Proteases as Therapeutic Targets Against the Parasitic Cnidarian Ceratonova shasta: Characterization of Molecules Key to Parasite Virulence In Salmonid Hosts

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.

Ceratonova shasta: a cnidarian parasite of annelids and salmonids

“…at least 6% of the C. shasta nematocyst proteome are proteases (Piriatinskiy et al ., 2017), and all main catalytic types of protease were found in C. shasta developmental stages (Alama-Bermejo et al ., 2020; Ahmad et al ., 2021). Differential expression, in silico characterization and annotation of 4 proteases and an inhibitor provided the first insights on the involvement of these enzymes in parasite virulence (Alama-Bermejo et al ., 2022). Virulent genotype IIR upregulates an aspartic protease (cathepsin D) in concert with the occurrence of increased parasite proliferation at 15 days post exposure (dpe), coincident with upregulation of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 (Taggart-Murphy et al ., 2021).…”

“…This suggested that C. shasta cathepsin D may induce an inflammatory response in the host, as has been shown for other parasite aspartic proteases (Cassone et al ., 2016). A cysteine protease inhibitor (stefin) in C. shasta was upregulated in IIR infections, with peaks of expression at 15 dpe and to a lesser extent at 29 dpe (Alama-Bermejo et al ., 2022). Stefins are usually involved in regulation of endopeptidases, especially during metabolically active proliferative stages in parasites (Lee et al ., 2013).…”

“…In genotype IIR, the stefin was present in proteomic data from ascites but absent from myxospores (Brekhman et al ., 2021), indicating some cell regulatory function in developmental stages. A contrasting expression profile between genotypes and the cathepsin D and the stefin 3D protein structures suggests these molecules are key for the rapid proliferation and metabolism of genotype IIR (Bartošová-Sojková et al ., 2021; Alama-Bermejo et al ., 2022).…”

Genome-Wide Analysis of Haemonchus contortus Proteases and Protease Inhibitors Using Advanced Informatics Provides Insights into Parasite Biology and Host–Parasite Interactions

“…Research on parasite-derived proteases has been an active area for more than 70 years (reviewed by [15][16][17][18][19][20][21]). Recent advances in this field have expanded our understanding of the diversity and specificity of parasite-derived proteases, with the identification of unique proteases in various species of parasitic helminths, such as Angiostrongylus cantonensis [22], Ascaris suum [23] and Meloidogyne incognita [24].…”

“…Recent advances in this field have expanded our understanding of the diversity and specificity of parasite-derived proteases, with the identification of unique proteases in various species of parasitic helminths, such as Angiostrongylus cantonensis [22], Ascaris suum [23] and Meloidogyne incognita [24]. These studies have highlighted the potential of proteases as potential drug and vaccine targets for the development of novel interventions against parasites (e.g., [18,21]). The use of advanced molecular technologies and informatics [25,26] is now providing improved insights into parasite-derived proteases, such as their classification and involvement in biological processes, as well as host-parasite interactions, with potential implications for the design of protease inhibitors.…”