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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Antimicrobial Peptides Containing D-Amino Acids with In VivoActivity against Plant Pathogenic BacteriaImma Güell 1 , Esther Badosa 2 , Montse Talleda 1 , Rafael Ferre 1 ,Jordi Cabrefiga 2 , Emili Montesinos 2 , Eduard Bardají 1 ,Lidia Feliu 1 , and Marta Planas 11 LIPPSO, Department of Chemistry; 2 Laboratory of Plant Pathology, Institute of Food andAgricultural Technology-CIDSAV-XaRTA, University of Girona, Girona, 17071, SpainIntroductionPhytopathogenic bacteria are responsible for a wide range of plant diseases causing largeeconomic losses in fruit and vegetable crops. Their control by chemical pesticides ishampered by environmental concerns and the emergence of antibiotic-resistant strains [1].In recent years, antimicrobial peptides have been described to be effective against plantpathogens.Up to now, we have identified linear undecapeptides with high in vitro activity againstthe plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae and Xanthomonasvesicatoria [2]. The best peptide, KKLFKKILKYL-NH2 (BP100), was also effective invivo to prevent infections of E. amylovora in flowers. However, the in vivo dose was 20 to40-fold higher than the MIC. This loss of activity could be attributed to peptide degradationby plant proteases. One strategy used to protect peptides against enzymatic hydrolysis is theincorporation of D-amino acids in the sequence. In the present study, we designed andsynthesized BP100 analogues containing D-amino acids [3].Results and DiscussionPeptides were designed based on the amino acid sequence of BP100 (KKLFKKILKYL-NH 2 ) (Table 1). We investigated the influence of incorporating D-amino acids at variouspositions on the biological activity of this peptide: (i) one D-amino acid (BP138-BP148);(ii) two or three D-amino acids (BP149-BP152, BP154); (iii) four to ten D-amino acids atthe C-terminus (BP155-BP161) and at the N-terminus (BP162-BP168); and (iv) allD-amino acids (BP153).Peptides were screened for their in vitro growth inhibition of the above bacteria (Table1). Single D-amino acid replacement had a pronounced effect on the peptide activityagainst X. vesicatoria and E. amylovora. In contrast, for P. syringae seven sequencesretained BP100 activity. BP143, BP145 and BP147 were the most active against the threebacteria with MIC values ranging from 2.5 to 7.5 µM. Double- or triple-D-amino acidreplacements led to peptides generally less active than BP100. For peptides incorporatingfour to ten D-amino acids at the C-terminus, activity increased with the number ofD-residues. In contrast, a decrease of activity was generally observed when five to eightD-amino acids were incorporated at the N-terminus, but peptides incorporating four, nineor ten D-amino acids were more active than BP100. The all D-isomer was more active thanthe parent peptide against the three pathogens.The toxicity to eukaryotic cells of the most active peptides was tested by their abilityto lyse human red blood cells (Table 1). Eighteen sequences displayed a hemolysis