Abstract
Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss, to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets.
Highlights
Host-parasite interactions involve an arsenal of different molecules, including proteases and their inhibitors, which are ubiquitous in parasites
Cathepsin D and aminopeptidase-N, both cysteine proteases were highly expressed by genotype 0 throughout the infection, with the highest relative change observed in this study
We assume that C. shasta cathepsin D could contribute to the rapid proliferation and virulence of this genotype by causing the destruction of the intestinal extracellular matrix in order to satisfy the high demand for nutrients by the developmental stages
Summary
Host-parasite interactions involve an arsenal of different molecules, including proteases and their inhibitors, which are ubiquitous in parasites These molecules have central roles in parasite life cycles, including invasion, migration, feeding and immunomodulation of host responses (McKerrow et al, 2006; Klotz et al, 2011), and represent attractive targets for drug design as antiparasite intervention strategies Myxozoans are microscopic cnidarian endoparasites that underwent morphological simplification from their free-living cnidarian ancestors almost 600 million years ago (Holzer et al, 2018) These parasites alternate between an invertebrate host, annelids or bryozoans, and a vertebrate host, usually fish; in each host they produce spores as waterborne transmission stages. The quantity and diversity of proteases in myxozoans opens a world of possibilities for exploring them as potential drug targets
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