A multigene family encoding surface glycoproteins in Trypanosoma congolense

Trypanosoma congolense, the causative agent of the most important livestock disease in Africa, expresses specific surface proteins involved in its parasitic lifestyle. Unfortunately, the complete repertoire of such molecules is far from being deciphered. As these membrane components are exposed to the host environment, they could be used as therapeutic or diagnostic targets. By mining the T. congolense genome database, we identified a novel family of lectin-like glycoproteins (TcoClecs). These molecules are predicted to have a transmembrane domain, a tandem repeat amino acid motif, a signal peptide and a C-type lectin-like domain (CTLD). This paper depicts several experimental arguments in favor of a surface localization in bloodstream forms of T. congolense. A TcoClec gene was heterologously expressed in U-2 OS cells and the product could be partially found at the plasma membrane. TcoClecs were also localized at the surface of T. congolense bloodstream forms. The signal was suppressed when the cells were treated with a detergent to remove the plasma membrane or with trypsin to « shave » the parasites and remove their external proteins. This suggests that TcoClecs could be potential diagnostic or therapeutic antigens of African animal trypanosomiasis. The potential role of these proteins in T. congolense as well as in other trypanosomatids is discussed

Confining Trypanosoma brucei in emulsion droplets reveals population variabilities in division rates and improves in vitro cultivation

Trypanosome parasites are infecting mammals in Sub-Saharan Africa and are transmitted between hosts through bites of the tsetse fly. The transmission from the insect vector to the mammal host causes a number of metabolic and physiological changes. A fraction of the population continuously adapt to the immune system of the host, indicating heterogeneity at the population level. Yet, the cell to cell variability in populations is mostly unknown. We develop here an analytical method for quantitative measurements at the single cell level based on encapsulation and cultivation of single-cell Trypanosoma brucei in emulsion droplets. We first show that mammalian stage trypanosomes survive for several hours to days in droplets, with an influence of droplet size on both survival and growth. We unravel various growth patterns within a population and find that droplet cultivation of trypanosomes results in 10-fold higher cell densities of the highest dividing cell variants compared to standard cultivation techniques. Some variants reach final cell titers in droplets closer to what is observed in nature than standard culture, of practical interest for cell production. Droplet microfluidics is therefore a promising tool for trypanosome cultivation and analysis with further potential for high-throughput single cell trypanosome analysis

Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness ( Trypanosoma brucei ), Chagas disease ( Trypanosoma cruzi ), and various forms of leishmaniasis ( Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including T. congolense ). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 ( Sp Cas9) and an in vitro -synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense , and Leishmania promastigotes. The functionality of Sp Cas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics. Importantly, this approach is adaptable to any wild-type parasite, including field isolates

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

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

PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation

Effect of upper- and lower-level baroclinicity on the persistence of the leading mode of midlatitude jet variability

The sensitivity of the variability of an eddy-driven jet to the upper- and lower-level baroclinicity of the mean state is analyzed using a three-level quasi-geostrophic model on the sphere. The model is forced by a relaxation in temperature to a steady, zonally symmetric profile with varying latitude and intensity of the maximum baroclinicity. The leading EOF of the zonally- and vertically-averaged zonal wind is characterized by a meridional shift of the eddy-driven jet. While changes in the upper-level baroclinicity have no significant impact on the persistence of this leading EOF, an increase in lower-level baroclinicity leads to a reduced persistence. For small lower-level baroclinicity, the leading EOF follows a classical zonal index regime, for which the meridional excursions of the zonal wind anomalies are maintained by a strong positive eddy feedback. For strong lower-level baroclinicity, the jet enters a poleward propagation regime, for which the eddy forcing continuously acts to push the jet poleward and prevents its maintenance at a fixed latitude. The enhanced poleward propagation when the lower-level baroclinicity increases is interpreted as resulting from the broader and weaker potential vorticity gradient which enables the waves to propagate equatorward and facilitates the poleward migration of the critical latitude. Finally, the decrease in EOF1 persistence as the lower-level baroclinicity increases is shown not to result from the impact of changes in the mean climatological jet latitude

Dynamical mechanisms acting on the persistence of meridional shifting and amplitude pulsing of eddy-driven jets

International audienceThe dynamics of mid-latitudes eddy-driven jets is investigated in a long-term integration of a dry three-level quasigeostrophic model on the sphere. As for most observed jets, the leading mode of variability (obtained using the Empirical Orthogonal Functions method) of the zonal-mean wind corresponds to latitudinal shifts of the jet, and the second mode to pulses of the jet speed. The first principal component (PC1) is also more persistent than the second one (PC2), showing that meridional shifts persists longer than amplitude pulses; this longer persistence arises from different eddy feedbacks both in the short term (i.e., within a few days following the peak of the PCs) and in the long term. The short-term eddy feedbacks come from two distinct mechanisms. First, a planetary waveguide effect acts as a negative feedback on both PCs. The positive phases of PC1 and PC2, which correspond to poleward-shifted and accelerated jets, respectively, are first driven then canceled by planetary waves reflecting on the equatorial flank of the jet. A similar process occurs for the negative phases when planetary waves reflect on the polar flank of the jet. Second, synoptic waves also exert a short-term negative feedback on PC2: when the jet accelerates, the enhanced meridional wind shear increases the barotropic sink of eddy energy and depletes it very rapidly, therefore preventing synoptic eddies from maintaining the accelerated jet. Finally, at lags longer than their typical time scale, synoptic eddies drive a positive feedback on PC1 only. This feedback can be explained by a baroclinic mechanism in which the jet shift modifies the baroclinicity, causing, first, eddy heat flux anomalies and then, momentum convergence anomalies. This feedback is absent for PC2, despite some changes in the baroclinicity

Positive and Negative Eddy Feedbacks Acting on Midlatitude Jet Variability in a Three-Level Quasigeostrophic Model

International audienceThe variability of midlatitude jets is investigated in a long-term integration of a dry three-level qua-sigeostrophic model on the sphere. As for most observed jets, the leading EOF of the zonal-mean wind corresponds to latitudinal shifts of the jet, and the second EOF to pulses of the jet speed. The first principal component (PC1) is also more persistent than the second one (PC2); this longer persistence arises from different eddy feedbacks both in the short term (i.e., within a few days following the peak of the PCs) and in the long term. The short-term eddy feedbacks come from two distinct mechanisms. First, a planetary waveguide effect acts as a negative feedback on both PCs. The positive phases of PC1 and PC2, which correspond to poleward-shifted and accelerated jets, respectively, are first driven then canceled by planetary waves reflecting on the equatorward flank of the jet. A similar process occurs for the negative phases when planetary waves reflect on the poleward flank of the jet. Second, synoptic waves also exert a short-term negative feedback on PC2: when the jet accelerates, the enhanced meridional wind shear increases the barotropic sink of eddy energy and depletes it very rapidly, therefore preventing synoptic eddies from maintaining the accelerated jet. Finally, at lags longer than their typical time scale, synoptic eddies drive a positive feedback on PC1 only. This feedback can be explained by a baroclinic mechanism in which the jet shift modifies the baroclinicity, causing, first, eddy heat flux anomalies and, then, momentum convergence anomalies. This feedback is absent for PC2, despite some changes in the baroclinicity