CLCA IS A METALLOPROTEASE INDUCED DURING PATHOGENESIS AND IS A POTENTIAL THERAPEUTIC MOLECULE TO DEAL WITH PISCINE DISEASE

Bacterial disease by Flavobacterium columnare, and Aeromonas hydrophila, cause significant economic loss to the aquaculture farmers. The stringent regulations on the use of antibiotics and low efficacy of available vaccines, creates the need for new solutions. Epithelial tissues play barrier and defensive functions in different organs to protect the fish from pathogens. Bacterial and parasitic infections damage the epithelial tissues and make the fish. Lot of studies have shown the role of epithelial junctional complexes in barrier function and a correlation with immune response. Our goal is to identify cost-effective molecules that can be used in fish feed or water as therapeutics to prevent and treat bacterial and parasitic diseases.

CLCAs are predominantly expressed in the epithelium and endothelium of chordates and is required for maintaining epithelial integrity. We have shown that CLCA2 interacts with EVA1, a protein that is expressed on epithelial and immune cells, both CLCA2 and EVA1 form a complex with TJ protein ZO-1 and CLCA2 alone forms a complex with beta-catenin. Both CLCA2 and EVA1 expression directly correlate with epithelial differentiation and loss of either protein results in cells to lose cell-cell junctions and undergo EMT and thereby compromise barrier function. On the other hand, there are ample studies showing the presence of proteases and metalloproteases in mucus. They directly kill the pathogens either by cleaving the bacterial proteins or by activating the innate or adaptive immune response, or producing antimicrobial peptides. Interestingly, we find that CLCA2 is a self-cleaving metalloprotease. It has a metal binding motif HEXXH which is conserved across species, including zebrafish. There is an auto cleavage site near amino acid 700. We found that Zn⁺² catalyzed CLCA2 cleavage (Fig 1) and the E to Q mutation in HEXXH abolished its proteolytic activity (Fig 2) (unpublished data). Thus, CLCA2 is a Zn⁺² dependent, self-cleaving metalloprotease expressed at cell-cell junctions. Our proposed model for CLCA2 is that along with maintaining epithelial integrity, during stress/pathogenic insult, CLCA undergoes self-cleavage, and activates immune cells and mucus secretion. Therefore, we speculate that sCLCA could be a potential therapeutic for piscine bacterial diseases.