 2010, 108(1):17-24. doi:JAM4394 [pii] 10.1111/j.1365-2672.2009.04394.xPubMedGoogle Scholar
- Schillaci D, Vitale M, Cusimano MG, Arizza V: Fragments of beta-thymosin from the sea urchin Paracentrotus lividus as potential antimicrobial peptides against staphylococcal biofilms. In Thymosins in Health and Disease Ii. Edited by: Goldstein AL, Garaci E. Blackwell Science Publ, Oxford; 2012:79-85.Google Scholar
- Schillaci D, Cusimano MG, Cunsolo V, Saletti R, Russo D, Vazzana M, Vitale M, Arizza V: Immune mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and anti-staphylococcal biofilm agents. AMB Express 2013, 3(1):35. doi:10.1186/2191-0855-3-35PubMed CentralPubMedGoogle Scholar
- Schumacher-Perdreau F, Heilmann C, Peters G, Götz F, Pulverer G: Comparative analysis of a biofilm-forming Staphylococcus epidermidis strain and its adhesion-positive, accumulation-negative mutant M7. FEMS Microbiol Lett 1994, 117: 71-78. 10.1111/j.1574-6968.1994.tb06744.xPubMedGoogle Scholar
- Shai Y: Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta Rev Biomembr 1999, 1462(1–2):55-70. http://dx.doi.org/10.1016/S0005-2736(99)00200-XGoogle Scholar
- Shai Y: Mode of action of membrane active antimicrobial peptides. Biopolymers 2002, 66(4):236-248. doi:10.1002/Bip.10260PubMedGoogle Scholar
- Sitaram N, Nagaraj R: The therapeutic potential of host-defense antimicrobial peptides. Curr Drug Targets 2002, 3(3):259-267. doi:10.2174/1389450023347786PubMedGoogle Scholar
- Skerlavaj B, Gennaro R, Bagella L, Merluzzi L, Risso A, Zanetti M: Biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities. J Biol Chem 1996, 271(45):28375-28381. 10.1074/jbc.271.45.28375PubMedGoogle Scholar
- Stabili L, Pagliara P: Antibacterial protection in Marthasterias glacialis eggs: characterization of lysozyme-like activity. Comp Biochem Physiol B Biochem Mol Biol 1994, 109(4):709-713. 10.1016/0305-0491(94)90134-1PubMedGoogle Scholar
- Stark M, Liu LP, Deber CM: Cationic hydrophobic peptides with antimicrobial activity. Antimicrob Agents 2002, 46(11):3585-3590. 10.1128/AAC.46.11.3585-3590.2002Google Scholar
- Storici P, Scocchi M, Tossi A, Gennaro R, Zanetti M: Chemical synthesis and biological-activity of a novel antibacterial peptide deduced from a pig myeloid cDNA. FEBS Lett 1994, 337(3):303-307. doi:10.1016/0014-5793(94)80214-9PubMedGoogle Scholar
- Tossi A, Scocchi M, Skerlavaj B, Gennaro R: Identification and characterization of a primary antibacterial domain in Cap18, a lipopolysaccharide-binding protein from rabbit leukocytes. FEBS Lett 1994, 339(1–2):108-112. doi:10.1016/0014-5793(94)80395-1PubMedGoogle Scholar
- Turner JD, Rouser G: Precise quantitative determination of human blood lipids by thin-layer and triethylaminoethylcellulose column chromatography. I Erythrocyte lipids. Anal Biochem 1970, 38(2):423-436. 10.1016/0003-2697(70)90467-7PubMedGoogle Scholar
- Verkleij AJ, Zwaal RFA, Roelofsen B, Comfurius P, Kastelijn D, van Deenen LLM: The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy. Biochim Biophys Acta Rev Biomembr 1973, 323(2):178-193. http://dx.doi.org/10.1016/0005-2736(73)90143-0Google Scholar
- Wang Z, Wang G: APD: the Antimicrobial Peptide Database. Nucleic Acids Res 2004, 32(Database issue):D590-D592. doi:10.1093/nar/gkh025PubMed CentralPubMedGoogle Scholar
- Wang G, Li X, Wang Z: APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Res 2009, 37(Database issue):D933-D937. doi:10.1093/nar/gkn823PubMed CentralPubMedGoogle Scholar
- Wu G, Wu H, Fan X, Zhao R, Li X, Wang S, Ma Y, Shen Z, Xi T: Selective toxicity of antimicrobial peptide S-thanatin on bacteria. Peptides 2010, 31: 1669-1673. doi:10.1016/j.peptides.2010.06.009PubMedGoogle Scholar
- Zasloff M: Antimicrobial peptides of multicellular organisms. Nature 2002, 415(6870):389-395. doi:10.1038/415389aPubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.