Molecular characterization and expression of the antimicrobial peptide defensin from the housefly (Musca domestica)

Abstract.

A 430-bp cDNA encoding the insect antimicrobial peptide defensin was cloned from the housefly, and designated Musca domestica defensin (Mdde). The open reading frame of the cDNA encoded a 92-amino acid peptide with an N-terminal signal sequence followed by a propeptide that is processed by cleavage to a 40-amino acid mature peptide. Northern analysis and in situ hybridization identified the corresponding mRNA in the fat body of bacterially challenged houseflies and in the epidermis of the body wall of naive and challenged houseflies. The Gram-negative bacterium (Escherichia coli) is a strong inducer of the gene. By RT-PCR, Mdde mRNA was also detected in naive and challenged insects. These findings suggest that the defensin gene is constitutively expressed in the epidermis of the housefly body wall. The predicted mature form of Mdde was expressed as a recombinant peptide in E. coli and Pichia pastoris. The recombinant Mdde expressed in Pichia was active against Gram-positive and some Gram-negative bacteria.