The TgsGP Gene Is Essential for Resistance to Human Serum in Trypanosoma brucei gambiense

Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense.     

Human and Animal Trypanosomes in C?te d'Ivoire Form a Single Breeding Population

Background

Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and contributes to the related veterinary disease, Nagana. T. brucei is segregated into three subspecies based on host specificity, geography and pathology. T. b. brucei is limited to animals (excluding some primates) throughout sub-Saharan Africa and is non-infective to humans due to trypanolytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species. T. b. gambiense is the more prevalent human, causing over 97% of human cases. Study of T. b. gambiense is complicated in that there are two distinct groups delineated by genetics and phenotype. The relationships between the two groups and local T. b. brucei are unclear and may have a bearing on the evolution of the human infectivity traits.

Methodology/Principal Findings

A collection of sympatric T. brucei isolates from Côte d’Ivoire, consisting of T. b. brucei and both groups of T. b. gambiense have previously been categorized by isoenzymes, RFLPs and Blood Incubation Infectivity Tests. These samples were further characterized using the group 1 specific marker, TgSGP, and seven microsatellites. The relationships between the T. b. brucei and T. b. gambiense isolates were determined using principal components analysis, neighbor-joining phylogenetics, STRUCTURE, F_ST, Hardy-Weinberg equilibrium and linkage disequilibrium.

Conclusions/Significance

Group 1 T. b. gambiense form a clonal genetic group, distinct from group 2 and T. b. brucei, whereas group 2 T. b. gambiense are genetically indistinguishable from local T. b. brucei. There is strong evidence for mating within and between group 2 T. b. gambiense and T. b. brucei. We found no evidence to support the hypothesis that group 2 T. b. gambiense are hybrids of group 1 and T. b. brucei, suggesting that human infectivity has evolved independently in groups 1 and 2 T. b. gambiense.     

A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models

Background

Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients.

Methodology/Principal findings

We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT.

Conclusions/Significance

We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin.     

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.     

Preclinical Assessment of the Treatment of Second-Stage African Trypanosomiasis with Cordycepin and Deoxycoformycin

Background

There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.).

Methodology/Principal Findings

Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.

Conclusions/Significance

Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.     

Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N′-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.     

Identification of peptide mimotopes of Trypanosoma brucei gambiense variant surface glycoproteins

Background

The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests.

Methodology/Principal Findings

PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05).

Conclusions/Significance

We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.     

Melarsoprol Sensitivity Profile of Trypanosoma brucei gambiense Isolates from Cured and Relapsed Sleeping Sickness Patients from the Democratic Republic of the Congo

Background

Sleeping sickness caused by Trypanosoma brucei (T.b.) gambiense constitutes a serious health problem in sub-Sahara Africa. In some foci, alarmingly high relapse rates were observed in patients treated with melarsoprol, which used to be the first line treatment for patients in the neurological disease stage. Particularly problematic was the situation in Mbuji-Mayi, East Kasai Province in the Democratic Republic of the Congo with a 57% relapse rate compared to a 5% relapse rate in Masi-Manimba, Bandundu Province. The present study aimed at investigating the mechanisms underlying the high relapse rate in Mbuji-Mayi using an extended collection of recently isolated T.b. gambiense strains from Mbuji-Mayi and from Masi-Manimba.

Methodology/Principal Findings

Forty five T.b. gambiense strains were used. Forty one were isolated from patients that were cured or relapsed after melarsoprol treatment in Mbuji-Mayi. In vivo drug sensitivity tests provide evidence of reduced melarsoprol sensitivity in these strains. This reduced melarsoprol sensitivity was not attributable to mutations in TbAT1. However, in all these strains, irrespective of the patient treatment outcome, the two aquaglyceroporin (AQP) 2 and 3 genes are replaced by chimeric AQP2/3 genes that may be associated with resistance to pentamidine and melarsoprol. The 4 T.b. gambiense strains isolated in Masi-Manimba contain both wild-type AQP2 and a different chimeric AQP2/3. These findings suggest that the reduced in vivo melarsoprol sensitivity of the Mbuji-Mayi strains and the high relapse rates in that sleeping sickness focus are caused by mutations in the AQP2/AQP3 locus and not by mutations in TbAT1.

Conclusions/Significance

We conclude that mutations in the TbAQP2/3 locus of the local T.b. gambiense strains may explain the high melarsoprol relapse rates in the Mbuji-Mayi focus but other factors must also be involved in the treatment outcome of individual patients.     

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.     

Cathepsin-L Can Resist Lysis by Human Serum in Trypanosoma brucei brucei

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence ‘hits’ were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors.     

A Primate APOL1 Variant That Kills Trypanosoma brucei gambiense

Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African T. b. rhodesiense, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite’s resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the in vitro lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (Papio papio), which is able to lyse examples of all sub-species of T. brucei including T. b. gambiense group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective T. brucei sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes.     

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions

Background

At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests.

Methodology/Principal Findings

A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79.

Conclusions/Significance

We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.     

A Single Amino Acid Substitution in the Group 1 Trypanosoma brucei gambiense Haptoglobin-Hemoglobin Receptor Abolishes TLF-1 Binding

Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.     

Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal

Background

The development of arsenical and diamidine resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 purine transporter as a result of alterations in the corresponding T. brucei adenosine transporter 1 gene (TbAT1). Previously, specific TbAT1 mutant type alleles linked to melarsoprol treatment failure were significantly more prevalent in T. b. gambiense from relapse patients at Omugo health centre in Arua district. Relapse rates of up to 30% prompted a shift from melarsoprol to eflornithine (α-difluoromethylornithine, DFMO) as first-line treatment at this centre. The aim of this study was to determine the status of TbAT1 in recent isolates collected from T. b. gambiense sleeping sickness patients from Arua and Moyo districts in Northwestern Uganda after this shift in first-line drug choice.

Methodology and results

Blood and cerebrospinal fluids of consenting patients were collected for DNA preparation and subsequent amplification. All of the 105 isolates from Omugo that we successfully analysed by PCR-RFLP possessed the TbAT1 wild type allele. In addition, PCR/RFLP analysis was performed for 74 samples from Moyo, where melarsoprol is still the first line drug; 61 samples displayed the wild genotype while six were mutant and seven had a mixed pattern of both mutant and wild-type TbAT1. The melarsoprol treatment failure rate at Moyo over the same period was nine out of 101 stage II cases that were followed up at least once. Five of the relapse cases harboured mutant TbAT1, one had the wild type, while no amplification was achieved from the remaining three samples.

Conclusions/significance

The apparent disappearance of mutant alleles at Omugo may correlate with melarsoprol withdrawal as first-line treatment. Our results suggest that melarsoprol could successfully be reintroduced following a time lag subsequent to its replacement. A field-applicable test to predict melarsoprol treatment outcome and identify patients for whom the drug can still be beneficial is clearly required. This will facilitate cost-effective management of HAT in rural resource-poor settings, given that eflornithine has a much higher logistical requirement for its application.     

High levels of genetic differentiation between Ugandan Glossina fuscipes fuscipes populations separated by Lake Kyoga

Background

Glossina fuscipes fuscipes is the major vector of human African trypanosomiasis, commonly referred to as sleeping sickness, in Uganda. In western and eastern Africa, the disease has distinct clinical manifestations and is caused by two different parasites: Trypanosoma brucei rhodesiense and T. b. gambiense. Uganda is exceptional in that it harbors both parasites, which are separated by a narrow 160-km belt. This separation is puzzling considering there are no restrictions on the movement of people and animals across this region.

Methodology and Results

We investigated whether genetic heterogeneity of G. f. fuscipes vector populations can provide an explanation for this disjunct distribution of the Trypanosoma parasites. Therefore, we examined genetic structuring of G. f. fuscipes populations across Uganda using newly developed microsatellite markers, as well as mtDNA. Our data show that G. f. fuscipes populations are highly structured, with two clearly defined clusters that are separated by Lake Kyoga, located in central Uganda. Interestingly, we did not find a correlation between genetic heterogeneity and the type of Trypanosoma parasite transmitted.

Conclusions

The lack of a correlation between genetic structuring of G. f. fuscipes populations and the distribution of T. b. gambiense and T. b. rhodesiense indicates that it is unlikely that genetic heterogeneity of G. f. fuscipes populations explains the disjunct distribution of the parasites. These results have important epidemiological implications, suggesting that a fusion of the two disease distributions is unlikely to be prevented by an incompatibility between vector populations and parasite.     

Cerebrospinal fluid neopterin as marker of the meningo-encephalitic stage of Trypanosoma brucei gambiense sleeping sickness

Background

Sleeping sickness, or human African trypanosomiasis (HAT), is a protozoan disease that affects rural communities in sub-Saharan Africa. Determination of the disease stage, essential for correct treatment, represents a key issue in the management of patients. In the present study we evaluated the potential of CXCL10, CXCL13, ICAM-1, VCAM-1, MMP-9, B2MG, neopterin and IgM to complement current methods for staging Trypanosoma brucei gambiense patients.

Methods and Findings

Five hundred and twelve T. b. gambiense HAT patients originated from Angola, Chad and the Democratic Republic of the Congo (D.R.C.). Their classification as stage 2 (S2) was based on the number of white blood cells (WBC) (>5/µL) or presence of parasites in the cerebrospinal fluid (CSF). The CSF concentration of the eight markers was first measured on a training cohort encompassing 100 patients (44 S1 and 56 S2). IgM and neopterin were the best in discriminating between the two stages of disease with 86.4% and 84.1% specificity respectively, at 100% sensitivity. When a validation cohort (412 patients) was tested, neopterin (14.3 nmol/L) correctly classified 88% of S1 and S2 patients, confirming its high staging power. On this second cohort, neopterin also predicted both the presence of parasites, and of neurological signs, with the same ability as IgM and WBC, the current reference for staging.

Conclusions

This study has demonstrated that neopterin is an excellent biomarker for staging T. b. gambiense HAT patients. A rapid diagnostic test for detecting this metabolite in CSF could help in more accurate stage determination.     

A Trypanosoma brucei Kinesin Heavy Chain Promotes Parasite Growth by Triggering Host Arginase Activity

Background

In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.

Methodology/Principal findings

By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.

Conclusion

A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.     

A Target-Based High Throughput Screen Yields Trypanosoma brucei Hexokinase Small Molecule Inhibitors with Antiparasitic Activity

Background

The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the γ-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.

Methodology/Principal Findings

Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were ∼20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03≤EC₅₀<3 µM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics.

Conclusions/Significance

The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.     

Phylogeography and taxonomy of Trypanosoma brucei

Background

Characterizing the evolutionary relationships and population structure of parasites can provide important insights into the epidemiology of human disease.

Methodology/Principal Findings

We examined 142 isolates of Trypanosoma brucei from all over sub-Saharan Africa using three distinct classes of genetic markers (kinetoplast CO1 sequence, nuclear SRA gene sequence, eight nuclear microsatellites) to clarify the evolutionary history of Trypanosoma brucei rhodesiense (Tbr) and T. b. gambiense (Tbg), the causative agents of human African trypanosomosis (sleeping sickness) in sub-Saharan Africa, and to examine the relationship between Tbr and the non-human infective parasite T. b. brucei (Tbb) in eastern and southern Africa. A Bayesian phylogeny and haplotype network based on CO1 sequences confirmed the taxonomic distinctness of Tbg group 1. Limited diversity combined with a wide geographical distribution suggested that this parasite has recently and rapidly colonized hosts across its current range. The more virulent Tbg group 2 exhibited diverse origins and was more closely allied with Tbb based on COI sequence and microsatellite genotypes. Four of five COI haplotypes obtained from Tbr were shared with isolates of Tbb, suggesting a close relationship between these taxa. Bayesian clustering of microsatellite genotypes confirmed this relationship and indicated that Tbr and Tbb isolates were often more closely related to each other than they were to other members of the same subspecies. Among isolates of Tbr for which data were available, we detected just two variants of the SRA gene responsible for human infectivity. These variants exhibited distinct geographical ranges, except in Tanzania, where both types co-occurred. Here, isolates possessing distinct SRA types were associated with identical COI haplotypes, but divergent microsatellite signatures.

Conclusions/Significance

Our data provide strong evidence that Tbr is only a phenotypic variant of Tbb; while relevant from a medical perspective, Tbr is not a reproductively isolated taxon. The wide distribution of the SRA gene across diverse trypanosome genetic backgrounds suggests that a large amount of genetic diversity is potentially available with which human-infective trypanosomes may respond to selective forces such as those exerted by drugs.     

Genetic control of resistance to Trypanosoma brucei brucei infection in mice

Background

Trypanosoma brucei brucei infects livestock, with severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to this parasite. However, no genes controlling these differences were mapped.

Methods

We studied the genetic control of survival after T. b. brucei infection using recombinant congenic (RC) strains, which have a high mapping power. Each RC strain of BALB/c-c-STS/A (CcS/Dem) series contains a different random subset of 12.5% genes from the parental “donor” strain STS/A and 87.5% genes from the “background” strain BALB/c. Although BALB/c and STS/A mice are similarly susceptible to T. b. brucei, the RC strain CcS-11 is more susceptible than either of them. We analyzed genetics of survival in T. b. brucei-infected F₂ hybrids between BALB/c and CcS-11. CcS-11 strain carries STS-derived segments on eight chromosomes. They were genotyped in the F₂ hybrid mice and their linkage with survival was tested by analysis of variance.

Results

We mapped four Tbbr (Trypanosoma brucei brucei response) loci that influence survival after T. b. brucei infection. Tbbr1 (chromosome 3) and Tbbr2 (chromosome 12) have effects on survival independent of inter-genic interactions (main effects). Tbbr3 (chromosome 7) influences survival in interaction with Tbbr4 (chromosome 19). Tbbr2 is located on a segment 2.15 Mb short that contains only 26 genes.

Conclusion

This study presents the first identification of chromosomal loci controlling susceptibility to T. b. brucei infection. While mapping in F₂ hybrids of inbred strains usually has a precision of 40–80 Mb, in RC strains we mapped Tbbr2 to a 2.15 Mb segment containing only 26 genes, which will enable an effective search for the candidate gene. Definition of susceptibility genes will improve the understanding of pathways and genetic diversity underlying the disease and may result in new strategies to overcome the active subversion of the immune system by T. b. brucei.