CONICET | Buscador de Institutos y Recursos Humanos

The ciliate Tetrahymena thermophila incorporates sterols from the environmental milieu that desaturates at positions C5(6), C7(8) and C22(23). Phytosterols are additionally modified by removing the ethyl group at C24. The enzymes involved are oxygen-, NAD(P)H- and cytochrome b5-dependent, reason why they were tentatively classified as members of the hydroxylases / desaturases superfamily. The ciliate genome revealed the presence of seven putative sterol desaturases of this family, two of which we have previously characterized as the deethylase and C5(6) desaturase. A Rieske oxygenase was also identified; this type of enzymes, with sterol C7(8) desaturase activity, was observed only in animals, called Neverland (Nvd) in insects and DAF-36 in nematodes. They perform the conversion of cholesterol in 7-dehydrocholesterol, first step in the synthesis of the essential hormones ecdysteroids and dafachronic acids. By adapting an RNA interference-by-feeding protocol, we easily screened six of the eight genes described above, identifying the Rieske-like oxygenase as the ciliate C7(8) desaturase (Des7p). The characterization was confirmed by obtaining a knockout mutant, becoming Des7p the first non-animal Rieske-sterol desaturase described. To our knowledge, this is the first time the feeding-RNAi technique was successfully applied in T. thermophila, enabling to consider such methodology for future reverse genetic-high throughput screenings in this ciliate. Bioinformatics analyses revealed the existence of Des7p-orthologous in other Oligohymenophorean ciliates as well as in non-animal Opisthokonta, like the protists Salpingoeca rosetta and Capsaspora owczarzaki. A horizontal gene transfer event from a unicellular Opisthokonta could explain the acquisition of the Rieske oxygenase by an ancient phagotrophic Oligohymenophorean.