Journal of Peptide Science J. Pept. Sci. 2008; 14: 670–682 Published online 29 November 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.967 Mapping the peptide and protein immune response in the larvae of the fleshfly Sarcophaga bullata ALICE CIENCIALOV ´ A, a TEREZA NEUBAUEROV ´ A, b MILOSLAV ˇ SANDA, a RADEK ˇ SINDELKA, c JOSEF CVA ˇ CKA, a ZDEN ˇ EK VOBURKA, a MILO ˇ S BUD ˇ E ˇ S ´ INSK ´ Y, a V ´ ACLAV KA ˇ SI ˇ CKA, a PETRA S ´ AZELOV ´ A, a VERONIKA ˇ SOL ´ INOV ´ A, a MARTINA MACKOV ´ A, b BOHUM ´ IR KOUTEK a and JI ˇ R ´ I JIR ´ A ˇ CEK a * a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n ´ am. 2, 166 10 Praha 6, Czech Republic b Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology, Technick ´ a 5, 166 28 Praha 6, Czech Republic c Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, V´ ıdeˇ nsk ´ a 1083, 142 20 Praha 4, Czech Republic Received 13 July 2007; Revised 25 September 2007; Accepted 30 September 2007 Abstract: We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide β-alanyl- L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified. Copyright  2007 European Peptide Society and John Wiley & Sons, Ltd. Keywords: fleshfly; Sarcophaga bullata; antimicrobial activity; sapecin; odorant-binding protein; transferrin INTRODUCTION Disease-causing microbes resistant to drug therapy are an increasing public health problem. Tuberculosis, gonorrhea, and childhood ear infections are just a few examples of diseases that have become difficult to treat with antibiotics. The World Health Organization (WHO, Geneva) estimates that 1 500 people die each hour from an infectious disease, half of these being children under 5 years of age. Today, nosocomial infections affect over 2 million patients annually in the USA, at a cost in excess of $4.5 billion [1]. The growing problem of microbial resistance to conventional antibiotics and the need for new antibiotics, especially with new mechanisms of action, has stimulated interest in the development of antimicrobial peptides as human therapeutics [2,3]. Peptides and polypeptides with antimicrobial activity can be classified on the basis of their biochemical and structural features, the largest group of which concerns the so-called cationic peptides, which are widely distributed in plants, animals and bacteria [4]. Currently, more than 1000 naturally occurring antimicrobial peptides are known [5–7] and are typically positively-charged and composed of 10–40 amino acids, approximately 50% of which consists of hydrophobic residues. These properties confer * Correspondence to: Jiˇ r´ ı Jir´ aˇ cek, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n´ am. 2, 166 10 Praha 6, Czech Republic; e-mail: jiracek@uochb.cas.cz their amphipathic character [6]. Cationic peptides are grouped into three families on the basis of their secondary structure: (i) linear peptides with an α- helical conformation (cecropins) [8], (ii) cyclic and open-ended cyclic peptides with pairs of cysteine residues (defensins) [9–11], and (iii) peptides with over- representation of amino acid proline (pyrrhocoricin) [12–14] or glycine (attacin) [1]. Three other classes of antimicrobial peptides have been recently isolated from different invertebrate and vertebrate species: anionic peptides, aromatic dipep- tides, and peptides derived from oxygen-binding pro- teins [4]. The anionic peptides are generally isolated from mammalian epithelia (e.g. enkelytin) [15], pep- tide B [16], or aspartic acid-rich peptides [17]. The aromatic dipeptides consist of low molecular weight compounds such as N -β -alanyl-5-S-glutathionyl-3,4- dihydroxyphenylalanine identified in fleshfly Sar- cophaga peregrina [18] and p-hydroxycinnamaldehyde that was isolated from the saw fly Acantholyda parki [19]. The dipeptide β -alanyl-tyrosine with modest antimicrobial activity has been constitutively found in the fleshfly Neobellieria bullata [20]. Hemocyanin derivatives, the peptides derived from oxygen-binding proteins, were recently isolated from the hemolymph of arthropods. The proteolyzed form of vertebrate hemoglobin with antimicrobial activity was detected in the tick Boophilus microplus [21]. There are different modes of action of antimicro- bial peptides. The majority of cationic antimicrobial Copyright  2007 European Peptide Society and John Wiley & Sons, Ltd.