Phosphatidylethanolamine and phosphatidylcholine biosynthesis by the Kennedy pathway occurs at different sites in Trypanosoma brucei

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway represent major and often essential routes for the production of PE and PC, with ethanolamine and choline/ethanolamine phosphotransferases (EPT and CEPT, respectively) catalysing the last reactions in the respective pathways. Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular parasite, Trypanosoma brucei, both branches of the Kennedy pathway are essential for cell growth in culture. We have previously reported that T. brucei EPT (TbEPT) catalyses the production of ether-type PE molecular species while T. brucei CEPT (TbCEPT) synthesizes diacyl-type PE and PC molecular species. We now show that the two enzymes localize to different sub-compartments of the ER. By expressing a series of tagged forms of the two enzymes in T. brucei parasites, in combination with sub-cellular fractionation and enzyme activity measurements, TbEPT was found exclusively in the perinuclear ER, a distinct area located close to but distinct from the nuclear membrane. In contrast, TbCEPT was detected in the bulk ER.

Biology of tRNA halves in Trypanosoma brucei

Although T. brucei has to challenge tremendous environment changes, e.g. switch from the bloodstream form in mammalian hosts to the mid gut form present in tsetse flies, there is no evidence for differential regulation of RNA Pol II transcription. Instead, constitutive transcription appears to occur. This observation indicates that protein levels have to be regulated by post-transcriptional mechanisms. It has been shown that non-protein coding RNAs (ncRNAs) are crucial in regulatory networks (e.g. chromosome remodelling; RNA polymerase activity; mRNA turnover; etc.), but all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. This is unexpected, since the ribosome has a central role during gene expression and due to the assumption that the primordial translation system most likely received direct regulatory input from small molecules including ncRNA cofactors. In our lab, it has been discovered that ncRNAs are able to directly bind to the ribosome, therefore influencing the translation rate in Haloferax volcanii and Saccharomyces cerevisiae. In order to extend this idea of ribosome-binding ncRNAs in mammalian parasites, we want to investigate this mechanism in T. brucei. Accordingly, we performed a genomic screen for small ribosome-associated RNAs followed by functional analyses of possible candidates. With the help of this genomic screen, we found tRNAs that are alternated and tRNA halves that are differentially expressed upon nutritional stress.

Biology of tRNA halves in Trypanosoma brucei

Although T. brucei has to challenge tremendous environment changes, e.g. switch from the bloodstream form in mammalian hosts to the mid gut form present in tsetse flies, there is no evidence for differential regulation of RNA Pol II transcription. Instead, constitutive transcription appears to occur. This observation indicates that protein levels have to be regulated by post-transcriptional mechanisms. It has been shown that non-protein coding RNAs (ncRNAs) are crucial in regulatory networks (e.g. chromosome remodelling; RNA polymerase activity; mRNA turnover; etc.), but all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. This is unexpected, since the ribosome has a central role during gene expression and due to the assumption that the primordial translation system most likely received direct regulatory input from small molecules including ncRNA cofactors. In our lab, it has been discovered that ncRNAs are able to directly bind to the ribosome, therefore influencing the translation rate in Haloferax volcanii and Saccharomyces cerevisiae. In order to extend this idea of ribosome-binding ncRNAs in mammalian parasites, we want to investigate this mechanism in T. brucei. Accordingly, we performed a genomic screen for small ribosome-associated RNAs followed by functional analyses of possible candidates. With the help of this genomic screen, we found tRNAs that are alternated and tRNA halves that are differentially expressed upon nutritional stress.

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth

myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na(+)- or H(+)-linked myo-inositol transporters. While Na(+)-coupled myo-inositol transporters are found exclusively in the plasma membrane, H(+)-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H(+)-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

Lipid remodelling of glycosylphosphatidylinositol (GPI) glycoconjugates in procyclic-form trypanosomes: biosynthesis and processing of GPIs revisited

The African trypanosome, Trypanosoma brucei, has been used as a model to study the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. In mammalian (bloodstream)-form parasites, diacyl-type GPI precursors are remodelled in their lipid moieties before attachment to variant surface glycoproteins. In contrast, the GPI precursors of insect (procyclic)-form parasites, consisting of lyso-(acyl)PI (inositol-acylated acyl-lyso-phosphatidylinositol) species, remain unaltered before protein attachment. By using a combination of metabolic labelling, cell-free assays and complementary MS analyses, we show in the present study that GPI-anchored glycoconjugates in T. congolense procyclic forms initially receive tri-acylated GPI precursors, which are subsequently de-acylated either at the glycerol backbone or on the inositol ring. Chemical and enzymatic treatments of [3H]myristate-labelled lipids in combination with ESI-MS/MS (electrospray ionization-tandem MS) and MALDI-QIT-TOF-MS3 (matrix-assisted laser-desorption ionization-quadrupole ion trap-time-of-flight MS) analyses indicate that the structure of the lipid moieties of steady-state GPI lipids from T. congolense procyclic forms consist of a mixture of lyso-(acyl)PI, diacyl-PI and diacyl-(acyl)PI species. Interestingly, some of these species are myristoylated at the sn-2 position. To our knowledge, this is the first demonstration of lipid remodelling at the level of protein- or polysaccharide-linked GPI anchors in procyclic-form trypanosomes.

An essential bacterial-type cardiolipin synthase mediates cardiolipin formation in a eukaryote

Cardiolipin is important for bacterial and mitochondrial stability and function. The final step in cardiolipin biosynthesis is catalyzed by cardiolipin synthase and differs mechanistically between prokaryotes and eukaryotes. To study the importance of cardiolipin synthesis for mitochondrial integrity, membrane protein complex formation, and cell proliferation in the human and animal pathogenic protozoan parasite, Trypanosoma brucei, we generated conditional cardiolipin synthase-knockout parasites. We found that cardiolipin formation in T. brucei procyclic forms is catalyzed by a bacterial-type cardiolipin synthase, providing experimental evidence for a prokaryotic-type cardiolipin synthase in a eukaryotic organism. Ablation of enzyme expression resulted in inhibition of de novo cardiolipin synthesis, reduction in cellular cardiolipin levels, alterations in mitochondrial morphology and function, and parasite death in culture. By using immunofluorescence microscopy and blue-native gel electrophoresis, cardiolipin synthase was shown to colocalize with inner mitochondrial membrane proteins and to be part of a large protein complex. During depletion of cardiolipin synthase, the levels of cytochrome oxidase subunit IV and cytochrome c1, reflecting mitochondrial respiratory complexes IV and III, respectively, decreased progressively.

Phosphatidylethanolamine is the precursor of the ethanolamine phosphoglycerol moiety bound to eukaryotic elongation factor 1A

In addition to its conventional role during protein synthesis, eukaryotic elongation factor 1A is involved in other cellular processes. Several regions of interaction between eukaryotic elongation factor 1A and the translational apparatus or the cytoskeleton have been identified, yet the roles of the different post-translational modifications of eukaryotic elongation factor 1A are completely unknown. One amino acid modification, which so far has only been found in eukaryotic elongation factor 1A, consists of ethanolamine-phosphoglycerol attached to two glutamate residues that are conserved between mammals and plants. We now report that ethanolamine-phosphoglycerol is also present in eukaryotic elongation factor 1A of the protozoan parasite Trypanosoma brucei, indicating that this unique protein modification is of ancient origin. In addition, using RNA-mediated gene silencing against enzymes of the Kennedy pathway, we demonstrate that phosphatidylethanolamine is a direct precursor of the ethanolamine-phosphoglycerol moiety. Down-regulation of the expression of ethanolamine kinase and ethanolamine-phosphate cytidylyltransferase results in inhibition of phosphatidylethanolamine synthesis in T. brucei procyclic forms and, concomitantly, in a block in glycosylphosphatidylinositol attachment to procyclins and ethanolamine-phosphoglycerol modification of eukaryotic elongation factor 1A.

Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.

Social motility of African trypanosomes is a property of a distinct life-cycle stage that occurs early in tsetse fly transmission

The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen-designated as early procyclic forms-express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4-7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

A subclass of eukaryotic proteins is subject to modification with fatty acids, the most common of which are palmitic and myristic acid. Protein acylation allows association with cellular membranes in the absence of transmembrane domains. Here we examine POMP39, a protein previously described to be present in the outer mitochondrial membrane proteome (POMP) of the protozoan parasite Trypanosoma brucei. POMP39 lacks canonical transmembrane domains, but is likely both myristoylated and palmitoylated on its N-terminus. Interestingly, the protein is also dually localized on the surface of the mitochondrion as well as in the flagellum of both insect-stage and the bloodstream form of the parasites. Upon abolishing of global protein acylation or mutation of the myristoylation site, POMP39 relocates to the cytosol. RNAi-mediated ablation of the protein neither causes a growth phenotype in insect-stage nor bloodstream form trypanosomes.

The Social Life of African Trypanosomes

The unicellular parasite Trypanosoma brucei shuttles between its definitive host, the tsetse fly, and various mammals including humans. In the fly digestive tract, T. brucei must first migrate to the ectoperitrophic space, establish a persistent infection of the midgut and then migrate to the salivary glands before being transmitted to a new mammalian host. In 2010, it was shown that insect stages of the parasite (procyclic forms) exhibit social motility (SoMo) when cultured on a semi-solid surface, and it was postulated that this behaviour might reflect a migration step in the tsetse fly. Now, almost 5 years after the initial report, several new publications shed some light on the biological function of SoMo and provide insights into the underlying signalling pathways.

A new approach to chemotherapy: drug-induced differentiation kills African trypanosomes

Human African trypanosomiasis (sleeping sickness) is a neglected tropical disease caused by Trypanosoma brucei spp. The parasites are transmitted by tsetse flies and adapt to their different hosts and environments by undergoing a series of developmental changes. During differentiation, the trypanosome alters its protein coat. Bloodstream form trypanosomes in humans have a coat of variant surface glycoprotein (VSG) that shields them from the immune system. The procyclic form, the first life-cycle stage to develop in the tsetse fly, replaces the VSG coat by procyclins; these proteins do not protect the parasite from lysis by serum components. Our study exploits the parasite-specific process of differentiation from bloodstream to procyclic forms to screen for potential drug candidates. Using transgenic trypanosomes with a reporter gene in a procyclin locus, we established a whole-cell assay for differentiation in a medium-throughput format. We screened 7,495 drug-like compounds and identified 28 hits that induced expression of the reporter and loss of VSG at concentrations in the low micromolar range. Small molecules that induce differentiation to procyclic forms could facilitate studies on the regulation of differentiation as well as serving as scaffolds for medicinal chemistry for new treatments for sleeping sickness.