Purification, characterization and gene expression of a glycine and proline-rich antibacterial protein family from larvae of a beetle, Allomyrina dichotoma

Two structurally related antibacterial proteins were isolated from larvae o f a beetle, Allomyrina dichotoma, immunized with Escherichia coil. The two proteins were designated A. dichotoma (A. d.) coleoptericin A and B. The ma ture portion of A. d. coleoptericins deduced from nucleotide sequences of t he cDNAs consists of seventy-two amino acids without cysteine residues and is rich in glycine (11.1%) and proline (11.1%). Comparison of the amino aci d sequences of the A. d. coleoptericins revealed that these antibacterial p roteins have 94%, 75%, 50% and 43% similarity to rhinocerosin, holotricin 2 , coleoptericin and acaloleptin A1. Recombinant A. d. coleoptericin A and B showed strong antibacterial activity against Staphylococcus aureus, methic illin resistant S. aureus (MRSA) and Bacillus subtilis. Recombinant A. d. c oleoptericin A and B were shown to not form pores through bacterial membran es of E. coil, but to hamper cell division. Results of Northern blotting sh owed that A. d. coleoptericin genes are inducible by bacteria and are expre ssed strongly in the fat bodies and haemocytes, and weakly in the Malpighia n tubules. Analysis of the evolutionary relationship of amino acid sequence s among A. d. coleoptericins and other antibacterial proteins suggests that A. d. coleoptericins, rhinocerosin and holotricin 2 are closely related an d form a gene family.