Purification, cDNA cloning and modification of a defensin from the coconutrhinoceros beetle, Oryctes rhinoceros

A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoce ros, immunized with Escherichia coli. A full-size cDNA was cloned by combin ing reverse-transcription PCR (RT-PCR), and 5'- and 3'-rapid amplification of cDNA ends (RACE). Analysis of the O. rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpigh ian tubules. O. rhinoceros defensin showed strong antibacterial activity ag ainst Staphylococcus aureus. A 9-mer peptide amidated at its C-terminus, AH CLAICRK-NH2, (Ala22-Lys30-NH2), was synthesized based on the deduced amino- acid sequence, assumed to be an active site sequence by analogy with the se quence of a defensin isolated from larvae of the beetle Allomyrina dichotom a. This peptide showed antibacterial activity against S. aureus, methicilli n-resistant S. aureus, E. coli and Pseudomonas aeruginosa. We further modif ied this oligopeptide and synthesized five 9 mer peptides, ALRLAIRKR-NH2, A LLLAIRKR-NH2, AWLLAIRKR-NH2, ALYLAIRKR-NH2 and ALWLAIRKR-NH2 These oligopep tides showed strong antibacterial activity against Gram-negative and Gram-p ositive bacteria. The antibacterial effect of Ala22-Lys30-NH2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. These Ala22-Lys30-NH2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast ce lls or macrophages, except for AWLLAIRKR-NH2.