Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this proces

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly

PLoS Biol

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met

Complete In Vitro Life Cycle of Trypanosoma congolense: Development of Genetic Tools

Trypanosoma congolense is a parasite responsible for severe disease of African livestock. Its life cycle is complex and divided into two phases, one in the tsetse fly vector and one in the bloodstream of the mammalian host. Molecular tools for gene function analyses in parasitic organisms are essential. Previous studies described the possibility of completing the entire T. congolense life cycle in vitro. However, the model showed major flaws including the absence of stable long-term culture of the infectious bloodstream forms, a laborious time-consuming period to perform the cycle and a lack of genetic tools. We therefore aimed to develop a standardized model convenient for genetic engineering. We succeeded in producing long-term cultures of all the developmental stages on long-term, to define all the differentiation steps and to finally complete the whole cycle in vitro. This improved model offers the opportunity to conduct phenotype analyses of genetically modified strains throughout the in vitro cycle and also during experimental infections

Erythrophagocytosis of desialylated red blood cells is responsible for anaemia during Trypanosoma vivax infection.

International audienceTrypanosomal infection-induced anaemia is a devastating scourge for cattle in widespread regions. Although Trypanosoma vivax is considered as one of the most important parasites regarding economic impact in Africa and South America, very few in-depth studies have been conducted due to the difficulty of manipulating this parasite. Several hypotheses were proposed to explain trypanosome induced-anaemia but mechanisms have not yet been elucidated. Here, we characterized a multigenic family of trans-sialidases in T. vivax, some of which are released into the host serum during infection. These enzymes are able to trigger erythrophagocytosis by desialylating the major surface erythrocytes sialoglycoproteins, the glycophorins. Using an ex vivo assay to quantify erythrophagocytosis throughout infection, we showed that erythrocyte desialylation alone results in significant levels of anaemia during the acute phase of the disease. Characterization of virulence factors such as the trans-sialidases is vital to develop a control strategy against the disease or parasite

Identification of Trans-Sialidases as a Common Mediator of Endothelial Cell Activation by African Trypanosomes

<div><p>Understanding African Trypanosomiasis (AT) host-pathogen interaction is the key to an “anti-disease vaccine”, a novel strategy to control AT. Here we provide a better insight into this poorly described interaction by characterizing the activation of a panel of endothelial cells by bloodstream forms of four African trypanosome species, known to interact with host endothelium. <i>T. congolense</i>, <i>T. vivax</i>, and <i>T. b. gambiense</i> activated the endothelial NF-κB pathway, but interestingly, not <i>T. b. brucei</i>. The parasitic TS (trans-sialidases) mediated this NF-κB activation, remarkably <i>via</i> their lectin-like domain and induced production of pro-inflammatory molecules not only <i>in vitro</i> but also <i>in vivo</i>, suggesting a considerable impact on pathogenesis. For the first time, TS activity was identified in <i>T. b. gambiense</i> BSF which distinguishes it from the subspecies <i>T. b. brucei.</i> The corresponding TS were characterized and shown to activate endothelial cells, suggesting that TS represent a common mediator of endothelium activation among trypanosome species with divergent physiopathologies.</p></div

Activation of BAE by African trypanosomes.

<p>(A) NF-κB immunofluorescent staining on BAE after 16 h of coculture with <i>T. congolense</i> IL3000, <i>T. vivax</i> Y486, <i>T. b. brucei</i> AnTat 1.1 and <i>T. b. gambiense</i> 1135 BSF. Scale bars = 20 µM. (B) Kinetics of BAE activation: percentage of activated BAE in presence of 1 µg/ml LPS and after 2, 6, 16 and 24 h of coculture with BSF of <i>T. congolense</i> IL3000, <i>T. vivax</i> Y486, <i>T. b. brucei</i> AnTat 1.1 and <i>T. b. gambiense</i> 1135. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#s2" target="_blank">Results</a> were similar with <i>T. congolense</i> STIB910 strain, <i>T. b. gambiense</i> LiTat strain, and <i>T. b. brucei</i> 427 strain. Each experiment was repeated at least three times independently. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#s2" target="_blank">Results</a> are expressed as mean-values±standard deviation (SD).</p

SA and TS activities of <i>T. b. gambiense</i> BSF and involvement in BAE activation.

<p>(A) SA and (B) TS activities were measured on crude extracts of <i>T. b. gambiense</i> 1135 and LiTat BSF and compared with previous results on <i>T. congolense</i>, <i>T. vivax</i> and <i>T. b. brucei</i> BSF <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#ppat.1003710-Coustou1" target="_blank">[21]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#ppat.1003710-Guegan1" target="_blank">[22]</a>. Activities are expressed as nmol of sialic acid released or transferred per min by 10⁹ lysed parasites. (C) Percentage of activated BAE by <i>T. b. brucei</i> 427 expressing heterologous TS of <i>T. b. gambiense T. b. brucei TbgSA B</i> and <i>T. b. brucei TbgSA B</i> compared to non-transfected and transfection control cell lines. (D) Effect of the TS inhibitor myricetin on BAE activation by <i>T. b. gambiense</i> 1135 BSF. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#s2" target="_blank">Results</a> are represented as percentage of activation capacity and normalized to the control (absence of myricetin). Myricetin concentration is indicated on the X-axis. Data are expressed as mean values±SD of three independent experiments. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#ppat.1003710.s005" target="_blank">Fig. S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#ppat.1003710.s006" target="_blank">S6</a>, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#ppat.1003710.s008" target="_blank">Table S2</a>.</p