Kinetics of methionine transport and metabolism by Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Methionine is an essential amino acid for both prokaryotic and eukaryotic organisms; however, little is known concerning its utilization in African trypanosomes, protozoa of the Trypanosoma brucei group. This study explored the Michaelis-Menten kinetic constants for transport and pool formation as well as metabolic utilization of methionine by two divergent strains of African trypanosomes, Trypanosoma brucei brucei (a veterinary pathogen), highly sensitive to trypanocidal agents, and Trypanosoma brucei rhodesiense (a human pathogenic isolate), highly refractory to trypanocidal arsenicals. The Michaelis-Menten constants derived by Hanes-Woolf analysis for transport of methionine for T. b. brucei and T. b. rhodesiense, respectively, were as follows: K(M) values, 1. 15 and 1.75 mM; V(max) values, 3.97 x 10(-5) and 4.86 x 10(-5) mol/L/min. Very similar values were obtained by Lineweaver-Burk analysis (K(M), 0.25 and 1.0 mM; V(max), 1 x 10(-5) and 2.0 x 10(-5) mol/L/min, T. b. brucei and T. b. rhodesiense, respectively). Cooperativity analyses by Hill (log-log) plot gave Hill coefficients (n) of 6 and 2 for T. b. brucei and T. b. rhodesiense, respectively. Cytosolic accumulation of methionine after 10-min incubation with 25 mM exogenous methionine was 1.8-fold greater in T. b. rhodesiense than T. b. brucei (2.1 vs 1.1 mM, respectively). In African trypanosomes as in their mammalian host, S-adenosylmethionine (AdoMet) is the major product of methionine metabolism. Accumulation of AdoMet was measured by HPLC analysis of cytosolic extracts incubated in the presence of increasing cytosolic methionine. In trypanosomes incubated for 10 min with saturating methionine, both organisms accumulated similar amounts of AdoMet (approximately 23 microM), but the level of trans-sulfuration products (cystathionine and cysteine) in T. b. rhodesiense was double that of T. b. brucei. Methionine incorporation during protein synthesis in T. b. brucei was 2.5 times that of T. b. rhodesiense. These results further confirm our belief that the major pathways of methionine utilization, for polyamine synthesis, protein transmethylation and the trans-sulfuration pathway, are excellent targets for chemotherapeutic intervention against African trypanosomes.     

Nonvariant antigens limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled approximately 3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.     

Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro

Apoptosis is a phenomenon previously associated exclusively with metazoan organisms. We show here that procyclic insect form Trypanosoma brucei rhodesiense, a protozoan parasite, when treated in vitro with concanavalin A displayed several features normally associated with apoptosis in metazoan cells. Lectin treatment induced cleavage of nuclear DNA into oligonucleosomal fragments, suggesting activation of an endogenous nuclease in the parasite. Treated trypanosomes, although agglutinated and non-motile, exhibited fluorescence after treatment with the vital stain fluorescein diacetate and retained (3)H-uridine indicating that their cell membranes remained intact during the period of DNA fragmentation. Electron micrographs showed characteristic morphology of cells undergoing apoptosis, including surface membrane vesiculation and migration of chromatin to the periphery of the nuclear membrane while mitochondria remained intact. These results suggest that treatment with concanavalin A triggers a cell death mechanism in T. b. rhodesiense similar to the process of apoptosis described in metazoa.     

Kinetics of S-adenosylmethionine cellular transport and protein methylation in Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

African trypanosomes of the Trypanosoma brucei group are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter for S-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates: Trypanosoma brucei brucei, a veterinary parasite, and Trypanosoma brucei rhodesiense, a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active against T. b. brucei. The apparent Km values for [methyl-3H]AdoMet transport, derived by Hanes-Woolf analysis, for T. b. brucei was 4.2 and 10 mM for T. b. rhodesiense, and the Vmax values were 124 and 400 micromol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/10(9) T. b. brucei and 3500 nmol/10(9) T. b. rhodesiense after 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation in T. b. brucei was fourfold greater than in T. b. rhodesiense. These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.     

Trypanosoma brucei rhodesiense transmitted by a single tsetse fly bite in vervet monkeys as a model of human African trypanosomiasis

We have investigated the pathogenicity of tsetse (Glossina pallidipes)-transmitted cloned strains of Trypanosoma brucei rhodesiense in vervet monkeys. Tsetse flies were confirmed to have mature trypanosome infections by xenodiagnosis, after which nine monkeys were infected via the bite of a single infected fly. Chancres developed in five of the nine (55.6%) monkeys within 4 to 8 days post infection (dpi). All nine individuals were successfully infected, with a median pre-patent period of 4 (range = 4-10) days, indicating that trypanosomes migrated from the site of fly bite to the systemic circulation rapidly and independently of the development of the chancre. The time lag to detection of parasites in cerebrospinal fluid (CSF) was a median 16 (range = 8-40) days, marking the onset of central nervous system (CNS, late) stage disease. Subsequently, CSF white cell numbers increased above the pre-infection median count of 2 (range = 0-9) cells/microl, with a positive linear association between their numbers and that of CSF trypanosomes. Haematological changes showed that the monkeys experienced an early microcytic-hypochromic anaemia and severe progressive thrombocytopaenia. Despite a 3-fold increase in granulocyte numbers by 4 dpi, leucopaenia occurred early (8 dpi) in the monkey infection, determined mainly by reductions in lymphocyte numbers. Terminally, leucocytosis was observed in three of nine (33%) individuals. The duration of infection was a median of 68 (range = 22-120) days. Strain and individual differences were observed in the severity of the clinical and clinical pathology findings, with two strains (KETRI 3741 and 3801) producing a more acute disease than the other two (KETRI 3804 and 3928). The study shows that the fly-transmitted model accurately mimics the human disease and is therefore a suitable gateway to understanding human African trypanosomiasis (HAT; sleeping sickness).     

Nonvariant Antigens Limited to Bloodstream Forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense1

ABSTRACT. The presence of nonvariant antigens (NVAs) limited to bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense was demonstrated for the first time by immunodiffusion and Immunoelectrophoresis. Noncloned and cloned populations were employed in preparation of polyclonal antisera in rabbits and of antigens to be used in the immunologic reactions. The NVAs could be shown best in systems in which hyperimmune rabbit sera (adsorbed with procyclic forms to eliminate antibodies against antigens common to bloodstream form and procyclic stages) were reacted with trypanosomes characterized by heterologous variant-specific antigens (VSAs). The NVAs demonstrated in this study are very likely different from the common parts of VSAs. As has been suggested by experiments with living trypanosomes, at least a part of the NVAs appears to be located on the surface of the bloodstream forms. In these experiments involving the quantitative indirect fluorescent antibody test, the amount of fluorescence recorded for the heterologous system, i.e. ETat 5 trypanosomes incubated with anti-AmTat 1.1 serum, equalled ～3.0% of the fluorescence emitted by the AmTat 1.1 bloodstream forms treated with their homologous antiserum. Evidently, only small amounts of NVAs are present on the surfaces of T. brucei bloodstream forms. In addition to the NVAs, the electrophoresis results suggested the presence of antigenic differences between procyclic stages belonging to different T. brucei stocks.     

Localization of serum resistance‐associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human‐infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance‐associated protein (SRA protein), protects against ApoL1‐mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA‐mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well‐characterized endosomal markers. By three‐dimensional deconvolved immunofluorescence single‐cell analysis, combined with double‐labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.     

De Novo Genome Assembly Shows Genome Wide Similarity between Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Background

Trypanosoma brucei is a eukaryotic pathogen which causes African trypanosomiasis. It is notable for its variant surface glycoprotein (VSG) coat, which undergoes antigenic variation enabled by a large suite of VSG pseudogenes, allowing for persistent evasion of host adaptive immunity. While Trypanosoma brucei rhodesiense (Tbr) and T. b gambiense (Tbg) are human infective, related T. b. brucei (Tbb) is cleared by human sera. A single gene, the Serum Resistance Associated (SRA) gene, confers Tbr its human infectivity phenotype. Potential genetic recombination of this gene between Tbr and non-human infective Tbb strains has significant epidemiological consequences for Human African Trypanosomiasis outbreaks.

Results

Using long and short read whole genome sequencing, we generated a hybrid de novo assembly of a Tbr strain, producing 4,210 scaffolds totaling approximately 38.8 megabases, which comprise a significant proportion of the Tbr genome, and thus represents a valuable tool for a comparative genomics analyses among human and non-human infective T. brucei and future complete genome assembly. We detected 5,970 putative genes, of which two, an alcohol oxidoreductase and a pentatricopeptide repeat-containing protein, were members of gene families common to all T. brucei subspecies, but variants specific to the Tbr strain sequenced in this study. Our findings confirmed the extremely high level of genomic similarity between the two parasite subspecies found in other studies.

Conclusions

We confirm at the whole genome level high similarity between the two Tbb and Tbr strains studied. The discovery of extremely minor genomic differentiation between Tbb and Tbr suggests that the transference of the SRA gene via genetic recombination could potentially result in novel human infective strains, thus all genetic backgrounds of T. brucei should be considered potentially human infective in regions where Tbr is prevalent.     

The Genome Sequence of Trypanosoma brucei gambiense,Causative Agent of Chronic Human African Trypanosomiasis

Background

Trypanosoma brucei gambiense is the causative agent of chronic Human African Trypanosomiasis or sleeping sickness, a disease endemic across often poor and rural areas of Western and Central Africa. We have previously published the genome sequence of a T. b. brucei isolate, and have now employed a comparative genomics approach to understand the scale of genomic variation between T. b. gambiense and the reference genome. We sought to identify features that were uniquely associated with T. b. gambiense and its ability to infect humans.

Methods and Findings

An improved high-quality draft genome sequence for the group 1 T. b. gambiense DAL 972 isolate was produced using a whole-genome shotgun strategy. Comparison with T. b. brucei showed that sequence identity averages 99.2% in coding regions, and gene order is largely collinear. However, variation associated with segmental duplications and tandem gene arrays suggests some reduction of functional repertoire in T. b. gambiense DAL 972. A comparison of the variant surface glycoproteins (VSG) in T. b. brucei with all T. b. gambiense sequence reads showed that the essential structural repertoire of VSG domains is conserved across T. brucei.

Conclusions

This study provides the first estimate of intraspecific genomic variation within T. brucei, and so has important consequences for future population genomics studies. We have shown that the T. b. gambiense genome corresponds closely with the reference, which should therefore be an effective scaffold for any T. brucei genome sequence data. As VSG repertoire is also well conserved, it may be feasible to describe the total diversity of variant antigens. While we describe several as yet uncharacterized gene families with predicted cell surface roles that were expanded in number in T. b. brucei, no T. b. gambiense-specific gene was identified outside of the subtelomeres that could explain the ability to infect humans.     

Diagnostic Accuracy of Loopamp Trypanosoma brucei Detection Kit for Diagnosis of Human African Trypanosomiasis in Clinical Samples

Background

Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT), but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME) DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP) in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC).

Methodology/Principal findings

142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9–91.8%) in the first run and 93.0% (95% CI 87.5–96.1%) in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4–96.3%) in the first run and 96.4% (95% CI 91.1–98.6%) in the second run. Reproducibility was excellent with a kappa value of 0.81.

Conclusions/Significance

In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions.     

Melarsoprol- and pentamidine-resistant Trypanosoma brucei rhodesiense populations and their cross-resistance

Resistance to melarsoprol and pentamidine was induced in bloodstream-form Trypanosoma brucei rhodesiense STIB 900 in vitro, and drug sensitivity was determined for melarsoprol, pentamidine and furamidine. The resistant populations were also inoculated into immunosuppressed mice to verify infectivity and to monitor whether rodent passage selects for clones with altered drug sensitivity. After proliferation in the mouse, trypanosomes were isolated and their IC(50) values to the three drugs were determined. To assess the stability of drug-induced resistance, drug pressure was ceased for 2 months and the drug sensitivity was determined again. Resistance was stable, with a few exceptions that are discussed. Drug IC(50)s indicated cross-resistance among all drugs, but to varying extents: resistance of the melarsoprol-selected and pentamidine-selected trypanosomes to pentamidine was the same, but the pentamidine-selected trypanosome population showed lower resistance to melarsoprol than the melarsoprol-selected trypanosomes. Interestingly, both resistant populations revealed the same intermediate cross-resistance to furamidine. Resistant trypanosome populations were characterised by molecular means, referring to the status of the TbAT1 gene. The melarsoprol-selected population apparently had lost TbAT1, whereas in the pentamidine-selected trypanosome population it was still present.     

Differential sensitivity of Trypanosoma brucei rhodesiense isolates to in vitro lysis by arsenicals.

Clinical isolates of Trypanosoma brucei rhodesiense, which were resistant to arsenical drugs in murine infections, were examined for resistance in vitro. A rapid lysis assay was developed which was able to predict in vivo sensitivity to melarsoprol (Mel B, Arsobal) and melarsen oxide. The assay was based on the finding that long slender bloodforms of drug-sensitive isolates would lyse in the presence of arsenicals upon incubation in heat-inactivated fetal bovine serum. On the basis of plots of decrease in the absorbance of trypanosome suspensions vs time of incubation with drug, L50 values, reflecting the drug concentration necessary for lysis of 50% of the cells within 30 min. were calculated for five strains. These values ranged from less than 30 microM for arsenical-sensitive strains to greater than 75 microM in proven arsenic refractory isolates. Calcium was essential for lysis, and the presence of the Ca2+ chelator EGTA (10 mM) in serum delayed lysis of sensitive strains. Ca2+ channel antagonists (Verapamil, Diltiazem), however, did not enhance lysis of refractory isolates when used at 20 to 30 microM. Intracellular concentrations of reduced trypanothione, the apparent target of arsenicals, were similar for all isolates, approximately 1.02 +/- 0.28 nmol/10(8) cells, as detected by monobromobimane derivitization and HPLC analysis. Uptake of melarsen oxide was found to be reduced in arsenical refractory strains. Uptake was judged by reduction of free reduced trypanothione as a result of formation of the trypanothione-arsenic complex Mel T. Little change was found in arsenical-resistant strains, but sensitive strains had 50 to 70% reductions in trypanothione levels after incubation with a low (1 microM) level of melarsen oxide.     

Cyclical transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by tsetse flies infected with culture-form procyclic trypanosomes

Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

A Search for Trypanosoma brucei rhodesiense Diagnostic Antigens by Proteomic Screening and Targeted Cloning

Background

The only available diagnostic method for East African trypanosomiasis is light microscopy of blood samples. A simple immunodiagnostic would greatly aid trypanosomiasis control.

Methodology and Principal Findings

To find trypanosome proteins that are specifically recognised by sera from human sleeping sickness patients, we have screened the Trypanosoma brucei brucei proteome by Western blotting. Using cytosolic, cytoskeletal and glycosomal fractions, we found that the vast majority of abundant trypanosome proteins is not specifically recognised by patient sera. We identified phosphoglycerate kinase (PGKC), heat shock protein (HSP70), and histones H2B and H3 as possible candidate diagnostic antigens. These proteins, plus paraflagellar rod protein 1, rhodesain (a cysteine protease), and an extracellular fragment of the Trypanosoma brucei nucleoside transporter TbNT10, were expressed in E. coli and tested for reactivity with patient and control sera. Only TbHSP70 was preferentially recognized by patient sera, but the sensitivity and specificity were insufficient for use of TbHSP70 alone as a diagnostic. Immunoprecipitation using a native protein extract revealed no specifically reacting proteins.

Conclusions

No abundant T. brucei soluble, glycosomal or cytoskeletal protein is likely to be useful in diagnosis. To find useful diagnostic antigens it will therefore be necessary to use more sophisticated proteomic methods, or to test a very large panel of candidate proteins.     

Cyclical Transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by Tsetse Flies Infected with Culture-Form Procyclic Trypanosomes*

SYNOPSIS. Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

A multiplex PCR that discriminates between Trypanosoma brucei brucei and zoonotic T. b. rhodesiense

Two subspecies of Trypanosoma brucei s.l. co-exist within the animal populations of Eastern Africa; T. b. brucei a parasite which only infects livestock and wildlife and T. b. rhodesiense a zoonotic parasite which infects domestic livestock, wildlife, and which in humans, results in the disease known as Human African Trypanosomiasis (HAT) or sleeping sickness. In order to assess the risk posed to humans from HAT it is necessary to identify animals harbouring potentially human infective parasites. The multiplex PCR method described here permits differentiation of human and non-human infective parasites T. b. rhodesiense and T. b. brucei based on the presence or absence of the SRA gene (specific for East African T. b. rhodesiense), inclusion of GPI-PLC as an internal control indicates whether sufficient genomic material is present for detection of a single copy T. brucei gene in the PCR reaction.     

The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense

Targett G. A. T. and Wilson V. C. L. C. 1973. The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense. International Journal for Parasitology, 3: 5–11. A simple test for distinguishing between the morphologically identical subspecies Trypanosoma brucei rhodesiense, which is infective to man, and T. brucei brucei, which by definition is not, has been described. This test, the blood incubation infectivity test (BIIT), is based on absolute differences in the infectivity to rats of the subspecies after exposure to human blood, and was applied to strains which are preserved in the laboratory as stabilates. Five T. brucei brucei strains were BIIT negative since their infectivity was destroyed by incubation in normal human blood but only five of the nine T. brucei rhodesiense strains tested were consistently BIIT positive. The other four gave equivocal results, indicating that the resistance of T. brucei rhodesiense strains to the trypanocidal effect of human blood can change, probably as a result of maintenance in the laboratory.