A study on the maturation of procyclic Trypanosoma brucei brucei in Glossina morsitans centralis and G. brevipalpis

Abstract. Teneral Glossina morsitans centralis and G. brevipalpis were fed in vitro upon medium containing procyclic Trypanosoma brucei brucei derived from the midguts of G. m. centralis or G. brevipalpis which had immature trypanosome infections. The tsetse were then maintained on rabbits and, on day 31, were dissected to determine the infection rates. In G. m. centralis the midgut and salivary gland infection rates by T. b. brucei were 46.0% and 27.0% with procyclic trypanosomes from G. m. centralis, and 45.4% and 24.7% with procyclic trypanosomes from G. brevipalpis, respectively. In G. brevipalpis the rates were 20.2% and 0.0% with procyclic trypanosomes from G. m. centralis, and 28.0% and 0.0% with procyclic trypanosomes from G. brevipalpis, respectively. Teneral G. m. centralis and G. brevipalpis were also fed similarly upon procyclic T. b. brucei derived from G.m.centralis or G. brevipalpis on day 31 of infection, the former tsetse species had mature infections while the latter were without infections in the salivary glands. In G.m.centralis the infection rates in the midgut and salivary glands were 48.9% and 17.0%, and 38.0% and 17.0% when fed on procyclic trypanosomes from G.m.centralis and G. brevipalpis, respectively. In G. brevipalpis the rates were 21.5% and 0.0%, and 10.7% and 0.0% with procyclic trypanosomes of G.m.centralis and G. brevipalpis origin, respectively. Thus, procyclic T. b. brucei from susceptible G.m.centralis could not complete cyclical development in refractory G. brevipalpis, whereas those from G. brevipalpis developed to metatrypanosomes in the salivary glands of G.m.centralis. Teneral and 15-day-old non-teneral G.m.centralis were fed in vitro upon heparinized goat's blood containing T. b. brucei bloodstream trypomastigotes, or upon medium containing procyclic T. b. brucei derived from G.m.centralis with mature infections. On day 31 their infection rates were determined. The infection rates by T. b. brucei in the midgut and salivary glands of G.m.centralis fed on the infected blood were 70.4% and 40.4% when fed as teneral tsetse, as against 15.3% and 4.0% when fed as non-teneral tsetse. Those tsetse which were fed on the medium containing procyclic trypanosomes showed rates of 50.0% and 25.6%, as against 11.6% and 2.5%, respectively. It would appear, therefore, that maturation of T. b. brucei in tsetse is probably not determined simply by an interaction between lectin and procyclic trypanosomes in the midgut of non-teneral tsetse, but it is the result of a complex interaction between many interrelated physiological factors of both the trypanosome and the tsetse vector.     

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.     

Major Surface Glycoproteins of Insect Forms of Trypanosoma brucei Are Not Essential for Cyclical Transmission by Tsetse

Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.     

Trypanosoma brucei: Reduction of GPI-phospholipase C protein during differentiation is dependent on replication of newly transformed cells

The protozoan parasite Trypanosoma brucei lives in the bloodstream of vertebrates or in a tsetse fly. Expression of a GPI-phospholipase C polypeptide (GPI-PLCp) in the parasite is restricted to the bloodstream form. Events controlling the amount of GPI-PLCp expressed during differentiation are not completely understood. Our metabolic “pulse-chase” analysis reveals that GPI-PLCp is stable in bloodstream form. However, during differentiation of bloodstream to insect stage (procyclic) T. brucei, translation GPI-PLC mRNA ceases within 8 h of initiating transformation. GPI-PLCp is not lost precipitously from newly transformed procyclic trypanosomes. Nascent procyclics contain 400-fold more GPI-PLCp than established insect stage T. brucei. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication. Sixteen cell divisions are required to reduce the amount of GPI-PLCp in newly differentiated procyclics to levels present in the established procyclic. GPI-PLCp is retained in strains of T. brucei that fail to replicate after differentiation of the bloodstream to the procyclic form. Thus, at least two factors control levels of GPI-PLCp during differentiation of bloodstream T. brucei; (i) repression of GPI-PLC mRNA translation, and (ii) sustained replication of newly transformed procyclic T. brucei. These studies illustrate the importance of repeated cell divisions in controlling the steady-state amount of GPI-PLCp during differentiation of the African trypanosome.     

Quantitative studies on trypanosomes in tsetse tissue culture

Tissues from pupae of Glossina morsitans of various ages were cultured in modified Trager's medium. Cellular outgrowths were produced from explants of proventriculus, brain, and imaginal body wall and large vesicles were extruded from pieces of midgut of young pupae. Complete alimentary tract from older pupae displayed rhythmic contractions for up to 3 weeks. When Trypanosoma brucei and T. congolense in mouse blood were added to hanging drop cultures of tsetse tissues and incubated at 28 C, the organisms multiplied and changed into forms morphologically similar to those found in the tsetse fly midgut. The trypanosomes were maintained for 30 days by serial passage at 5-day intervals. The growth of T. brucei in the presence of different pupal tissues was studied. Of all the tissues tested the complete alimentary tract from pupae older than 21 days gave the best results. Growth also occurred when the trypanosomes were separated from the insect tissue by a semipermeable membrane. The trypanosomes failed to grow in (a) culture medium alone, (b) media containing extracts of alimentary canal and (c) medium in which alimentary tract had been cultured for 3 or 4 days.     

Targeting the tsetse-trypanosome interplay using genetically engineered Sodalis glossinidius

Sodalis glossinidius, a secondary bacterial symbiont of the tsetse fly, is currently considered as a potential delivery system for anti-trypanosomal components interfering with African trypanosome transmission (i.e. paratransgenesis). Nanobodies (Nbs) have been proposed as potential candidates to target the parasite during development in the tsetse fly. In this study, we have generated an immune Nb-library and developed a panning strategy to select Nbs against the Trypanosoma brucei brucei procyclic developmental stage present in the tsetse fly midgut. Selected Nbs were expressed, purified, assessed for binding and tested for their impact on the survival and growth of in vitro cultured procyclic T. b. brucei parasites. Next, we engineered S. glossinidius to express the selected Nbs and validated their ability to block T. brucei development in the tsetse fly midgut. Genetically engineered S. glossinidius expressing Nb_88 significantly compromised parasite development in the tsetse fly midgut both at the level of infection rate and parasite load. Interestingly, expression of Nb_19 by S. glossinidius resulted in a significantly enhanced midgut establishment. These data are the first to show in situ delivery by S. glossinidius of effector molecules that can target the trypanosome-tsetse fly crosstalk, interfering with parasite development in the fly. These proof-of-principle data represent a major step forward in the development of a control strategy based on paratransgenic tsetse flies. Finally, S. glossinidius-based Nb delivery can also be applied as a powerful laboratory tool to unravel the molecular determinants of the parasite-vector association.     

Molecular characterization of a tsetse fly midgut proteolytic lectin that mediates differentiation of African trypanosomes

Differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms is an important first step in the establishment of an infection within the tsetse fly. This complex process is mediated by a wide variety of factors, including those associated with the vector itself, the trypanosomes and the bloodmeal. As part of an on-going project in our laboratory, we recently isolated and characterized a bloodmeal-induced molecule with both lectin and trypsin activities from midguts of the tsetse fly, Glossina longipennis [Osir, E.O., Abubakar, L., Imbuga, M.O., 1995. Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly, Glossina longipennis. Parasitol. Res. 81, 276-281]. The protein (lectin-trypsin complex) was found to be capable of stimulating differentiation of bloodstream trypanosomes in vitro. Using polyclonal antibodies to the complex, we screened a G. fuscipes fuscipes cDNA midgut expression library and identified a putative proteolytic lectin gene. The cDNA encodes a putative mature polypeptide with 274 amino acids (designated Glossina proteolytic lectin, Gpl). The deduced amino acid sequence includes a hydrophobic signal peptide and a highly conserved N-terminal sequence motif. The typical features of serine protease trypsin family of proteins found in the sequence include the His/Asp/Ser active site triad with the conserved residues surrounding it, three pairs of cysteine residues for disulfide bridges and an aspartate residue at the specificity pocket. Expression of the gene in a bacterial expression system yielded a protein (M(r) approximately 32,500). The recombinant protein (Gpl) bound d(+) glucosamine and agglutinated bloodstream-form trypanosomes and rabbit red blood cells. In addition, the protein was found to be capable of inducing transformation of bloodstream-form trypanosomes into procyclic forms in vitro. Antibodies raised against the recombinant protein showed cross-reactivity with the alpha subunit of the lectin-trypsin complex. These results support our earlier hypothesis that this molecule is involved in the establishment of trypanosome infections in tsetse flies.     

Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei

Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4–5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4–5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse.     

Development of Real Time PCR to Study Experimental Mixed Infections of T. congolense Savannah and T. b. brucei in Glossina morsitans morsitans

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut.     

A comparative analysis of trypanosomatid SNARE proteins

The Kinetoplastida are flagellated protozoa evolutionary distant and divergent from yeast and humans. Kinetoplastida include trypanosomatids, and a number of important pathogens. Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. inflict significant morbidity and mortality on humans and livestock as the etiological agents of human African trypanosomiasis, Chagas' disease and leishmaniasis respectively. For all of these organisms, intracellular trafficking is vital for maintenance of the host–pathogen interface, modulation/evasion of host immune system responses and nutrient uptake. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are critical components of the intracellular trafficking machinery in eukaryotes, mediating membrane fusion and contributing to organelle specificity. We asked how the SNARE complement evolved across the trypanosomatids. An in silico search of the predicted proteomes of T. b. brucei and T. cruzi was used to identify candidate SNARE sequences. Phylogenetic analysis, including comparisons with yeast and human SNAREs, allowed assignment of trypanosomatid SNAREs to the Q or R subclass, as well as identification of several SNAREs orthologous with those of opisthokonts. Only limited variation in number and identity of SNAREs was found, with Leishmania major having 27 and T. brucei 26, suggesting a stable SNARE complement post-speciation. Expression analysis of T. brucei SNAREs revealed significant differential expression between mammalian and insect infective forms, especially within R and Qb-SNARE subclasses, suggesting possible roles in adaptation to different environments. For trypanosome SNAREs with clear orthologs in opisthokonts, the subcellular localization of TbVAMP7C is endosomal while both TbSyn5 and TbSyn16B are at the Golgi complex, which suggests conservation of localization and possibly also function. Despite highly distinct life styles, the complement of trypanosomatid SNAREs is quite stable between the three pathogenic lineages, suggesting establishment in the last common ancestor of trypanosomes and Leishmania. Developmental changes to SNARE mRNA levels between blood steam and procyclic life stages suggest that trypanosomes modulate SNARE functions via expression. Finally, the locations of some conserved SNAREs have been retained across the eukaryotic lineage.     

Trypanosoma brucei brucei: Effects of ferrous iron and heme on ecto-nucleoside triphosphate diphosphohydrolase activity

Trypanosoma brucei brucei is the causative agent of animal African trypanosomiasis, also called nagana. Procyclic vector form resides in the midgut of the tsetse fly, which feeds exclusively on blood. Hemoglobin digestion occurs in the midgut resulting in an intense release of free heme. In the present study we show that the magnesium-dependent ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of procyclic T. brucei brucei is inhibited by ferrous iron and heme. The inhibition of E-NTPDase activity by ferrous iron, but not by heme, was prevented by pre-incubation of cells with catalase. However, antioxidants that permeate cells, such as PEG-catalase and N-acetyl-cysteine prevented the inhibition of E-NTPDase by heme. Ferrous iron was able to induce an increase in lipid peroxidation, while heme did not. Therefore, both ferrous iron and heme can inhibit E-NTPDase activity of T. brucei brucei by means of formation of reactive oxygen species, but apparently acting through distinct mechanisms.     

Trypanosoma brucei AMP-activated kinase subunit homologs influence surface molecule expression

The African trypanosome, Trypanosoma brucei, can gauge its environment by sensing nutrient availability. For example, procyclic form (PF) trypanosomes monitor changes in glucose levels to regulate surface molecule expression, which is important for survival in the tsetse fly vector. The molecular connection between glycolysis and surface molecule expression is unknown. Here we partially characterize T. brucei homologs of the β and γ subunits of the AMP-activated protein kinase (AMPK), and determine their roles in regulating surface molecule expression. Using flow cytometry and mass spectrometry, we found that TbAMPKβ or TbAMPKγ-deficient parasites express both of the major surface molecules, EP- and GPEET-procyclin, with the latter being a form that is expressed when glucose is low such as in the tsetse fly. Last, we have found that the putative scaffold component of the complex, TbAMPKβ, fractionates with organellar components and colocalizes in part with a glycosomal marker as well as the flagellum of PF parasites.     

The Trypanosoma brucei Life Cycle Switch TbPTP1 Is Structurally Conserved and Dephosphorylates the Nucleolar Protein NOPP44/46

Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development. Moreover, inactivation of T. brucei protein-tyrosine phosphatase 1 (TbPTP1) triggers differentiation of bloodstream stumpy forms into tsetse procyclic forms through unknown downstream effects. Here, we link these events by showing that NOPP44/46 is a major substrate of TbPTP1. TbPTP1 substrate-trapping mutants selectively enrich NOPP44/46 from procyclic stage cell lysates, and TbPTP1 efficiently and selectively dephosphorylates NOPP44/46 in vitro. To provide insights into the mechanism of NOPP44/46 recognition, we determined the crystal structure of TbPTP1. The TbPTP1 structure, the first of a kinetoplastid protein-tyrosine phosphatase (PTP), emphasizes the conservation of the PTP fold, extending to one of the most diverged eukaryotes. The structure reveals surfaces that may mediate substrate specificity and affords a template for the design of selective inhibitors to interfere with T. brucei transmission.     

Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina

The activity of lectins in different species of tsetse was compared in vivo by the time taken to remove all trypanosomes from the midgut following an infective feed and in vitro by agglutination tests. Teneral male Glossina pallidipes Austen, G. austeni Newstead and G. p. palpalis R-D. removed 50% of all Trypanosoma brucei rhodesiense Stephens & Fantham infections within 60 h. A 'refractory' line of G. m. morsitans Westwood took 170 h to kill 50% infections while a 'susceptible' line of the same species failed to kill 50%. Agglutination tests with midgut homogenates showed differences between fly stocks which accorded with differences in rate of trypanosome killing in vivo. Flies fed before an infective feed were able to remove trypanosomes from their midguts more quickly than flies infected as tenerals. Increasing the period of starvation before infection increased the susceptibility to trypanosome infection of non-teneral flies. Teneral flies showed little agglutinating activity in vitro, suggesting that lectin is produced in response to the bloodmeal. Feeding flies before infection also abolished the differences in rate of trypanosome killing found between teneral 'susceptible' and 'refractory' G. m. morsitans, suggesting that maternally inherited susceptibility to trypanosome infection is a phenomenon limited to teneral flies. Electron micrographs of midguts of G. m. morsitans suggest that procyclic trypanosomes are killed by cell lysis, presumably the result of membrane damage caused by lectin action.     

Tsetse EP Protein Protects the Fly Midgut from Trypanosome Establishment

African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis.     

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.     

Trypanosoma brucei s.l.: Microsatellite markers revealed high level of multiple genotypes in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus of Cameroon

To identify Trypanosoma brucei genotypes which are potentially transmitted in a sleeping sickness focus, microsatellite markers were used to characterize T. brucei found in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus in Cameroon. For this study, two entomological surveys were performed during which 2685 tsetse flies were collected and 1596 (59.2%) were dissected. Microscopic examination revealed 1.19% (19/1596) mid-gut infections with trypanosomes; the PCR method identified 4.7% (75/1596) infections with T. brucei in the mid-guts. Of these 75 trypanosomes identified in the mid-guts, Trypanosoma brucei gambiense represented 0.81% (13/1596) of them, confirming the circulation of human infective parasite in the Fontem focus. Genetic characterization of the 75 T. brucei samples using five microsatellite markers revealed not only multiple T. brucei genotypes (47%), but also single genotypes (53%) in the mid-guts of the wild tsetse flies. These results show that there is a wide range of trypanosome genotypes circulating in the mid-guts of wild tsetse flies from the Fontem sleeping sickness focus. They open new avenues to undertake investigations on the maturation of multiple infections observed in the tsetse fly mid-guts. Such investigations may allow to understand how the multiple infections evolve from the tsetse flies mid-guts to the salivary glands and also to understand the consequence of these evolutions on the dynamic (which genotype is transmitted to mammals) of trypanosomes transmission.     

Expression of the human RNA-binding protein HuR in Trypanosoma brucei increases the abundance of mRNAs containing AU-rich regulatory elements

The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian ‘bloodstream form’ trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.