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.     

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.     

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.     

Differences between Trypanosoma brucei gambiense groups 1 and 2 in their resistance to killing by trypanolytic factor 1

Background

The three sub-species of Trypanosoma brucei are important pathogens of sub-Saharan Africa. T. b. brucei is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. T. b. rhodesiense and T. b. gambiense are able to resist lysis by TLF. There are two distinct sub-groups of T. b. gambiense that differ genetically and by human serum resistance phenotypes. Group 1 T. b. gambiense have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 T. b. gambiense are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (HpHbR)) gene. Here we investigate if this is also true in group 2 parasites.

Methodology

Isogenic resistant and sensitive group 2 T. b. gambiense were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the HpHbR gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to T. b. brucei. Both resistant and sensitive group 2, as well as group 1 T. b. gambiense, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed.

Conclusions

Our data indicate that, despite group 1 T. b. gambiense avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 T. b. gambiense is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 T. b. gambiense variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of HpHbR. Thus there are differences in the mechanism of human serum resistance between T. b. gambiense groups 1 and 2.     

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.     

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.     

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.     

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.     

Genetic analysis of the human infective trypanosome <Emphasis Type="Italic">Trypanosoma brucei gambiense</Emphasis>: chromosomal segregation,crossing over,and the construction of a genetic map

Background  

Trypanosoma brucei is the causative agent of human sleeping sickness and animal trypanosomiasis in sub-Saharan Africa, and it has been subdivided into three subspecies: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause sleeping sickness in humans, and the nonhuman infective Trypanosoma brucei brucei. T. b. gambiense is the most clinically relevant subspecies, being responsible for more than 90% of all trypanosomal disease in humans. The genome sequence is now available, and a Mendelian genetic system has been demonstrated in T. brucei, facilitating genetic analysis in this diploid protozoan parasite. As an essential step toward identifying loci that determine important traits in the human-infective subspecies, we report the construction of a high-resolution genetic map of the STIB 386 strain of T. b. gambiense.     

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.     

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.     

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.     

Differential Gene Repertoire in Mycobacterium ulcerans Identifies Candidate Genes for Patho-Adaptation

Background

Based on large genomic sequence polymorphisms, several haplotypes belonging to two major lineages of the human pathogen Mycobacterium ulcerans could be distinguished among patient isolates from various geographic origins. However, the biological relevance of insertional/deletional diversity is not understood.

Methodology

Using comparative genomics, we have investigated the genes located in regions of difference recently identified by DNA microarray based hybridisation analysis. The analysed regions of difference comprise ∼7% of the entire M. ulcerans genome.

Principal Findings

Several different mechanisms leading to loss of functional genes were identified, ranging from pseudogenization, caused by frame shift mutations or mobile genetic element interspersing, to large sequence polymorphisms. Four hot spot regions for genetic instability were unveiled. Altogether, 229 coding sequences were found to be differentially inactivated, constituting a repertoire of coding sequence variation in the rather monomorphic M. ulcerans.

Conclusions/Significance

The differential gene inactivation patterns associated with the M. ulcerans haplotypes identified candidate genes that may confer enhanced adaptation upon ablation of expression. A number of gene conversions confined to the classical lineage may contribute to particular virulence of this group comprising isolates from Africa and Australia. Identification of this spectrum of anti-virulence gene candidates expands our understanding of the pathogenicity and ecology of the emerging infectious disease Buruli ulcer.     

Genome size, karyotype polymorphism and chromosomal evolution in Trypanosoma cruzi

Background

The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites.

Methodology/Principal Findings

The genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (José-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively.

Conclusions

Despite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.     

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background

Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.

Methodology/Principal Findings

The T. rangeli haploid genome is ∼24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins.

Conclusions/Significance

Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.     

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.     

A CATT Negative Result after Treatment for Human African Trypanosomiasis Is No Indication for Cure

Background

Cure after treatment for human African trypanosomiasis (HAT) is assessed by examination of the cerebrospinal fluid every 6 months, for a total period of 2 years. So far, no markers for cure or treatment failure have been identified in blood. Trypanosome-specific antibodies are detectable in blood by the Card Agglutination Test for Trypanosomiasis (CATT). We studied the value of a normalising, negative post-treatment CATT result in treated Trypanosoma brucei (T.b.) gambiense sleeping sickness patients as a marker of cure.

Methodology/Principal Findings

The CATT/T.b. gambiense was performed on serum of a cohort of 360 T.b. gambiense patients, consisting of 242 primary and 118 retreatment cases. The CATT results during 2 years of post-treatment follow-up were studied in function of cure or treatment failure. At inclusion, sensitivity of CATT was 98% (234/238) in primary cases and only 78% (91/117) in retreatment cases. After treatment, the CATT titre decreased both in cured patients and in patients experiencing treatment failure.

Conclusions/Significance

Though CATT is a good test to detect HAT in primary cases, a normalising or negative CATT result after treatment for HAT does not indicate cure, therefore CATT cannot be used to monitor treatment outcome.