Paul Capewell

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.

Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor

Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1–resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucei is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1–resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1–resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense.

The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes

The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology. DOI: http://dx.doi.org/10.7554/eLife.17716.001

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.

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.

Whole-Genome Sequencing of Trypanosoma brucei Reveals Introgression between Subspecies That Is Associated with Virulence

ABSTRACT Human African trypanosomiasis is caused by two subspecies of Trypanosoma brucei. Trypanosoma brucei rhodesiense is found in East Africa and frequently causes acute disease, while Trypanosoma brucei gambiense is found in West Africa and is associated with chronic disease. Samples taken from a single focus of a Ugandan outbreak of T. b. rhodesiense in the 1980s were associated with either chronic or acute disease. We sequenced the whole genomes of two of these isolates, which showed that they are genetically distinct from each other. Analysis of single nucleotide polymorphism markers in a panel of 31 Ugandan isolates plus 32 controls revealed a mixture of East African and West African haplotypes, and some of these haplotypes were associated with the different virulence phenotypes. It has been shown recently that T. b. brucei and T. b. rhodesiense populations undergo genetic exchange in natural populations. Our analysis showed that these strains from the Ugandan epidemic were intermediate between the reference genome sequences of T. b. gambiense and T. b. brucei and contained haplotypes that were present in both subspecies. This suggests that the human-infective subspecies of T. brucei are not genetically isolated, and our data are consistent with genomic introgression between East African and West African T. b. brucei subspecies. This has implications for the control of the parasite, the spread of drug resistance, and understanding the variation in virulence and the emergence of human infectivity. IMPORTANCE We present a genetic study of the acute form of “sleeping sickness” caused by the protozoan parasite Trypanosoma brucei rhodesiense from a single outbreak in Uganda. This represents an advance in our understanding of the relationship between the T. b. rhodesiense and Trypanosoma brucei gambiense subspecies that have previously been considered geographically distinct. Our data suggest that introgression of West African-derived T. brucei haplotypes may be associated with differences in disease presentation in the East African disease. These findings are not only of scientific interest but also important for parasite control, as they suggest that the human-infective T. brucei subspecies are not genetically isolated. We present a genetic study of the acute form of “sleeping sickness” caused by the protozoan parasite Trypanosoma brucei rhodesiense from a single outbreak in Uganda. This represents an advance in our understanding of the relationship between the T. b. rhodesiense and Trypanosoma brucei gambiense subspecies that have previously been considered geographically distinct. Our data suggest that introgression of West African-derived T. brucei haplotypes may be associated with differences in disease presentation in the East African disease. These findings are not only of scientific interest but also important for parasite control, as they suggest that the human-infective T. brucei subspecies are not genetically isolated.

The post-transcriptional trans-acting regulator, TbZFP3, co-ordinates transmission-stage enriched mRNAs in Trypanosoma brucei

Post-transcriptional gene regulation is essential to eukaryotic development. This is particularly emphasized in trypanosome parasites where genes are co-transcribed in polycistronic arrays but not necessarily co-regulated. The small CCCH protein, TbZFP3, has been identified as a trans-acting post-transcriptional regulator of Procyclin surface antigen expression in Trypanosoma brucei. To investigate the wider role of TbZFP3 in parasite transmission, a global analysis of associating transcripts was carried out. Examination of a subset of the selected transcripts revealed their increased abundance through mRNA stabilization upon TbZFP3 ectopic overexpression, dependent upon the integrity of the CCCH zinc finger domain. Reporter assays demonstrated that this regulation was mediated through 3′-UTR sequences for two target transcripts. Global developmental expression profiling of the cohort of TbZFP3-selected transcripts revealed their significant enrichment in transmissible stumpy forms of the parasite. This analysis of the specific mRNAs selected by the TbZFP3mRNP provides evidence for a developmental regulon with the potential to co-ordinate genes important in parasite transmission.

Haptoglobin-hemoglobin receptor independent killing of African trypanosomes by human serum and trypanosome lytic factors

The haptoglobin-hemoglobin receptor (HpHbR) of African trypanosomes plays a critical role in human innate immunity against these parasites. Localized to the flagellar pocket of the veterinary pathogen Trypanosoma brucei brucei this receptor binds Trypanosome Lytic Factor-1 (TLF-1), a subclass of human high-density lipoprotein (HDL) facilitating endocytosis, lysosomal trafficking and subsequent killing. Recently, we found that group 1 Trypanosoma brucei gambiense does not express a functional HpHbR. We now show that loss of the TbbHpHbR reduces the susceptibility of T. b. brucei to human serum and TLF-1 by 100- and 10,000-fold, respectively. The relatively high concentrations of human serum and TLF-1 needed to kill trypanosomes lacking the HpHbR indicates that high affinity TbbHpHbR binding enhances the cytotoxicity; however, in the absence of TbbHpHbR, other receptors or fluid phase endocytosis are sufficient to provide some level of susceptibility. Human serum contains a second innate immune factor, TLF-2, that has been suggested to kill trypanosomes independently of the TbbHpHbR. We found that T. b. brucei killing by TLF-2 was reduced in TbbHpHbR-deficient cells but to a lesser extent than TLF-1. This suggests that both TLF-1 and TLF-2 can be taken up via the TbbHpHbR but that alternative pathways exist for the uptake of these toxins. Together the findings reported here extend our previously published studies and suggest that group 1 T. b. gambiense has evolved multiple mechanisms to avoid killing by trypanolytic human serum factors.

Population genomics reveals the origin and asexual evolution of human infective trypanosomes

Evolutionary theory predicts that the lack of recombination and chromosomal re-assortment in strictly asexual organisms results in homologous chromosomes irreversibly accumulating mutations and thus evolving independently of each other, a phenomenon termed the Meselson effect. We apply a population genomics approach to examine this effect in an important human pathogen, Trypanosoma brucei gambiense. We determine that T.b. gambiense is evolving strictly asexually and is derived from a single progenitor, which emerged within the last 10,000 years. We demonstrate the Meselson effect for the first time at the genome-wide level in any organism and show large regions of loss of heterozygosity, which we hypothesise to be a short-term compensatory mechanism for counteracting deleterious mutations. Our study sheds new light on the genomic and evolutionary consequences of strict asexuality, which this pathogen uses as it exploits a new biological niche, the human population. DOI: http://dx.doi.org/10.7554/eLife.11473.001

Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host.

The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically ‘slender forms’ to transmissible ‘stumpy forms’ through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.