Tetrahymanol Synthesis Research Articles

Phagocytic and pinocytic uptake of cholesterol in Tetrahymena thermophila impact differently on gene regulation for sterol homeostasis

The ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet. This metabolic shift is mainly driven by transcriptional regulation of genes for tetrahymanol synthesis (TS) and sterol bioconversion (SB). The mechanistic details of sterol uptake, intracellular trafficking and the associated gene expression changes are unknown. By following cholesterol incorporation over time in a conditional phagocytosis-deficient mutant, we found that although phagocytosis is the main sterol intake route, a secondary endocytic pathway exists. Different expression patterns for TS and SB genes were associated with these entry mechanisms. Squalene synthase was down-regulated by a massive cholesterol intake only attainable by phagocytosis-proficient cells, whereas C22-sterol desaturase required ten times less cholesterol and was up-regulated in both wild-type and mutant cells. These patterns are suggestive of at least two different signaling pathways. Sterol trafficking beyond phagosomes and esterification was impaired by the NPC1 inhibitor U18666A. NPC1 is a protein that mediates cholesterol export from late endosomes/lysosomes in mammalian cells. U18666A also produced a delay in the transcriptional response to cholesterol, suggesting that the regulatory signals are triggered between lysosomes and the endoplasmic reticulum. These findings could hint at partial conservation of sterol homeostasis between eukaryote lineages.

Squalene-Tetrahymanol Cyclase Expression Enables Sterol-Independent Growth of Saccharomyces cerevisiae.

Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast Saccharomyces cerevisiae therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of containing sterols, contain the pentacyclic triterpenoid "sterol surrogate" tetrahymanol, which is formed by cyclization of squalene. Here, we demonstrate that expression of the squalene-tetrahymanol cyclase gene TtTHC1 from the ciliate Tetrahymena thermophila enables synthesis of tetrahymanol by S. cerevisiae Moreover, expression of TtTHC1 enabled exponential growth of anaerobic S. cerevisiae cultures in sterol-free synthetic media. After deletion of the ERG1 gene from a TtTHC1-expressing S. cerevisiae strain, native sterol synthesis was abolished and sustained sterol-free growth was demonstrated under anaerobic as well as aerobic conditions. Anaerobic cultures of TtTHC1-expressing S. cerevisiae on sterol-free medium showed lower specific growth rates and biomass yields than ergosterol-supplemented cultures, while their ethanol yield was higher. This study demonstrated that acquisition of a functional squalene-tetrahymanol cyclase gene offers an immediate growth advantage to S. cerevisiae under anaerobic, sterol-limited conditions and provides the basis for a metabolic engineering strategy to eliminate the oxygen requirements associated with sterol synthesis in yeasts.IMPORTANCE The laboratory experiments described in this report simulate a proposed horizontal gene transfer event during the evolution of strictly anaerobic fungi. The demonstration that expression of a single heterologous gene sufficed to eliminate anaerobic sterol requirements in the model eukaryote Saccharomyces cerevisiae therefore contributes to our understanding of how sterol-independent eukaryotes evolved in anoxic environments. This report provides a proof of principle for a metabolic engineering strategy to eliminate sterol requirements in yeast strains that are applied in large-scale anaerobic industrial processes. The sterol-independent yeast strains described in this report provide a valuable platform for further studies on the physiological roles and impacts of sterols and sterol surrogates in eukaryotic cells.

Genome-wide Transcriptional Analysis of Tetrahymena thermophila Response to Exogenous Cholesterol.

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 2h. 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 distinct pathway for tetrahymanol synthesis in bacteria

Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol.

Lipid Composition of the Marine Ciliates Pleuronema sp. and Fabrea salina: Shifts in Response to Changes in Diet1

ABSTRACT. The composition and incorporation of lipids in two marine ciliates, Pleuronema sp. and Fabrea salina, was examined following growth on either an algal or bacterial diet. When allowed to feed on a natural bacterial community, Pleuronema sp. synthesized the triterpenoid alcohol gammaceran‐3β‐ol (tetrahymanol) and two hopanoids (hopan‐3β‐ol and one uncharacterized hopanoid). When fed the marine alga Isochrysis galbana, F. salina contained the major algal sterol 24‐methylcholesta‐5, 22‐dien‐3β‐ol and several long chain ketones specific to the alga. In both ciliates, fatty acids composition showed a general correspondence to that of the diet. Using a series of antibiotic treatments to alter the bacterial prey community, and thus fatty acid composition of the ciliate's diet, promoted changes in the fatty acid composition of Pleuronema sp. to resemble that of the bacterial prey. The addition of a mixture of algal sterols to a bacterized culture of another scuticociliate, Parauronema acutum, inhibited tetrahymanol synthesis and resulted in the incorporation of sterols into the ciliate.

The Effect of the Lipogenic Inhibitor Cerulenin, on Macrostomal Cell Formation in Tetrahymena vorax

ABSTRACT. Macrostomal cell formation is blocked by the antibiotic cerulenin at levels of 15 μg/ml or higher. Inhibition can be reversed up to 4 h following cerulenin addition by washing and resuspending cells in new, noncerulenin‐treated transforming principle. In these latter cases, additional time equal to the time spent in the inhibitor, is needed for cells to reach control values of transformation. Neither the addition of saturated or unsaturated fatty acids, cholesterol added alone or in combination with stearic acid, nor a mixture of lipids extracted from Tetrahymena vorax reversed the cerulenin effect. Radioisotope incorporation data showed while protein synthesis was reduced by the end of 1 h and tetrahymanol synthesis by the end of 2 h, little or no effect of this inhibitor occurred on RNA or fatty acid synthesis during these times. One interpretation of these results is that cerulenin, by preventing first protein synthesis and later tetrahymanol synthesis, interferes with synthesis and formation of membranes required for the microstome to macrostome transition.

The Sterol Requirement of Tetrahymena setosa HZ‐11

ABSTRACTThe growth rate of Tetrahymena setosa cells is stimulated significantly by as little as 0.1 μg and optimally by about 1 μg of ergosterol per ml of medium. Cell yields in the stationary phase are, however, not perceptibly affected by increasing sterol concentrations. Ergosterol, in concentrations that stimulate growth optimally, does not cause a reduction of tetrahymanol synthesis. The latter process is impaired only at much higher ergosterol concentrations. Epicholesterol and coprostanol inhibit ergosterol‐stimulated growth competitively. It is concluded that the trace amounts of sterol needed by T. setosa do not serve to replace tetrahymanol but function in some other manner, probably unrelated to the control of membrane fluidity. This conclusion supports the views advanced earlier by Holz, Erwin, Wagner & Rosenbaum.

The steric requirements for sterol inhibition of tetrahymanol biosynthesis.

AbstractMany naturally occurring sterols are accumulated and metabolized byTetrahymena pyriformis. In most cases, the sterols are desaturated to giveΔ5,7,22‐derivatives. Compounds with an ethyl, but not with a methyl, substituent at C‐24 are dealkylated. Exposure of the ciliates to the appropriate sterol sharply curtails the synthesis of the native pentacyclic triterpenoid alcohols, tetrahymanol and diplopterol. An analysis with modified sterols has revealed several additional features that are required for desaturation at C‐7,8 and C‐22,23 and for inhibition of tetrahymanol biosynthesis. The presence of atrans‐17(20)‐double bond, which eliminates free rotation at C‐20 and fixes C‐22 to the right of the nucleus, does not interfere with desaturation, while thecis‐ or left‐handed isomer is not metabolized. Thecis‐Δ17(20)‐isomer is, however, an effective inhibitor of tetrahymanol biosynthesis, although less so that thetrans‐counterpart. When a methyl or hydroxyl group at C‐20 protrudes to the front of the molecule in the right‐handed conformation, metabolism is reduced or abolished. Shortening (by one C‐atom) or lengthening of the sterol side chain has little effect on the ability of the compounds to inhibit tetrahymanol biosynthesis or to support growth, as long as the overall length of the side chain does not exceed seven carbons from C‐20. The presence of a 7α‐, 7β‐, 20α‐, 20β‐, or a 25‐hydroxy group in the cholesterol molecule sharply inhibits desaturation and curtails the effectiveness of the compound as an inhibitor of tetrahymanol biosynthesis. The 7‐ or 22‐keto derivatives seem to act in a fashion similar to the hydroxy derivatives, but these compounds show greater inhibition of growth. 20‐Methylcholesterol, however, is a potent inhibitor of synthesis, which suggests that the polarity of the substituent of C‐20 is more important than bulk. Many sterols can effectively replace tetrahymanol in the membranes of these ciliates. However, several of the compounds, which inhibit synthesis, appear to be physiologically inappropriate, and poor growth results. An example of the latter class is 20‐methylcholesterol. Finally, a class of sterols, represented by 20α‐hydroxycholesterol and 7‐ketocholesterol, does not severly inhibit tetrahymanol synthesis but leads to growth inhibition and surface abnormalities. These sterols apparently lead to a disordered membrane, even in the presence of tetrahymanol.

Inhibition of diplopterol synthesis inTetrahymena pyriformis by a hypocholesteremic compound

The hypocholesteremic compound, 3beta-(beta-dimethylaminoethoxy)-androst-5-en-17-one was earlier shown to inhibit the synthesis of tetrahymanol and two undentified lipids. It now has been demonstrated that one of the unidentified compounds is diplopterol.

A Comparison of the effects of some hypocholesteremic compounds on squalene metabolism in Tetrahymena pyriformis and rat liver

1. 1. Six dialkylaminoethoxy compounds, five of which exhibit hypocholesteremic activity, have been compared for their effects on the growth of Tetrahymena pyriformis and on the in vitro cyclization of squalene to tetrahymanol by this organism. With one exception, there was good correlation between ability of the compounds to inhibit growth and squalene cyclization. There was also good correlation between inhibition of T. pyriformis squalene cyclase and hypocholesteremic activity. 2. 2. The above compounds have also been compared for their effects on the conversion of 2,3-oxidosqualene to C 30 and C 27, sterols by rat liver homogenate. Only two of the six (one of which did not display hypocholesteremic activity) inhibited this process at the concentrations employed. The effect of these two agents on the conversion of squalene to C 30 and C 27 sterols by rat liver homogenate was also examined. 3. 3. Cholesterol had no effect on either in vivo or in vitro squalene cyclization by T. pyriformis, nor did it overcome the in vitro inhibition of squalene cyclization by 3β-(β-dimethylaminoethoxy)-androst-5-en-17-one (DMAE-DHA). 4. 4. A lag period is associated with tetrahymanol synthesis by a cell-free preparation of T. pyriformis. Two fat-soluble antioxidants inhibited squalene cyclization by this preparation; one water-soluble one did not. Sulfhydryl-blocking agents had little effect on squalene cyclase activity.

Environmentally produced alterations of the tetra-hymanol: Phospholipid ratio in Tetrahymena pyriformis membranes

Whereas the sterol-like triterpenoid tetrahymanol constitutes a fixed proportion of membrane lipids in growing and early stationary phase Tetrahymena cells, starved or senescent cultures increase their tetrahymanol-phospholipid ratios many fold. We detected two causes of tetrahymanol enrichment, both related to the absolute resistance of the compound to catabolic attack. Surviving cells of senescent cultures ingest fragments of dead cells, degrading the phospholipids but accumulating tetrahymanol in large amounts. On the other hand, growing cells placed in a nutrient-free medium receive no exogenous lipids. Rather, they gradually utilize their own structural components for energy, decreasing to 20% of their normal size during the process. There is a concomitant reduction of the cellular phospholipid content, but tetrahymanol is not degraded. Consequently, after 4 days of starvation the membrane tetrahymanol: phospholipid ratio is 3 times the normal value despite a virtual absence of tetrahymanol synthesis. We discuss the apparently successful adjustment of the cells to altered proportions of a lipid whose concentration is considered critical for proper membrane function.

On the mechanism of growth inhibition of Tetrahymena pyriformis by steroids

1. 1. Studies have been made of the effects of 2,4-dinitrophenol and three steroids on respiration and the synthesis of RNA, protein and lipids from 14C-labeled precursors by Tetrahymena pyriformis. Concentrations of each compound were employed which would customarily cause a 30 to 50% inhibition of growth. 2. 2. 2,4-Dinitrophenol at first increased, then decreased, oxygen uptake. Drastic inhibition of the synthesis of RNA, protein and lipids was noted. 3. 3. Dehydroisoandrosterone (50 μM) and progesterone (10 μM) inhibited respiration by 10–15% and blocked RNA synthesis within 30 min by 95 and 40%, respectively. Synthesis of protein and bulk lipids was affected more slowly and to a lesser extent. When impairment of tetrahymanol biosynthesis was noted, it was accompanied by reduced levels of squalene. 4. 4. 3β-(β-Dimethylaminoethoxy)-androst-5-en-17-one, a derivative of dehydroisoandrosterone with potent hypocholesteremic properties, exerted no effect on respiration or on incorporation of [2- 14C]acetate into bulk lipids. Some inhibition of protein and RNA synthesis was noted after 2 h. However, synthesis of tetrahymanol was sharply curtailed within 30 min, in conjunction with the accumulation of squalene. In some experiments the synthesis of two unidentified lipids was also adversely affected.

EFFECT OF TRIPARANOL ON SYNTHESIS OF SQUALENE AND TETRAHYMANOL BY TETRAHYMENA PYRIFORMIS.

SummaryIn triparanol-inhibited cultures of T. pyriformis (strains W, H and S), squalene is greatly increased in amount and tetra-hymanol is decreased. In contrast, squalene appears in low concentration in uninhibited cultures. It appears that squalene might be converted directly to the pentacyclic compound, tetrahymanol, and that the cyclization of squalene is blocked by triparanol. Cholesterol was not identified in control or inhibited cultures and appears neither to be a sterol of major importance in this organism, nor an intermediate in the synthesis of tetrahymanol. Methods of purification and identification of tetrahymanol are given.