Genome‐wide Transcriptional Analysis of Tetrahymena thermophila Response to Exogenous Cholesterol

Abstract

The ciliate Tetrahymena thermophila does not require sterols for growth and synthesizes pentacyclic triterpenoid alcohols, mainly tetrahymanol, as sterol surrogates. However, when sterols are present in the environment, T. thermophila efficiently incorporates and modifies them. These modifications consist of desaturation reactions at positions C5(6), C7(8), and C22(23), and de‐ethylation at C24 of 29‐carbon sterols (i.e. phytosterols). Three out of four of the enzymes involved in the sterol modification pathway have been previously identified. However, identification of the sterol C22 desaturase remained elusive, as did other basic aspects of this metabolism. To get more insights into this peculiar metabolism, we here perform a whole transcriptome analysis of T. thermophila in response to exogenous cholesterol. We found 356 T. thermophila genes to be differentially expressed after supplementation with cholesterol for 2 h. Among those that were upregulated, we found two genes belonging to the long spacing family of desaturases that we tentatively identified by RNAi analysis as sterol C22 desaturases. Additionally, we determined that the inhibition of tetrahymanol synthesis after supplementation with cholesterol occurs by a transcriptional downregulation of genes involved in squalene synthesis and cyclization. Finally, we identified several uncharacterized genes that are likely involved in sterols transport and signaling.

A novel Tetrahymena thermophila sterol C-22 desaturase belongs to the fatty acid hydroxylase/desaturase superfamily

“…A differential genome-wide transcriptomic analysis of the ciliate, growing in the absence or presence of cholesterol, detected numerous upregulated genes by the sterol. Interestingly, the second and third most upregulated genes code for oxygenases, which were selected as putative sterol desaturases ( DES22A and DES22B ) ( 24 ).…”

“…DES22A and DES22B genes are the second (75.4-fold) and third (70.1-fold) most upregulated genes in the differential genome-wide transcriptomic previously reported. (24).…”

“…In T. thermophila , the sterol C-22 desaturase activity was shown to be induced by the presence of sterols in the culture media ( 15 ). Based on this feature, in a previous work, we identified putative sequences using a transcriptional analysis of the ciliate exposed to exogenous cholesterol ( 24 ). In this work, we confirmed and identified two paralogous genes coding for two proteins with sterol C-22 desaturase activity belonging to a different enzyme family, the FAD.…”

Phytosterol Profiles, Genomes and Enzymes – An Overview

“…Tetrahymena spp. and Paramecium tetraurelia are sterol auxotrophs that develop on dietary sterols unless degraded environmental conditions like starvation switches on a capacity to make tetrahymanol ( Raederstorff and Rohmer, 1988 ; Najle et al, 2020 ). The enzymes recruited for metabolization of exogenous sterols are a canonical sterol-C5-desaturase ( Poklepovich et al, 2012 ) belonging to the fatty acid hydroxylase group ( Sperling et al, 2003 ), a C24-dealkylase (or sterol-24-de ethylase) acting on the sterol side chain ( Tomazic et al, 2011 , 2014 ), and a Rieske Oxygenase performing a sterol-C7-desaturation ( Nusblat et al, 2005 ; Najle et al, 2013 ).…”

“…This observation adds on typical cases of convergent evolution, here for a functional remodeling of phytosterols, by enzymes most probably acquired by horizontal gene transfer ( Darnet et al, 2020 ). Genome wide investigations in Tetrahymena led to the identification of a sterol-22-desaturase belonging to the super family of fatty acid hydroxylases, and not to the canonical P450 monooxygenases ( Najle et al, 2020 ). The multiple desaturations of exogenous cholesterol by Tetrahymena thermophila ( Figure 6 ) is reminiscent of the conversion of dietary cholesterol into dafachronic acids – a class of signaling steroids- by Caenorhabditis elegans , a distantly related sterol-auxotroph ( Zhou et al, 2020 ).…”