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.     

Regulators of Trypanosoma brucei Cell Cycle Progression and Differentiation Identified Using a Kinome-Wide RNAi Screen

The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite''s protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite''s 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2), depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.     

Structural characterization reveals a novel bilobed architecture for the ectodomains of insect stage expressed Trypanosoma brucei PSSA‐2 and Trypanosoma congolense ISA

African trypanosomiasis, caused by parasites of the genus Trypanosoma, is a complex of devastating vector‐borne diseases of humans and livestock in sub‐Saharan Africa. Central to the pathogenesis of African trypanosomes is their transmission by the arthropod vector, Glossina spp. (tsetse fly). Intriguingly, the efficiency of parasite transmission through the vector is reduced following depletion of Trypanosoma brucei Procyclic‐Specific Surface Antigen‐2 (TbPSSA‐2). To investigate the underlying molecular mechanism of TbPSSA‐2, we determined the crystal structures of its ectodomain and that of its homolog T. congolense Insect Stage Antigen (TcISA) to resolutions of 1.65 Å and 2.45 Å, respectively using single wavelength anomalous dispersion. Both proteins adopt a novel bilobed architecture with the individual lobes displaying rotational flexibility around the central tether that suggest a potential mechanism for coordinating a binding partner. In support of this hypothesis, electron density consistent with a bound peptide was observed in the inter‐lob cleft of a TcISA monomer. These first reported structures of insect stage transmembrane proteins expressed by African trypanosomes provide potentially valuable insight into the interface between parasite and tsetse vector.     

Vitamin B6 Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B₆) as a cofactor. This vitamin can be synthesized by tsetse''s obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B₆ for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse''s reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B₆ metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B₆ in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B₆ production.     

An Investigation into the Protein Composition of the Teneral Glossina morsitans morsitans Peritrophic Matrix

Background

Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism.

Methods

PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS.

Results

Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM.

Conclusion

To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.     

Trypanosome Infection Establishment in the Tsetse Fly Gut Is Influenced by Microbiome-Regulated Host Immune Barriers

Tsetse flies (Glossina spp.) vector pathogenic African trypanosomes, which cause sleeping sickness in humans and nagana in domesticated animals. Additionally, tsetse harbors 3 maternally transmitted endosymbiotic bacteria that modulate their host''s physiology. Tsetse is highly resistant to infection with trypanosomes, and this phenotype depends on multiple physiological factors at the time of challenge. These factors include host age, density of maternally-derived trypanolytic effector molecules present in the gut, and symbiont status during development. In this study, we investigated the molecular mechanisms that result in tsetse''s resistance to trypanosomes. We found that following parasite challenge, young susceptible tsetse present a highly attenuated immune response. In contrast, mature refractory flies express higher levels of genes associated with humoral (attacin and pgrp-lb) and epithelial (inducible nitric oxide synthase and dual oxidase) immunity. Additionally, we discovered that tsetse must harbor its endogenous microbiome during intrauterine larval development in order to present a parasite refractory phenotype during adulthood. Interestingly, mature aposymbiotic flies (Gmm ^Apo) present a strong immune response earlier in the infection process than do WT flies that harbor symbiotic bacteria throughout their entire lifecycle. However, this early response fails to confer significant resistance to trypanosomes. Gmm ^Apo adults present a structurally compromised peritrophic matrix (PM), which lines the fly midgut and serves as a physical barrier that separates luminal contents from immune responsive epithelial cells. We propose that the early immune response we observe in Gmm ^Apo flies following parasite challenge results from the premature exposure of gut epithelia to parasite-derived immunogens in the absence of a robust PM. Thus, tsetse''s PM appears to regulate the timing of host immune induction following parasite challenge. Our results document a novel finding, which is the existence of a positive correlation between tsetse''s larval microbiome and the integrity of the emerging adult PM gut immune barrier.     

Identification of Tsetse (Glossina spp.) Using Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry

Glossina (G.) spp. (Diptera: Glossinidae), known as tsetse flies, are vectors of African trypanosomes that cause sleeping sickness in humans and nagana in domestic livestock. Knowledge on tsetse distribution and accurate species identification help identify potential vector intervention sites. Morphological species identification of tsetse is challenging and sometimes not accurate. The matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF MS) technique, already standardised for microbial identification, could become a standard method for tsetse fly diagnostics. Therefore, a unique spectra reference database was created for five lab-reared species of riverine-, savannah- and forest- type tsetse flies and incorporated with the commercial Biotyper 3.0 database. The standard formic acid/acetonitrile extraction of male and female whole insects and their body parts (head, thorax, abdomen, wings and legs) was used to obtain the flies'' proteins. The computed composite correlation index and cluster analysis revealed the suitability of any tsetse body part for a rapid taxonomical identification. Phyloproteomic analysis revealed that the peak patterns of G. brevipalpis differed greatly from the other tsetse. This outcome was comparable to previous theories that they might be considered as a sister group to other tsetse spp. Freshly extracted samples were found to be matched at the species level. However, sex differentiation proved to be less reliable. Similarly processed samples of the common house fly Musca domestica (Diptera: Muscidae; strain: Lei) did not yield any match with the tsetse reference database. The inclusion of additional strains of morphologically defined wild caught flies of known origin and the availability of large-scale mass spectrometry data could facilitate rapid tsetse species identification in the future.     

Modulation of cellular insulin signaling and PTP1B effects by lipid metabolites in skeletal muscle cells

Normal glucose regulation is achieved by having adequate insulin secretion and effective glucose uptake/disposal. Excess lipids in peripheral tissues — skeletal muscle, liver and adipose tissue — may attenuate insulin signaling through the protein kinase B (AKt) pathway and up-regulate protein tyrosine phosphatase 1B (PTP1B), a negative regulator of insulin signaling. We studied accumulation of lipid metabolites [triglycerides (TAGs), diglycerides (DAGs)] and ceramides in relation to insulin signaling and expression and phosphorylation of PTP1B by preincubating rat skeletal muscle cells (L6 myotubes) with three saturated and three unsaturated free fatty acids (FFAs) (200 μM). Cells were also evaluated in the presence of wortmannin, an inhibitor of phosphatidylinositol 3-kinases and thus AKt (0–100 nM). Unsaturated FFAs increased DAGs, TAGs and PTP1B expression significantly, but cells remained insulin sensitive as assessed by robust AKt and PTP1B phosphorylation at serine (Ser) 50, Ser 398 and tyrosine 152. Saturated palmitic and stearic acids increased ceramides, up-regulated PTP1B, and had AKt and PTP1B phosphorylation at Ser 50 impaired. We show a significant correlation between phosphorylation levels of AKt and of PTP1B at Ser 50 (R²=0.84, P<.05). The same was observed with increasing wortmannin dose (R²=0.73, P<.05). Only FFAs that increased ceramides caused impairment of AKt and PTP1B phosphorylation at Ser 50. PTP1B overexpression in the presence of excess lipids may not directly cause insulin resistance unless it is accompanied by decreased PTP1B phosphorylation. A clear relationship between PTP1B phosphorylation levels at Ser 50 and its negative effect on insulin signaling is shown.     

Tsetse blood-meal sources,endosymbionts and trypanosome-associations in the Maasai Mara National Reserve,a wildlife-human-livestock interface

African trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Knowledge on tsetse fly vertebrate hosts and the influence of tsetse endosymbionts on trypanosome presence, especially in wildlife-human-livestock interfaces, is limited. We identified tsetse species, their blood-meal sources, and correlations between endosymbionts and trypanosome presence in tsetse flies from the trypanosome-endemic Maasai Mara National Reserve (MMNR) in Kenya. Among 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosome DNA, most (17/28) being of Trypanosoma vivax species. Blood-meal analyses based on high-resolution melting analysis of vertebrate cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 354) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Flies positive for trypanosome DNA had fed on hippopotamus and buffalo. Tsetse flies were more likely to be positive for trypanosomes if they had the Sodalis glossinidius endosymbiont (P = 0.0002). These findings point to complex interactions of tsetse flies with trypanosomes, endosymbionts, and diverse vertebrate hosts in wildlife ecosystems such as in the MMNR, which should be considered in control programs. These interactions may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes. Although the African buffalo is a key reservoir of AT, the higher proportion of hippopotamus blood-meals in flies with trypanosome DNA indicates that other wildlife species may be important in AT transmission. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis risk. Our results add to existing data suggesting that Sodalis endosymbionts are associated with increased trypanosome presence in tsetse flies.     

Diversity of trypanosomes in humans and cattle in the HAT foci Mandoul and Maro,Southern Chad—A matter of concern for zoonotic potential?

BackgroundAfrican trypanosomes are parasites mainly transmitted by tsetse flies. They cause trypanosomiasis in humans (HAT) and animals (AAT). In Chad, HAT/AAT are endemic. This study investigates the diversity and distribution of trypanosomes in Mandoul, an isolated area where a tsetse control campaign is ongoing, and Maro, an area bordering the Central African Republic (CAR) where the control had not started.Methods717 human and 540 cattle blood samples were collected, and 177 tsetse flies were caught. Trypanosomal DNA was detected using PCR targeting internal transcribed spacer 1 (ITS1) and glycosomal glyceraldehyde-3 phosphate dehydrogenase (gGAPDH), followed by amplicon sequencing.ResultsTrypanosomal DNA was identified in 14 human samples, 227 cattle samples, and in tsetse. Besides T. b. gambiense, T. congolense was detected in human in Maro. In Mandoul, DNA from an unknown Trypanosoma sp.-129-H was detected in a human with a history of a cured HAT infection and persisting symptoms. In cattle and tsetse samples from Maro, T. godfreyi and T. grayi were detected besides the known animal pathogens, in addition to T. theileri (in cattle) and T. simiae (in tsetse). Furthermore, in Maro, evidence for additional unknown trypanosomes was obtained in tsetse. In contrast, in the Mandoul area, only T. theileri, T. simiae, and T. vivax DNA was identified in cattle. Genetic diversity was most prominent in T. vivax and T. theileri.ConclusionTsetse control activities in Mandoul reduced the tsetse population and thus the pathogenic parasites. Nevertheless, T. theileri, T. vivax, and T. simiae are frequent in cattle suggesting transmission by other insect vectors. In contrast, in Maro, transhumance to/from Central African Republic and no tsetse control may have led to the high diversity and frequency of trypanosomes observed including HAT/AAT pathogenic species. Active HAT infections stress the need to enforce monitoring and control campaigns. Additionally, the diverse trypanosome species in humans and cattle indicate the necessity to investigate the infectivity of the unknown trypanosomes regarding their zoonotic potential. Finally, this study should be widened to other trypanosome hosts to capture the whole diversity of circulating trypanosomes.     

Protein tyrosine phosphatase TbPTP1: A molecular switch controlling life cycle differentiation in trypanosomes

Differentiation in African trypanosomes (Trypanosoma brucei) entails passage between a mammalian host, where parasites exist as a proliferative slender form or a G0-arrested stumpy form, and the tsetse fly. Stumpy forms arise at the peak of each parasitaemia and are committed to differentiation to procyclic forms that inhabit the tsetse midgut. We have identified a protein tyrosine phosphatase (TbPTP1) that inhibits trypanosome differentiation. Consistent with a tyrosine phosphatase, recombinant TbPTP1 exhibits the anticipated substrate and inhibitor profile, and its activity is impaired by reversible oxidation. TbPTP1 inactivation in monomorphic bloodstream trypanosomes by RNA interference or pharmacological inhibition triggers spontaneous differentiation to procyclic forms in a subset of committed cells. Consistent with this observation, homogeneous populations of stumpy forms synchronously differentiate to procyclic forms when tyrosine phosphatase activity is inhibited. Our data invoke a new model for trypanosome development in which differentiation to procyclic forms is prevented in the bloodstream by tyrosine dephosphorylation. It may be possible to use PTP1B inhibitors to block trypanosomatid transmission.     

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.     

Structural characterization and epitope mapping of the glutamic acid/alanine-rich protein from Trypanosoma congolense: defining assembly on the parasite cell surface

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.     

Protein Tyrosine Phosphatase 1B Regulates Pyruvate Kinase M2 Tyrosine Phosphorylation

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and adiposity and is a drug target for the treatment of obesity and diabetes. Here we identify pyruvate kinase M2 (PKM2) as a novel PTP1B substrate in adipocytes. PTP1B deficiency leads to increased PKM2 total tyrosine and Tyr¹⁰⁵ phosphorylation in cultured adipocytes and in vivo. Substrate trapping and mutagenesis studies identify PKM2 Tyr-105 and Tyr-148 as key sites that mediate PTP1B-PKM2 interaction. In addition, in vitro analyses illustrate a direct effect of Tyr-105 phosphorylation on PKM2 activity in adipocytes. Importantly, PTP1B pharmacological inhibition increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity. Moreover, PKM2 Tyr-105 phosphorylation is regulated nutritionally, decreasing in adipose tissue depots after high-fat feeding. Further, decreased PKM2 Tyr-105 phosphorylation correlates with the development of glucose intolerance and insulin resistance in rodents, non-human primates, and humans. Together, these findings identify PKM2 as a novel substrate of PTP1B and provide new insights into the regulation of adipose PKM2 activity.     

Forward motility is essential for trypanosome infection in the tsetse fly

African trypanosomes are flagellated protozoan parasites transmitted by the bite of tsetse flies and responsible for sleeping sickness in humans. Their complex development in the tsetse digestive tract requires several differentiation and migration steps that are thought to rely on trypanosome motility. We used a functional approach in vivo to demonstrate that motility impairment prevents trypanosomes from developing in their vector. Deletion of the outer dynein arm component DNAI1 results in strong motility defects but cells remain viable in culture. However, although these mutant trypanosomes could infect the tsetse fly midgut, they were neither able to reach the foregut nor able to differentiate into the next stage, thus failing to complete their parasite cycle. This is the first in vivo demonstration that trypanosome motility is essential for the accomplishment of the parasite cycle.     

Adaptations in the Glucose Metabolism of Procyclic Trypanosoma brucei Isolates from Tsetse Flies and during Differentiation of Bloodstream Forms

Procyclic forms of Trypanosoma brucei isolated from the midguts of infected tsetse flies, or freshly transformed from a strain that is close to field isolates, do not use a complete Krebs cycle. Furthermore, short stumpy bloodstream forms produce acetate and are apparently metabolically preadapted to adequate functioning in the tsetse fly.African trypanosomatids comprise various pleomorphic trypanosome species that proliferate in the bloodstream of their mammalian hosts as long slender bloodstream form (BSF) trypanosomes, and at the peak of parasitemia they differentiate into nondividing short stumpy form trypanosomes (1). After being ingested during a bloodmeal by a tsetse fly (Glossina sp.), short stumpy form trypanosomes differentiate into procyclic form (PCF) trypanosomes, which actively multiply and colonize the midgut of the fly. Subsequently, PCF Trypanosoma brucei migrates to the salivary glands while undergoing a complex differentiation (22). Here, attached epimastigote forms start multiplying, after which nondividing metacyclic trypomastigotes develop. The life cycle of T. brucei is completed when these metacyclic trypomastigotes are injected into a mammal through the bite of an infected fly, after which they transform into long slender BSF trypanosomes. During this life cycle, trypanosomes encounter different environments to which they have adapted, resulting in distinct stages, characterized by morphological as well as metabolic changes. Long slender BSF trypanosomes degrade glucose by glycolysis and excrete pyruvate as the sole metabolic end product (12, 13, 23). On the other hand, PCF trypanosomes do not excrete pyruvate but degrade glucose to acetate and succinate as main end products (25). Krebs cycle activity was thought previously to be present in trypanosomatids, at least in insect stages of some African trypanosomatids (3, 9, 10, 12, 21). However, this presumed flux through the Krebs cycle is supported only poorly by direct experimental evidence and was based mainly on the presence of certain enzyme activities. Although genes for all enzymes of this cycle are indeed present in the genome and expressed in the insect stages, recent studies revealed that at least in T. brucei, the cycle is not used for the complete oxidation of acetyl-coenzyme A (CoA) to carbon dioxide (2, 26). Instead, parts of the cycle are most likely used in anabolic pathways, such as gluconeogenesis and fatty acid formation, and also for the final steps in the degradation of amino acids (26). It is possible that the reported discrepancies on the presence or absence of full-circle Krebs cycle activity are caused by differences in the number of passages through mice after the isolation of the strain from the field. Such passages may have been ongoing for many years, during which the parasites were continuously propagated as BSF trypanosomes. Furthermore, most insect form trypanosomes that were investigated up to now have been propagated for many years as PCF trypanosomes in rich culture media. Hence, the reported discrepancies could be due to differences between freshly differentiated PCF trypanosomes and those well adapted to in vitro culture, and the absence of an active Krebs cycle in PCF trypanosomes could be the result of an adaptation caused by the prolonged in vitro culturing. To investigate these possibilities, we analyzed the glucose metabolism of PCF T. brucei directly after isolation from the midguts of tsetse flies. We also studied freshly differentiated PCF trypanosomes from the AntAR 1 strain, a T. brucei strain that has had a minor history of animal passaging since its field isolation (15, 17).To investigate the cause of the conflicting reports on Krebs cycle activity in PCF trypanosomes, we first analyzed the effect of environmental factors by comparing the carbohydrate metabolism of PCF trypanosomes well adapted to in vitro culturing and PCF trypanosomes isolated from their natural environment, the midguts of tsetse flies. These experiments were performed with PCF TREU 927 T. brucei, a pleomorphic strain that has been thoroughly characterized and is still able to infect Glossina morsitans, performing a complete physiological life cycle (2). For the infection of tsetse flies, male G. morsitans flies originating from the colony maintained at the Institute of Tropical Medicine in Antwerp, Belgium, were infected with procyclic TREU 927 T. brucei by in vitro membrane feeding and subsequently maintained for 10 days by feeding on rabbit blood (15). Then, flies were dissected on a sterile glass slide and the infected midguts were isolated and incubated for at least 30 min at 28°C in SDM-79 medium that was gently rotated. After sedimentation of the midguts by gravity, insect gut debris was removed by centrifugation at 300 × g for 5 min. PCF trypanosomes were then isolated from the collected supernatant by centrifugation at 1,500 × g for 10 min. Since PCF trypanosomes could not be isolated from the midgut without minor amounts of contaminating insect gut material, such as gut cells and debris, we also investigated the glucose metabolism of this fraction. Analysis of metabolic end products produced from [6-¹⁴C]glucose in this control incubation of insect gut debris, which also contained minor amounts of trypanosome cells, showed the formation of ¹⁴C-labeled pyruvate, CO₂, acetate, and lactate (Fig. ​(Fig.1A).1A). Minor amounts of lactate were also produced in the incubations with PCF trypanosomes isolated from the midgut, which also contained minor amounts of insect gut debris. Since lactate is not an excreted end product of T. brucei, this labeled lactate is indicative for the glucose degradation activity of insect gut debris. Therefore, end product formation in the incubations with PCF trypanosomes isolated from the midgut was corrected for end products produced by the contaminating insect gut debris by subtracting all produced lactate and the calculated accompanying amounts of other end products produced in the insect gut debris incubation. The metabolic incubations with PCF trypanosomes directly after isolation from the tsetse midgut showed that these trypanosomes degrade glucose to the same metabolic end products, acetate, succinate, and pyruvate, as the in vitro culture-adapted PCF trypanosomes (Fig. ​(Fig.1A).1A). Furthermore, the ratio of acetate and succinate produced by PCF trypanosomes isolated from the midgut were similar to that of in vitro-cultured PCF trypanosomes (Fig. ​(Fig.1A).1A). On the other hand, a major difference was observed in the amount of glucose consumed since the PCF trypanosomes isolated from the midguts of tsetse flies consumed 16-fold less glucose than PCF trypanosomes that were derived from in vitro cultures. This difference in glucose consumption can probably be explained by our observation that both motility and especially growth of PCF trypanosomes isolated from the midgut were significantly reduced compared to the in vitro culture-derived PCF trypanosomes. Apparently, the environmental conditions in the midgut of the fly did affect the PCF trypanosomes, but they did not significantly alter the metabolic pathways used for energy metabolism. However, PCF trypanosomes isolated from the midgut of the fly excreted more pyruvate (Fig. ​(Fig.1A),1A), which suggests that pyruvate is a more important metabolic end product for PCF trypanosomes under physiological conditions than acknowledged thus far. Most importantly, however, just like continuously in vitro-cultured ones, PCF trypanosomes isolated from the midgut of the fly did not degrade [6-¹⁴C]glucose to labeled CO₂ (Fig. ​(Fig.1A),1A), which demonstrates the absence of a functional Krebs cycle in these tsetse fly-derived PCF trypanosomes.Open in a separate windowFIG. 1.Radioactive end products of [6-¹⁴C]glucose metabolism of procyclic TREU 927 T. brucei cells grown in vitro or isolated from the midguts of tsetse flies (A) and that of AntAR 1 T. brucei during differentiation of BSF to PCF trypanosomes (B). (A) The results of a single experiment for PCF trypanosomes isolated from the midgut and for insect gut debris and the mean + the standard deviation (SD) of three parallel incubations for in vitro-cultured PCF trypanosomes are shown. Total end product formation from [6-¹⁴C]glucose was 2.08 ± 0.19 μmol/h per 10⁸ cells and 0.23 μmol/h per 10⁸ cells for in vitro-cultured and midgut-isolated PCF trypanosomes, respectively, and was calculated using the number of trypanosome cells present at the beginning of the incubation. End product formation in the incubation with PCF trypanosomes isolated from the midgut was corrected for end products produced by contaminating insect gut debris (see text for details). (B) Metabolic incubations using 6-¹⁴C-labeled glucose were performed during differentiation from short stumpy BSF trypanosomes to insect stage PCF trypanosomes. Incubations with PCF trypanosomes were started at 24, 48, and 96 h after induction of differentiation (PCF trypanosomes on day 1, PCF trypanosomes on day 2, and PCF trypanosomes on day 4, respectively); means + SDs of three parallel incubations are shown (for the short stumpy form, six incubations in two independent experiments). Total glucose consumption in incubations with long slender BSF trypanosomes, short stumpy BSF trypanosomes, PCF trypanosomes on day 1, PCF trypanosomes on day 2, and PCF trypanosomes on day 4 was 4.8, 3.4, 1.5, 1.1, and 0.79 μmol/h per 10⁸ cells, respectively. Excreted labeled end products shown in panels A and B were analyzed as described previously (25) and are expressed as the percentage of the total amount of radioactive end products produced (in the incubation of gut debris, one other unidentified end product was produced, which explains why this total in the figure does not add up to 100%). The decrease in pyruvate production between long slender and short stumpy BSF trypanosomes as well as the increase in acetate production is significant as calculated using an unpaired t test (P < 0.01 for pyruvate and P < 0.001 for acetate).Although TREU 927 T. brucei is a pleomorphic trypanosome strain, it cannot be excluded that these trypanosomes have adapted their energy metabolism during the substantial period that this strain has been cultured in vitro. Therefore, we also studied the carbohydrate metabolism of freshly transformed PCF of the T. brucei AntAR 1 strain, a well-characterized pleomorphic strain that is close to the wild isolate (17). To investigate the energy metabolism of these freshly differentiated PCF trypanosomes, AntAR 1 BSF trypanosomes were harvested from the blood of infected immune-suppressed NMRI mice as described previously (16) and either directly incubated with [6-¹⁴C]glucose or differentiated to PCF trypanosomes, by addition of 6 mM cis-aconitate and incubation at 27°C (7). These trypanosomes were then incubated with [6-¹⁴C]glucose at different time points after the initiation of differentiation. Our experiments (Fig. ​(Fig.1B)1B) confirmed that differentiation of trypanosomes from BSF to PCF is accompanied by a metabolic shift in excreted end products from pyruvate to acetate and succinate (3, 14, 25). This metabolic shift during differentiation of BSF to PCF trypanosomes was complete after 1 to 2 days (Fig. ​(Fig.1B),1B), which is in agreement with previous observations (9). A subsequent switch in medium from HMI-9, a medium used to culture BSF T. brucei, to SDM-79, a medium used for the culture of PCF T. brucei, did not result in further changes in excreted end products (data not shown).Our experiments, however, did not show any significant production of labeled CO₂ and certainly not the massive increase in CO₂ formation upon differentiation of BSF into PCF trypanosomes that was reported in a comparable study by Durieux et al. (9). We cannot exclude that this difference in Krebs cycle activity between our study and that of Durieux et al. is caused by a strain difference, but since the AntAR 1 strain we used can be considered to be close to the field isolate, the results presented here are indicative of wild-type T. brucei metabolism and strongly suggest that a functional Krebs cycle is absent in PCF T. brucei cells in vivo.Next to the absence of carbon dioxide formation via Krebs cycle activity during differentiation of BSF to PCF trypanosomes, our metabolic experiments also demonstrated that acetate accounted for 30% of the glucose-derived excreted labeled end products in freshly isolated BSF AntAR 1 T. brucei cells (Fig. ​(Fig.1B).1B). This is a surprising observation since BSF trypanosomes are reported to rely on glycolysis only and to excrete pyruvate and minor amounts of glycerol (12, 13, 23). However, the BSF trypanosomes that we tested in our incubations were predominantly short stumpy BSF cells, whereas nearly all previously performed metabolic studies of BSF trypanosomes were performed with long slender BSF cells. In order to investigate whether differentiation from long slender to short stumpy form trypanosomes indeed shifts the metabolism toward acetate formation, we analyzed the energy metabolism of BSF trypanosomes harvested from mice at two different time points after infection. At day 4 after infection, predominantly long slender BSF trypanosomes were isolated (94% long slender versus 6% short stumpy), whereas at day 7 after infection, predominantly short stumpy BSF trypanosomes were isolated (92% short stumpy versus 8% long slender). Analysis of glucose-derived metabolic end products from incubations with BSF AntAR 1 trypanosomes isolated at day 4 or at day 7 after infection showed that short stumpy BSF trypanosomes indeed produce significant amounts of acetate as an end product of glucose metabolism (Fig. ​(Fig.1B).1B). In the incubations with predominantly long slender BSF AntAR 1 T. brucei cells, some acetate was also produced, but this relatively small amount of acetate formation can be explained by the presence of a certain amount of short stumpy cells. Although the incubations were started with nearly 95% long slender BSF cells, BSF cells from the AntAR 1 strain are highly pleomorphic and rapidly differentiate to short stumpy forms during in vitro culture conditions. Therefore, increasing amounts of short stumpy form T. brucei were formed during our incubations (up to 40 to 50% at the end of incubation), which accounts for the amount of acetate formed during these incubations.Since acetate production in Trypanosomatidae is catalyzed by the mitochondrial enzyme acetate:succinate CoA transferase (ASCT), which was previously shown not to be expressed in in vitro-cultured BSF T. brucei (20), we examined the ASCT enzyme activity in lysates derived from either over 92% short stumpy cells or 94% long slender cells. These experiments showed that the ASCT enzyme is present in short stumpy BSF trypanosomes in an amount equivalent to around 15% of that of PCF trypanosomes (Fig. ​(Fig.2).2). This is in agreement with the observation that acetate is a more prominent excreted end product in PCF trypanosomes than in short stumpy BSF cells. On the other hand, ASCT activity was nearly absent in long slender BSF trypanosomes (Fig. ​(Fig.2),2), which confirms the conclusion that in our incubations acetate is not produced by long slender BSF trypanosomes but by short stumpy BSF trypanosomes.Open in a separate windowFIG. 2.ASCT activity in total lysates of T. brucei AntAR 1. Enzymatic activity of ASCT was determined in total lysates derived from cultures containing predominantly long slender BSF trypanosomes (BSF LS; 94%), predominantly short stumpy BSF trypanosomes (BSF SS; 92%), or exclusively PCF trypanosomes (PCF). Shown are the means + standard deviations of three experiments.Hence, our experiments show that short stumpy BSF trypanosomes do not only degrade glucose by glycolysis but additionally produce acetate. Acetate formation in trypanosomes occurs via the mitochondrial enzyme ASCT and involves transfer of a CoA moiety from acetyl-CoA to succinate, yielding succinyl-CoA (24). This succinyl-CoA can then be converted back into succinate by succinyl-CoA synthetase, a reaction concomitantly converting ADP in ATP (6, 24). Therefore, our observations that short stumpy BSF trypanosomes produce acetate and express ASCT demonstrate that these stages in addition to glycolysis also use a mitochondrial pathway for the degradation of glucose and production of ATP.Multiple mitochondrial adaptations have been reported to occur during the transition from long slender BSF to short stumpy BSF T. brucei. Differential gene expression and the formation of cristea in the inner mitochondrial membrane have been shown to occur during this transition (8, 11, 19). Furthermore, the trypanosomal homologue of complex I of the respiratory chain is expressed in short stumpy BSF trypanosomes (4, 5, 18). Our experiments show that this more elaborate composition of the electron transport chain is also used by this stage, as the production of acetate implies that acetyl-CoA is formed, which is catalyzed by the pyruvate dehydrogenase complex and results in the production of NADH inside the mitochondrion. This means that either complex I or the alternative NADH dehydrogenase is active in this stage (18). Moreover, our experiments show that the previously reported mitochondrial adaptations in short stumpy BSF trypanosomes are not restricted to morphological changes and to changes in the composition of the electron transport chain but also result in a functionally altered energy metabolism.In conclusion, the data described in this paper demonstrate the absence of a functional Krebs cycle in the mitochondria of PCF T. brucei, isolated from the tsetse midgut or freshly differentiated from BSF trypanosomes. Furthermore, we show that short stumpy BSF T. brucei cells produce large amounts of acetate. Therefore, the mitochondria of short stumpy trypanosomes are metabolically divergent from the mitochondria in long slender BSF T. brucei cells. These results are consistent with prior work (4, 5, 8, 11). The functional changes might be a preadaptation that allows short stumpy BSF T. brucei to function in the intestines of infected tsetse flies and enables them to differentiate further into PCF trypanosomes.     

Tsetse Fly (G.f. fuscipes) Distribution in the Lake Victoria Basin of Uganda

Tsetse flies transmit trypanosomes, the causative agent of human and animal African trypanosomiasis. The tsetse vector is extensively distributed across sub-Saharan Africa. Trypanosomiasis maintenance is determined by the interrelationship of three elements: vertebrate host, parasite and the vector responsible for transmission. Mapping the distribution and abundance of tsetse flies assists in predicting trypanosomiasis distributions and developing rational strategies for disease and vector control. Given scarce resources to carry out regular full scale field tsetse surveys to up-date existing tsetse maps, there is a need to devise inexpensive means for regularly obtaining dependable area-wide tsetse data to guide control activities. In this study we used spatial epidemiological modelling techniques (logistic regression) involving 5000 field-based tsetse-data (G. f. fuscipes) points over an area of 40,000 km², with satellite-derived environmental surrogates composed of precipitation, temperature, land cover, normalised difference vegetation index (NDVI) and elevation at the sub-national level. We used these extensive tsetse data to analyse the relationships between presence of tsetse (G. f. fuscipes) and environmental variables. The strength of the results was enhanced through the application of a spatial autologistic regression model (SARM). Using the SARM we showed that the probability of tsetse presence increased with proportion of forest cover and riverine vegetation. The key outputs are a predictive tsetse distribution map for the Lake Victoria basin of Uganda and an improved understanding of the association between tsetse presence and environmental variables. The predicted spatial distribution of tsetse in the Lake Victoria basin of Uganda will provide significant new information to assist with the spatial targeting of tsetse and trypanosomiasis control.     

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.     

OmpA-Mediated Biofilm Formation Is Essential for the Commensal Bacterium Sodalis glossinidius To Colonize the Tsetse Fly Gut

Many bacteria successfully colonize animals by forming protective biofilms. Molecular processes that underlie the formation and function of biofilms in pathogenic bacteria are well characterized. In contrast, the relationship between biofilms and host colonization by symbiotic bacteria is less well understood. Tsetse flies (Glossina spp.) house 3 maternally transmitted symbionts, one of which is a commensal (Sodalis glossinidius) found in several host tissues, including the gut. We determined that Sodalis forms biofilms in the tsetse gut and that this process is influenced by the Sodalis outer membrane protein A (OmpA). Mutant Sodalis strains that do not produce OmpA (Sodalis ΔOmpA mutants) fail to form biofilms in vitro and are unable to colonize the tsetse gut unless endogenous symbiotic bacteria are present. Our data indicate that in the absence of biofilms, Sodalis ΔOmpA mutant cells are exposed to and eliminated by tsetse''s innate immune system, suggesting that biofilms help Sodalis evade the host immune system. Tsetse is the sole vector of pathogenic African trypanosomes, which also reside in the fly gut. Acquiring a better understanding of the dynamics that promote Sodalis colonization of the tsetse gut may enhance the development of novel disease control strategies.