Diptericin

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within 1 h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

Drosophila haemocytes play a key role in defence against microbial aggression. Their capacity to sense and dispose of bacteria and also to signal to other immune tissues is probably vital to overcome an infection. In this work we used the haemocyte-like mbn-2 cell line to investigate how expression of the antimicrobial peptide diptericin is affected after a high dose bacterial challenge with diaminopimelic acid (DAP)-peptidoglycan Gram-positive and Gram-negative bacteria. We report that diptericin expression is negatively affected by high infection dose and rapid bacterial growth regardless of the type of infection and bacterial virulence and occurs in the absence of mbn-2 cell death. Furthermore we show that the mbn-2 cell population is heterogeneous, containing both phagocytic and nonphagocytic cells and that contact with large numbers of bacteria decreases diptericin expression in the phagocytic cell population.

Insects produce various anti-microbial peptides in response to injury and infection. In Drosophila, diptericin has previously been studied as an anti-bacterial immune response gene. Here, we report the cloning of the diptericin-like protein (dptlp) gene as a paralog of Drosophila diptericin. By comparison of their sequences, we found that the dptlp gene has all of the functional domains conserved in the diptericin gene and other anti-bacterial proteins. The dptlp gene was rapidly induced by bacterial infections and showed different time-dependent gene expression patterns from those of diptericin. Like diptericin, dptlp was specifically produced from the fat body, and its expression was strictly dependent on bacterial infections. In addition, the dptlp gene expression was almost completely abolished in the imd mutant, which implicates that its expression is regulated by the anti-bacterial arm of the Drosophila innate immune regulatory pathways. In support of this, we found GATA, interferon consensus responding element, and kappa B binding sites, which is known to be important for the proper expression of anti-bacterial genes, in the proximal promoter region of the dptlp gene. Taken together, our findings support that dptlp is a novel anti-bacterial peptide whose expression is regulated by the anti-bacterial immune response mechanism.