Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis

Hemolymph from partially fed virgin Dermncentor variabilis females was coll ected following Borrelia burgdorferi challenge and assayed for antimicrobia l activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1 h post challenge. Following purification by a three-step pro cedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2 kDa, was determined by MALDI-TOF mass sp ectrometry. N-terminal sequencing by the Edman degradation method gave a se quence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I -(R)-(R)-(R)-G-G-Y-S-Q-I-I-K. A computer search of databases showed that th e peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70 degreesC. Although the tick defensin alone was not immediately effec tive against B. burgdorferi, tick defensin plus lysozyme killed more than 6 5% of cultured B. burgdorferi within 1 h. (C) 2001 Elsevier Science Ltd. Al l rights reserved.