RESUMO
Small cationic antimicrobial peptides (AMPs) are host defense molecules detected in virtually all groups of organisms. To investigate the immune response mechanisms of Mytilus galloprovincialis, primary and suppression subtractive hybridization libraries were prepared from hemolymph of mussels injected with heat-inactivated bacteria or poly I:C, the latter mimicking viral infection. After DNA sequencing, sequence processing and similarity searching, a remarkable abundance of AMP mRNAs were identified. In detail, 25.9% and 32.4% AMP sequences from mussels infected with bacteria and 43.4% and 40.6% from mussels stimulated with poly I:C were detected by selective amplification of 180 differentially expressed genes and random sequencing of 967 cDNA clones, respectively. The 232 ESTs matching with myticin A and B (Mytilus spp.) displayed considerable sequence variability and revealed a third cluster proposed here as myticin C. Phenetic analysis of the translated myticin ESTs yielded 74 and 25 variants of the precursor and active peptide, respectively, and confirmed the high polymorphism of the new form. Myticin C shows typical features of the CSalphabeta AMP family (eight-cysteine array and secretory signal peptide) as well as amino acid variation, mainly in the anionic C-terminal region. The sequencing of one intronic region from genomic DNA, allowed us to detect 13 variants in 9 individual mussels referring them to one gene only. In addition to hemolymph, myticin C transcripts were detected in various mussel tissues, oocytes and early larval stages. The striking sequence variability and expression levels of myticins in mussels confirm the fundamental role of these natural antibiotics in the ancient host-pathogen interplay of mutual inhibition, evasion and adaptation strategies.