607 Research Article Received: 30 December 2008 Revised: 7 April 2009 Accepted: 15 April 2009 Published online in Wiley Interscience: 08 June 2009 (www.interscience.com) DOI 10.1002/psc.1148 Esculentin 1 – 21: a linear antimicrobial peptide from frog skin with inhibitory effect on bovine mastitis-causing bacteria ‡ Alfonso Enrique Islas-Rodrìguez, a§ Ludovica Marcellini, b§ Barbara Orioni, c Donatella Barra, b Lorenzo Stella c and Maria Luisa Mangoni b* Mastitis, or inflammation of the mammary gland, is the most common and expensive illness of dairy cows throughout the world. Although stress and physical injuries may give rise to inflammation of the udders, infections by bacteria or other microorganisms remain the major cause, and infusion of antibiotics is the main treatment approach. However, the increased emergence of multidrug-resistant pathogens and the production of milk contaminated with antibiotics has become a serious threat in the livestock. Hence, there is an urgent need for the discovery of new therapeutic agents with a new mode of action. Gene-encoded AMPs, which represent the first line of defence in all living organisms, are considered as promising candidates for the development of new anti-infective agents. This paper reports on the antibacterial activities in vitro and in an animal model, of the frog skin AMP esculentin 1–21 [Esc(1–21)], along with a plausible mode of action. Our data revealed that this peptide (i) is highly potent against the most common mastitis-causing microbes (e.g. Streptococcus agalactiae); and (ii) is active in vivo, causing a visible regression of the clinical stage of mastitis in dairy cows, after 1 week of peptide treatment. Biophysical characterisation revealed that the peptide adopts an α-helical structure in microbial mimicking membranes and is able to permeate the membrane of S. agalactiae in a dose-dependent manner. Overall, these data suggest Esc(1 – 21) as an attractive AMP for the future design of new antibiotics to cure mastitis in cattle. Copyright c  2009 European Peptide Society and John Wiley & Sons, Ltd. Keywords: frog skin; antimicrobial peptide; multidrug-resistant strains; mastitis; anti-infective agent; esculentin; peptide-membrane interaction; mode of action Introduction The widespread and often empirical use of conventional antibiotics to defeat microbial infections has led to a drastic reduction in their therapeutic efficacy and to the emergence of multidrug-resistant (MDR) strains. This represents a worldwide concern in human and veterinary practice. In the latter case, bovine mastitis remains one of the most costly sicknesses in dairy industry with economic losses estimated at 1.2–1.7 billion dollars per year in the United States alone [1,2]. Note that clinical and subclinical mastitis are the two major forms of inflammation of the mammary glands in response to bacterial invasion, in dairy cows. Clinical mastitis is characterised by a decreased production of milk, with considerable alterations of its composition (e.g. decrease of protein, fat, lactose contents, increase in leucocytes counts [3]) and appearance (e.g. formation of flakes and clots). Furthermore, the infected udder becomes swollen, red, hot, sometimes painful to touch [4], whereas in severe cases the animal shows signs of generalised reaction such as fever, loss of appetite, diarrhoea, weakness, depression and shock. In contrast, subclinical mastitis is more difficult to detect, because of the absence of visible abnormalities in milk and udders, as well as of systemic signs of illness. In this case, leucocyte count in milk, which is positively correlated to the level of infection (see Materials and Methods), represents the best diagnostic method. Intra-mammary infusion of antibiotics is the main approach to treat mastitis on many farms. However, the increased number of MDR pathogens and the production of milk contaminated with antibiotics (not suggested during lactation) have become a serious threat in the livestock [5]. ∗ Correspondence to: Maria Luisa Mangoni, Unit` a di Diagnostica Molecolare avanzata, II Facolt` a di Medicina e Chirurgia, Azienda Ospedaliera S. Andrea, via di Grottarossa, 1035-00189 Roma, Italy. E-mail: marialuisa.mangoni@uniroma1.it a Laboratorio de P´ eptidos Naturales, Departamento de Biología Celular y Molecular, Universidad de Guadalajara, Centro Universitario de Ciencias Biol´ ogicas y Agropecuarias, Zapopan, Jalisco, M´ exico b Istituto Pasteur-Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche, Azienda Ospedaliera S. Andrea, Universit` a ‘La Sapienza’, 00185 Roma, Italy c Dipartimento di Scienze e Tecnologie Chimiche, Universit` a di Roma Tor Vergata, 00133 Roma, Italy ‡ Special issue devoted to contributions presented at the 1st Italy-Korea Symposium on Antimicrobial Peptides, 24-25 July 2008. § The authors Alfonso Enrique Islas-Rodrìguez and Ludovica Marcellini con- tributed equally to this work. Abbreviations used: AMP, antimicrobial peptide; Esc(1–21), esculentin- 1a(1–21); CFU, colony-forming units; LB, Luria-Bertani; LC, lethal concentra- tion;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide;NaPB, sodium phosphate buffer; PBS, phosphate buffered saline; PG, phosphatidyl- glycerol; PE, phosphatidylethanolamine. J. Pept. Sci. 2009; 15: 607–614 Copyright c  2009 European Peptide Society and John Wiley & Sons, Ltd.