Financial support: INCO-DEV: IMMUNAQUA ICA4-CT-2001-10023 and DI-PUCV 122.779/2004.

Keywords: antimicrobial peptides, biochemical characterization, gill tissues, in vitro activity, mussels.

Antimicrobial peptides are small-sized, cationic molecules able to neutralize pathogenic microorganisms through mechanisms directed related to the immune system of most organisms (http://www.bbcm.univ.trieste.it/~tossi/antimic.html; http://aps.unmc.edu/AP/main.php). We have obtained antimicrobial peptides from gill tissues of the mussel Mytilus edulis chilensis. Considering the chemical nature of these molecules we used an acid extraction procedure followed by consecutive cationic exchange and hydrophobic interaction chromatography steps for peptide enrichment. Active fractions were characterized by SDS-containing protein gel electrophoresis demonstrating that were particularly enriched with low molecular weight peptides displaying neutralizing growth activity against Gram positive and Gram negative bacteria as well as over fungal pathogens. Active fractions resulted to be thermostable and non cytotoxic to eukaryotic cells. Considering these results, industrial waste gills of bivalves arise as a new source for antimicrobial molecules.

Comparing the polypeptide profiles along the purification procedure, the 20% and 40% ACN eluted fractions from the hydrophobic interaction chromatography, are clearly enriched in low molecular weight peptides. Nonetheless, the content of the 20% fraction is the only one in which low molecular weight peptides are the prominent species observed. Notwithstanding the 40% fraction is also enriched, specific activity of the former drove our priority for its characterization.

All post Sep-Pak C-18 fractions were tested for antimicrobial activity over standard strains of bacteria (G+ and G-) as well as over selected fungi strains. In general, testing required 10 higher concentrations for bacteria than for fungi. For bacteria, the 20% ACN fraction was the most efficient with a minimal growth inhibition of 65% although inhibition over 75% was obtained over M. luteus and A. hydrophila. For fungi, both fractions 20% and 40% ACN were equally efficient, also with a minimal growth inhibition of 65%.

To evaluate another physicochemical parameter aliquots of the 20% ACN fraction were heated at either 50ºC or 100ºC for 30 or 60 min showing that this treatment did not affect the antimicrobial activity of these novel molecules.

Finally, to rule out cytotoxicity, samples from the 20% ACN fractions were exposed for 3 hrs to the fish cell line CHSE-214 with no effect over cell viability. 

Based upon our results and considering that mussel gills represent roughly 13% of the smooth body weight portion, we consider this tissue as a reasonable future source for new antimicrobial molecule isolation. If this is the case, this approach could be extended to other tissues, especially to those involving tissues discarded in the industrial processing of commercial marine invertebrates. Thus, we have demonstrated in this report that some slight modifications added to existing protocols broaden the range of extractable AMPs. Additionally we have foresee a potential use for byproducts in the aquaculture industry as a source for new pharmaceutical molecules. We are in the process of designing strategies to prove the feasibility of these alternatives.