The identification and characterization of novel antimicrobial peptides from the Atlantic cod (Gadus morhua) genome

Browne, Mitchell J. (Mitchell James) (2010) The identification and characterization of novel antimicrobial peptides from the Atlantic cod (Gadus morhua) genome. Masters thesis, Memorial University of Newfoundland.

[English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (10MB)

Abstract

Antimicrobial peptides (AMPs) are short peptides that exhibit broad-spectrum activity against a variety of microbes including bacteria, viruses, fungi and protozoa. Based on previously generated Atlantic cod (Gadus morhua) expressed sequence tags (ESTs), I identified sequences representing four novel AMP-like transcripts [a peptide with sequence similarity to lipopolysaccharide binding protein (LBP), a transcript with sequence similarity to the potato (Solanum tuberosum) AMP snakin-2, as well as two piscidin-like peptides. The two peptides with similarity to the piscidins, a family of small cationic AMPs from fish, were selected for further study. I obtained full-length cDNA sequences for two paralogous piscidin-like transcripts using bi-directional rapid amplification of cDNA ends (RACE). The Atlantic cod paralogues were termed gaduscidins (GAD-1 and GAD-2), derived from the genus name Gadus. Quantitative reverse transcription - polymerase chain reaction (QPCR) was used in transcript expression studies of GAD-1 and GAD-2. I examined the constitutive expression of these transcripts in several tissues from non-stressed juvenile cod. Transcript expression of GAD-1 and GAD-2 was also examined in immune tissues following intraperitoneal (IP) injection of formalin-killed atypical Aeromonas salmonicidia (ASAL), or phosphate-buffered saline (PBS injection control). Putative GAD-1 and GAD-2 mature peptides were chemically synthesized for structural characterization, first using circular dichroism (CD) spectroscopy, followed by solution nuclear magnetic resonance (NMR). In addition to structural characterization, functional characterization was also carried out to determine hemolytic and antimicrobial activity of GAD-1 and GAD-2. A hemolytic assay against Atlantic cod red blood cells (RBCs) was performed, as well as a minimal inhibitory concentration (MIC) assay for both GAD-1 and GAD-2 with Staphylococcus intermedius and Escherichia coil.

Actions (login required)

View Item