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bioRxiv · 10.1101/2025.10.24.684163

Exploring the Repertoire of Rhomboid Proteases in Cryptosporidium parvum Parasite: Phylogenesis, Structural motifs and Cellular Localization in Sporozoite Cells

Vanni, I.·Pizzi, E.·Bertuccini, L.·Ludovisi, A.·Tosini, F.
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Abstract

Cryptosporidium parvum is an apicomplexan parasite and an important pathogen of mammals and humans, which can be infected by zoonotic transmission or directly by human-to-human contacts. This parasite attacks the small intestine, and the main symptom is a watery diarrhoea that can be particularly severe in newborns and deadly in immunodeficient subjects. Rhomboids are ubiquitous proteases embedded in cell membranes that act by cleaving other membrane proteins in or near their transmembrane domains. Apicomplexan rhomboids play an important role in approaching and invading the host cell. This study analysed the phylogenetic origin, the structural motifs and the subcellular localization of C. parvum rhomboids. Altogether, C. parvum possesses three rhomboids, namely CpRom1, CpRom2 and CpRom3. The similarity search in Cryptosporidium genus revealed that C. parvum as well as other "intestinal" species lacks a PARL-like rhomboid whereas this type of mitochondrial rhomboid was present in "gastric" species like Cryptosporidium muris and Cryptosporidium andersoni. At the genome level this was revealed by a precise excision of the PARL-like gene in intestinal species whereas the rest of chromosomal synteny was well conserved among the Cryptosporidium species. The analysis of the structural domains revealed that C. parvum rhomboids can be classified as mixed secretases and the comparison with orthologs from Toxoplasma gondii and Plasmodium falciparum showed that C. parvum rhomboids can be distinguished in two separate clusters based on similarities at the level of the catalytic sites. The three rhomboids were expressed simultaneously in the invasive stage of sporozoite, but each of them had a different spatial distribution. Indeed, CpRom1 had a dual localization: this rhomboid was internal at the apical complex, and it was also accumulated at the posterior pole of the sporozoite. Otherwise, CpRom2 was prevalently contained in the apical complex, and a point of accumulation was on the surface of the apical end. Differently from CpRom1 and CpRom2, CpRom3 is distributed along the entire surface of sporozoites. Finally, we listed 10 membrane proteins as candidate substrates for the C. parvum rhomboids based on the similarities with some proven substrates of apicomplexan rhomboids and the copresence in subcellular structures with the three rhomboids.

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Vanni, I., Pizzi, E., Bertuccini, L., Ludovisi, A., Tosini, F.. 2025-10-24. Exploring the Repertoire of Rhomboid Proteases in Cryptosporidium parvum Parasite: Phylogenesis, Structural motifs and Cellular Localization in Sporozoite Cells. https://doi.org/10.1101/2025.10.24.684163

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