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

Sterols in eukaryotic cells play important roles in modulating membrane fluidity and in cell signaling and trafficking. During evolution, a combination of gene losses and acquisitions gave rise to an extraordinary diversity of sterols in different organisms. The sterol C-22 desaturase identified in plants and fungi as a cytochrome P-450 monooxygenase evolved from the first eukaryotic cytochrome P450 and was lost in many lineages. Although the ciliate Tetrahymena thermophila desaturates sterols at the C-22 position, no cytochrome P-450 orthologs are present in the genome. Here, we aim to identify the genes responsible for the desaturation as well as their probable origin. We used gene knockout and yeast heterologous expression approaches to identify two putative genes, retrieved from a previous transcriptomic analysis, as sterol C-22 desaturases. Furthermore, we demonstrate using bioinformatics and evolutionary analyses that both genes encode a novel type of sterol C-22 desaturase that belongs to the large fatty acid hydroxylase/desaturase superfamily and the genes originated by genetic duplication prior to functional diversification. These results stress the widespread existence of nonhomologous isofunctional enzymes among different lineages of the tree of life as well as the suitability for the use of T. thermophila as a valuable model to investigate the evolutionary process of large enzyme families.

Functional characterization of the first lipoyl-relay pathway from a parasitic protozoan.

Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as "lipoyl-relay", requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.

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 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.

Structural determinant of functionality in acyl lipid desaturases.

Little is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of Bacillus cereus DesA desaturase improves Bacillus subtilis Des activity. In addition, the replacement of the first two transmembrane domains from Bacillus licheniformis inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was sufficient to resurrect this enzyme. Unexpectedly, we were able to restore the activity of ORF BL02692 with a single substitution (Cys40Tyr) of a cysteine localized in the first transmembrane domain close to the lipid-water interface. Substitution of eight residues (Gly90, Trp104, Lys172, His228, Pro257, Leu275, Tyr282, and Leu284) by site-directed mutagenesis produced inactive variants of DesA. Homology modeling of DesA revealed that His228 is part of the metal binding center, together with the canonical His boxes. Trp104 shapes the hydrophobic tunnel, whereas Gly90 and Lys172 are probably involved in substrate binding/recognition. Pro257, Leu275, Tyr282, and Leu284 might be relevant for the structural arrangement of the active site or interaction with electron donors. This study reveals the role of the N-terminal region of Δ5 phospholipid desaturases and the individual residues necessary for the activity of this class of enzymes.

Lipoic acid metabolism in Trypanosoma cruzi as putative target for chemotherapy.

Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC50 values one order of magnitude lower (62-66 µM). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [14C]-octanoic acid was taken up 12 fold less efficiently than [14C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments.

Biosynthesis of very long chain fatty acids in Trypanosoma cruzi.

Trypanosoma brucei and Trypanosoma cruzi showed similar fatty acid (FA) compositions, having a high proportion of unsaturated FAs, mainly 18:2Δ9,12 (23-39%) and 18:1Δ9 (11-17%). C22 polyunsaturated FAs are in significant amounts only in T. brucei (12-20%) but represent a mere 2% of total FAs in T. cruzi. Both species have also similar profiles of medium- and long-chain saturated FAs, from 14:0 to 20:0. Interestingly, procyclic and bloodstream forms of T. brucei lack very long chain FAs (VLCFAs), whereas epimastigotes and trypomastigotes of T. cruzi contain 22:0 (0.1-0.2%), 24:0 (1.5-2%), and 26:0 (0.1-0.2%). This is in agreement with the presence of an additional FA elongase gene (TcELO4) in T. cruzi. TcELO4 was expressed in a Saccharomyces cerevisiae mutant lacking the endogenous ScELO3, rescuing the synthesis of saturated and hydroxylated C26 FAs in the yeast. Expression of TcELO4 also rescued the synthetic lethality of a ScELO2, ScELO3 double mutation, and the VLCFA profile of the transformed yeast was similar to that found in T. cruzi. By identifying TcELO4 as the enzyme responsible for the elongation of FA from 16:0 and 18:0 up to 26:0, with 24:0 being the preferred product, this work completed the characterization of FA elongases in Trypanosoma spp.

The Sterol-C7 desaturase from the ciliate Tetrahymena thermophila is a Rieske Oxygenase, which is highly conserved in animals.

The ciliate Tetrahymena thermophila incorporates sterols from its environment that desaturates at positions C5(6), C7(8), and C22(23). Phytosterols are additionally modified by removal of the ethyl group at carbon 24 (C24). The enzymes involved are oxygen-, NAD(P)H-, and cytochrome b5 dependent, reason why they were classified as members of the hydroxylases/desaturases superfamily. The ciliate's genome revealed the presence of seven putative sterol desaturases belonging to this family, two of which we have previously characterized as the C24-de-ethylase and C5(6)-desaturase. A Rieske oxygenase was also identified; this type of enzyme, with sterol C7(8)-desaturase activity, was observed only in animals, called Neverland in insects and DAF-36 in nematodes. They perform the conversion of cholesterol into 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 earlier, allowing the characterization of the Rieske-like oxygenase as the ciliate's C7(8)-desaturase (Des7p). This characterization was confirmed by obtaining the corresponding knockout mutant, making Des7p the first nonanimal Rieske-sterol desaturase described. To our knowledge, this is the first time that the feeding-RNAi technique was successfully applied in T. thermophila, enabling to consider such methodology for future reverse genetics high-throughput screenings in this ciliate. Bioinformatics analyses revealed the presence of Des7p orthologs in other Oligohymenophorean ciliates and in nonanimal Opisthokonts, like the protists Salpingoeca rosetta and Capsaspora owczarzaki. A horizontal gene transfer event from a unicellular Opisthokont to an ancient phagotrophic Oligohymenophorean could explain the acquisition of the Rieske oxygenase by Tetrahymena.

The presence of plasmenyl ether lipids in Capsaspora owczarzaki suggests a premetazoan origin of plasmalogen biosynthesis in animals.

Plasmalogens are glycerophospholipids with a vinyl ether bond, rather than an ester bond, at sn-1 position. These lipids were described in anaerobic bacteria, myxobacteria, animals and some protists, but not in plants or fungi. Anaerobic and aerobic organisms synthesize plasmalogens differently. The aerobic pathway requires oxygen in the last step, which is catalyzed by PEDS1. CarF and TMEM189 were recently identified as the PEDS1 from myxobacteria and mammals, which could be of valuable use in exploring the distribution of this pathway in eukaryotes. We show the presence of plasmalogens in Capsaspora owczarzaki, one of the closest unicellular relatives of animals. This is the first report of plasmalogens in non-metazoan opisthokontas. Analysis of its genome revealed the presence of enzymes of the aerobic pathway. In a broad BLAST search, we found PEDS1 homologs in Opisthokonta and some genera of Amoebozoa and Excavata, consistent with the restricted distribution of plasmalogens reported in eukaryotes. Within Opisthokonta, PEDS1 is limited to Filasterea (Capsaspora and Pigoraptor), Metazoa and a small group of fungi comprising three genera of ascomycetes. A phylogenetic analysis of PEDS1 traced the acquisition of plasmalogen synthesis in animals to a filasterean ancestor and suggested independent acquisition events for Amoebozoa, Excavata and Ascomycetes.

Synergistic effect of inhibitors of fatty acid desaturases on Trypanosoma parasites.

The pathway for unsaturated fatty acid biosynthesis is essential in trypanosomatid parasites and has been a key target in our work on the discovery and analysis of several inhibitory compounds. Here, we show the effect of novel inhibitors of stearoyl-CoA desaturase (SCD) and oleate desaturase (OD), alone and in combination, on the growth rate of parasite cultures. GS-456332, an inhibitor of human Δ9 desaturase, efficiently inhibited growth of both Trypanosoma cruzi epimastigotes and Trypanosoma brucei bloodstream form cells, with EC50 values of 136.9 ± 24.2 and 9.4 ± 3.1 nM, respectively. This effect was specific for SCD. Stearolic acid (9-octadecynoic acid) was also able to arrest T. cruzi and T. brucei growth by specific inhibition of their OD, with EC50 values of 1.0 ± 0.2 µM and 0.1 ± 0.01 µM, respectively. When these compounds were administered simultaneously, a clearly synergistic effect was observed for both Trypanosoma species, with EC50 values in the low nanomolar range. These results demonstrate the feasibility of using combinations of drugs, inhibiting different enzymes on the same metabolic pathway, for the development of more efficient chemotherapeutic strategies against neglected diseases caused by these parasites.

A novel sterol desaturase-like protein promoting dealkylation of phytosterols in Tetrahymena thermophila.

The gene TTHERM_00438800 (DES24) from the ciliate Tetrahymena thermophila encodes a protein with three conserved histidine clusters, typical of the fatty acid hydroxylase superfamily. Despite its high similarity to sterol desaturase-like enzymes, the phylogenetic analysis groups Des24p in a separate cluster more related to bacterial than to eukaryotic proteins, suggesting a possible horizontal gene transfer event. A somatic knockout of DES24 revealed that the gene encodes a protein, Des24p, which is involved in the dealkylation of phytosterols. Knocked-out mutants were unable to eliminate the C-24 ethyl group from C(29) sterols, whereas the ability to introduce other modifications, such as desaturations at positions C-5(6), C-7(8), and C-22(23), were not altered. Although C-24 dealkylations have been described in other organisms, such as insects, neither the enzymes nor the corresponding genes have been identified to date. Therefore, this is the first identification of a gene involved in sterol dealkylation. Moreover, the knockout mutant and wild-type strain differed significantly in growth and morphology only when cultivated with C(29) sterols; under this culture condition, a change from the typical pear-like shape to a round shape and an alteration in the regulation of tetrahymanol biosynthesis were observed. Sterol analysis upon culture with various substrates and inhibitors indicate that the removal of the C-24 ethyl group in Tetrahymena may proceed by a mechanism different from the one currently known.

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei.

Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC(50)) of PCF was 1.0 ± 0.2 µM for Isoxyl and 5 ± 2 µM for 10-TS, whereas BSF appeared more susceptible with EC(50) values 0.10 ± 0.03 µM (Isoxyl) and 1.0 ± 0.6 µM (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.

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.

C-5(6) sterol desaturase from tetrahymena thermophila: Gene identification and knockout, sequence analysis, and comparison to other C-5(6) sterol desaturases.

The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

Elongation of polyunsaturated fatty acids in trypanosomatids.

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.

Functional characterization of front-end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan.

A survey of the three kinetoplastid genome projects revealed the presence of three putative front-end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Delta8, Delta5 and Delta6 desaturases for L. major, and Delta6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Delta6 desaturases could have Delta4 desaturase activity, based mainly on the conserved HX(3)HH motif for the second histidine box, when compared with Delta4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front-end desaturases present in L. major and T. brucei. Our prediction about the presence of Delta4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Delta5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Delta8 desaturase turned out to be a functional Delta6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareDelta6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very-long-chain polyunsaturated fatty acid biosynthesis is functional in L. major using Delta6, Delta5 and Delta4 desaturases. In trypanosomes, only Delta4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms.

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.

Sterol metabolism in the filasterean Capsaspora owczarzaki has features that resemble both fungi and animals.

Sterols are essential for several physiological processes in most eukaryotes. Sterols regulate membrane homeostasis and participate in different signalling pathways not only as precursors of steroid hormones and vitamins, but also through its role in the formation of lipid rafts. Two major types of sterols, cholesterol and ergosterol, have been described so far in the opisthokonts, the clade that comprise animals, fungi and their unicellular relatives. Cholesterol predominates in derived bilaterians, whereas ergosterol is what generally defines fungi. We here characterize, by a combination of bioinformatic and biochemical analyses, the sterol metabolism in the filasterean Capsaspora owczarzaki, a close unicellular relative of animals that is becoming a model organism. We found that C. owczarzaki sterol metabolism combines enzymatic activities that are usually considered either characteristic of fungi or exclusive to metazoans. Moreover, we observe a differential transcriptional regulation of this metabolism across its life cycle. Thus, C. owczarzaki alternates between synthesizing 7-dehydrocholesterol de novo, which happens at the cystic stage, and the partial conversion-via a novel pathway-of incorporated cholesterol into ergosterol, the characteristic fungal sterol, in the filopodial and aggregative stages.

Acquisition and biosynthesis of saturated and unsaturated fatty acids by trypanosomatids.

As components of phospholipids and glycosylphosphatidylinositol anchors, fatty acids are responsible for forming the core of biological membranes and the correct localization of proteins within membranes. They also contribute to anchoring proteins by direct acylation of specific amino acids. Fatty acids can be used as energy sources and serve as signaling molecules or precursors for their synthesis. All these processes highlight the important role of fatty acids in cell physiology, justifying the diverse strategies for their acquisition evolved by different organisms. This review describes several recent findings in the salvage and biosynthesis of fatty acids by parasitic protists belonging to the class Kinetoplastea. They include two biosynthetic routes, the mitochondrial one and a peculiar membrane-associated pathway, the synthesis of polyunsaturated fatty acids, and the scavenging of lysophospholipids and lipoproteins from host plasma. These different processes are also explored as putative targets for chemotherapy.

Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography-electrospray tandem mass spectrometry.

Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2Δ mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2Δ mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time.

Highly specific methyl-end fatty-acid desaturases of trypanosomatids.

A detailed analysis of the trypanosomatids' genome projects revealed the presence of genes predicted to encode fatty-acid desaturases of the methyl-end type (MED). After cloning and functional characterization of all identified genes, it can be concluded that Trypanosoma cruzi contains two MEDs with oleate desaturase (OD) activities whereas Leishmania major contains one OD and two active linoleate desaturases (LD). All characterized ODs are highly specific for oleate (18:1Δ9) as substrate, presenting a ν+3 regioselectivity, although palmitoleate (16:1Δ9) can be desaturated as well, but to a lesser extent. L. major LD appears to use exclusively linoleate (18:2n-6), converting it into α-linolenate (18:3n-3). This strong specificity assures no further conversion of polyunsaturated fatty acids (PUFAs) of the n-6 series into the n-3 series, downstream in the PUFA biosynthesis pathway. This characterization completes the identification of all enzymes involved in PUFA biosynthesis in a parasitic protist. Differently from their Trypanosoma brucei orthologue, T. cruzi and L. major ODs were more active when expressed either, in the presence of trienoic fatty acids or at higher temperatures. This could be evidence for a differential post-translational regulation of these enzymes as a result of direct sensing of environmentally dependent parameters such as membrane fluidity.