Abstract
In Drosophila melanogaster, seven distinct families of antimicrobial peptides with different structures and specificities are synthesized by the fat body and released into the hemolymph during the immune response. Using microscale high performance liquid chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Edman degradation, we have isolated and characterized from immune-challenged Drosophila two novel induced molecules, under the control of the Imd pathway, that correspond to post-translationally modified antimicrobial peptides or peptide fragments. The first molecule is a doubly glycosylated form of drosocin, an O-glycosylated peptide that kills Gram-negative organisms. The second molecule represents a truncated form of the pro-domain of the Drosophila attacin C carrying two post-translational modifications and has significant structural similarities to proline-rich antibacterial peptides including drosocin. We have synthesized this peptide and found that it is active against Gram-negative bacteria. Furthermore, this activity is potentiated when the peptide is used in combination with the Drosophila antimicrobial peptide cecropin A. The synergistic action observed between these two molecules suggests that the truncated post-translationally modified pro-domain of attacin C by itself may play an important role in the antimicrobial defense of Drosophila.
Highlights
The fruit fly Drosophila melanogaster has recently become a recognized genetic tool for studying innate immunity
Purification and Primary Structure Determination of the Pro-domain of Attacin C—Using differential display by matrix-assisted laser desorption/ionization (MALDI)-TOF mass spectrometry (MS) RP high-performance liquid chromatography (HPLC), and Edman sequencing, we have purified and characterized DIM-15 (2767.6 Da) and DIM-16 (2971.1 Da), two peptides that are under the control of the Imd pathway
As first step of purification, 10 runs of micro-RPHPLC were made on hemolymph collected from a total of 1,200 challenged flies to isolate a sufficient amount of material for structural identification
Summary
Insect Immunization and Hemolymph Collection—Two- to three-dayold Drosophila melanogaster Oregon R adults were experimentally infected with a mixture of two bacteria (Micrococcus luteus as Grampositive strain and Escherichia coli as Gram-negative strain), and the hemolymph (20 batches of 60 flies) of 24-h challenged flies was collected according to a procedure reported previously (10). During the second step of purification of DIM-15 and -16, the same column was used, but the fractions were eluted using linear biphasic gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid at a flow rate of 80 l/min and at 35 °C, from 2 to 22% over 10 min and from 22 to 37% over 50 min. For the final purification of DIM-16, the same column as for the first step was used, but the fractions were eluted with a linear biphasic gradient of acetonitrile in 0.05% aqueous trifluoroacetic acid at 35 °C and at a flow rate of 80 l/min from 2 to 22% over 10 min and from 22 to 32% over 50 min. The strains used were from private and public collections (25)
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