Attacin-C

We described the cDNA cloning of two antimicrobial peptides (AMPs), cecropin (BdCec), and attacin C (BdAttC), from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious insect pest of fruit trees. Using rapid amplification of cDNA ends, fragments encompassing the entire open reading frames of BdCec and BdAttC were cloned and sequenced. The complete 425 bp cDNA of BdCec encodes a protein of 64 amino acids with a predicted molecular weight of 6.84 kDa. The 931 bp cDNA of BdAttC encodes a protein of 239 residues with a predicted molecular weight of 24.97 kDa. Real-time quantitative RT-PCR demonstrated that the developmental transcription profiles of BdCec and BdAttC were similar in each larvae, pupae, and adults. The constitutive expression levels of both AMPs were high in the first-instar and late third-instar larvae, suggesting that their antimicrobial activity is active in the newly hatched larvae and just before pupation. The basal expression levels were not significant different in adult fat bodies. The expression of BdCec and BdAttC was upregulated after bacterial challenge in adult fat bodies. The ratio of inducible expression to constitutive expression was lower in males compared to females.

Attacin gene exists as paralogous conversion and is being used for identification of strain variations in insects based on the sequence variation. Hence, a study was undertaken to analyze the sequence variation of the attacin gene isoforms in the tasar silkworm Anthereae mylitta that exists in the form of different ecoraces depending upon the environment, food plant and location. Comparison of the previously reported attacin sequences with the DNA sequences of attacin A and B genes revealed six amino acid substitutions among the sequences of the ecoraces which however did not affect the functional domain of Attacin. The generated dendrogram clearly indicated unique branches for each ecorace with two separate gene clusters for attacin A and B. The Sarihan ecorace formed a separate sub-group under both the gene clusters. The present study also revealed the presence of Attacin_N Superfamily domain exclusively in Exon I separated from the Attacin_C Superfamily domain that was present in Exon II and part of Exon III, a prominent character of attacin gene. The phylogenetic reconstruction analysis of attacin gene in A.mylitta supported the common evolutionary origin of attacin genes belonging to the Lepidoteran and Dipteran families that formed two separate clusters.

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.