Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians

Abstract

The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.

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

“…The four protease protein sequences were used as queries to find homologous genes in seven myxozoan transcriptomes: Tetracapsuloides bryosalmonae, Sphaerospora molnari, Myxobolus cerebralis, Myxobolus squamalis, Henneguya salminicola and Kudoa iwatai to compare their protease repertoire with that of our model organism transcriptome, C. shasta (genotype IIR). Similar searches were done for myxozoan stefins in a previous publication (Bartosǒva-Sojkováet al, 2021) and are discussed herein. Transcriptomes were downloaded from public data repositories (figshare, Dryad and Transcriptome Shotgun Assembly Sequence Database or TSA at NCBI, see Table 2).…”

“…Stefins are tight binding intracellular inhibitors of the papain family of cysteine proteases that generally lack a signal peptide, and hence are usually involved in regulation of endopeptidases (Turk et al, 2008). Myxozoan stefins are atypical molecules with a chimeric structure combining motifs of type 1 cystatin or classical stefins and type 2 cystatins (Bartosǒva-Sojkováet al, 2021). While some myxozoan stefins posses a signal peptide, C. shasta stefin lacks this feature, which suggests it has intracellular/ cytoplasmic localization.…”

“…2.5% of proteins (422 proteases) in the Thelohanellus kitauei genome (Yang et al, 2014), 2.6% (235 proteases) in the Sphaerospora molnari trancriptome (Hartigan et al, 2020) or 6.1% (7 proteases) in the Ceratonova shasta nematocyst proteome (Piriatinskiy et al, 2017). It further has been demonstrated that these proteins are genetically diversified (Eszterbauer et al, 2020) or structurally modified (Hartigan et al, 2020;Bartosǒva-Sojkováet al, 2021) in some myxozoan species, underlining the importance and specificity of these molecules in parasite survival and successful host interaction. Thus, the quantity and diversity of proteases in myxozoans opens a world of possibilities for exploring them as potential drug targets.…”

Ceratonova shasta: a cnidarian parasite of annelids and salmonids

“…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).…”

Proteases—From Basic Structure to Function to Drug Design as Targeted Therapy

“…The team set out hypotheses to describe the driving forces for the origins of this unique cystatin subtype and propose evolutionary scenarios elucidating the current existence of cystatins in the Metazoa, especially in their early emerging lineages. Their study identified molecules for which future functional studies could help to identify their roles in parasites and host–parasite interactions [ 5 ].…”