engleski

Targeted amplicon sequencing for obtaining novel antimicrobial peptide sequences

AMPs are endogenous antibiotics present in all organisms acting directly towards pathogens and also showing immunomodulatory properties – altering gene expression to facilitate and modulate immune response1, 2. Skin secretions of many tested anurans have been shown to contain a variety of bioactive peptides that often exhibit antibacterial activity, also against multidrug resistant isolates3. Identifying novel peptides normally requires handling several individuals from each species, which are either sacrificed or held in captivity and treated with electric shocks to obtain small amounts of crude peptide. This is followed by several rounds of purification using different precipitation and chromatographic techniques combined with activity testing of fractions to identify the active principles4. Here we report a different, potentially faster, less invasive and efficient approach to selectively amplify transcripts encoding for AMPs, thus providing their sequences. After RNA extraction from tissue samples, cDNA synthesis followed by PCR amplification was performed. For this purpose forward degenerate primers were designed based on highly conserved signal peptide regions5, 6 together with a reverse primer designed on the poly-A tail of mRNA. Signal peptide regions themselves were derived from sequences already available in DADP database7 and transcriptome data in SRA8 databases. Resulting amplicons were size-selected and processed by ion semiconductor sequencing, obtaining several thousand sequencing reads which were then assembled into contigs representing nearly full-length AMP-encoding transcripts. More than 100 different peptides were identified from 5 different specimens belonging to 5 different frog species indicating big peptide diversity and confirming the effectiveness of the method. Applying knowledge-based criteria and on-line tools resulted in selection of the best candidates for chemical synthesis and extensive characterization.

Amplicon sequencing ; Antimicrobial peptides ; Anura

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