Smolstatin1, a unique cystatin-like stefin of Sphaerospora molnari, is essential for parasite development

Stefins, members of the cystatin superfamily, regulate cysteine proteases, yet their roles in early-branching metazoans remain poorly defined. Here, we characterize Smolstatin1, a cystatin-like stefin from the myxozoan Sphaerospora molnari , a common carp pathogen. Phylogenetic analysis places Smolstatin1 within a monophyletic clade of myxozoan cystatin-like stefins that groups with homologues from foodborne flukes and canonical metazoan stefins. Smolstatin1 is expressed throughout intrapiscine development, is upregulated during sporogony, and localizes to membrane-bound compartments and the blood-stage surface. Recombinant Smolstatin1 inhibits parasite and host cysteine proteases, with strongest activity toward cathepsin L-like enzymes and weaker activity against legumain, while showing negligible effects on serine proteases. RNAi-mediated knockdown reduces parasite motility and alters cell integrity, suggesting a role in parasite fitness and development. Structural analysis reveals an unusual domain-swapped dimer. Although antigenic, Smolstatin1 vaccination does not protect fish from infection. Together, these findings identify Smolstatin1 as a key protease regulator and expand our understanding of cystatin evolution and function.

Czech Academy of Sciences, Institute of Microbiology
University of South Bohemia in České Budějovice
Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir
Czech Academy of Sciences, Biology Centre