Project description:Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in that parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunodominant proteins, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission.
Project description:The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically examined the protein complexes of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that had no available experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying candidates potentially involved in the life stage specific post-transcriptional regulation in T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins.
Project description:Abstract: The Kinetoplastida (Euglenozoa) are unicellular flagellates that include the trypanosomatid parasites, most notably Trypanosoma brucei, T.cruzi and Leishmania spp. These organisms cause substantial mortality and morbidity in humans and their livestock worldwide as the causative agents of African sleeping sickness, Chagas disease and leishmaniasis respectively. Draft genome sequences are available for several species of both Trypanosoma and Leishmania. Bodo saltans is a free-living heterotroph found worldwide in freshwater and marine habitats, and it is among the closest bodonid relatives of the trypanosomatids. The purpose of a B. saltans genome sequence is to provide an 'out-group' for comparative genomic analysis of the trypanosomatid parasites. It will provide a model of the ancestral trypanosomatid to distinguish those derived parts of the parasite genomes (i.e., unique trypanosomatid adaptations) from those which are a legacy of the free-living ancestor. To aid annotation of the B.saltans genome sequence, total genomic RNA was extracted on four occasions from the total cellular mass of 160ml of B.saltans cell culture, for the purposes of transcription profiling by high throughput sequencing. Cells were unmodified. B.saltans cells were grown in water at 4oC. Total genomic RNA was extracted from a cell pellet using TRIZOL reagent and ethanol precipitated. Poly A+ mRNA was purified from total RNA using oligo dT dyna bead selection and libraries were created using the Illumina RNA-seq protocol. The samples were sequenced on an Illumina HiSeq 2000. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:The availability of genome sequence data has greatly enhanced our understanding of the adaptations of trypanosomatid parasites to their respective host environments. However, these studies remain somewhat restricted by modest taxon sampling, generally due to focus on the most important pathogens of humans. To address this problem, at least in part, we are releasing a draft genome sequence for the African crocodilian trypanosome, Trypanosoma grayi ANR4. This dataset comprises genomic DNA sequences assembled de novo into contigs, encompassing over 10,000 annotated putative open reading frames and predicted protein products. Using phylogenomic approaches we demonstrate that T. grayi is more closely related to Trypanosoma cruzi than it is to the African trypanosomes T. brucei, T. congolense and T. vivax, despite the fact T. grayi and the African trypanosomes are each transmitted by tsetse flies. The data are deposited in publicly accessible repositories where we hope they will prove useful to the community in evolutionary studies of the trypanosomatids.
Project description:This project has the main aim to determine the protein composition of extracellular vesicles released by the African sleep sickness parasite, Trypanosoma brucei.
Project description:T. brucei PF cells were treated with several chemical reagents and anti-trypanosomatid drugs. The effect of each chemical perturbation on the transcriptome of T. brucei was examined by transcript profiling of treated vs. control cells. The results indicated widespread changes, suggesting that the transcriptome of T. brucei is highly responsive to environmental factors that perturb its metabolic and biological pathways.
Project description:African trypanosomes have been recently shown to colonise the skin in a process critical for parasite transmission. However, the tissue responses to infection, especially in the lead to parasite transition from the host skin to the vector remain unresolved. Here, using a combination of spatial and single cell transcriptomics, coupled with imaging mass cytometry and genetic models, we investigated the local immune response of the skin in both a murine model of infection and in human samples from the Democratic Republic of Congo (DRC). Our results provide several novel key findings previously unappreciated in the context of parasitic infections in the skin. Firstly, we identified that the skin stromal cells, in particular interstitial preadipocytes located in the subcutis, upregulate several genes involved in inflammatory signalling and antigen presentation, including several molecules involved in T cell activation and survival. Secondly, we detected a significant expansion of a population of IL-17 producing Vg6 gdT cells in the infected murine skin compared to naïve controls, that occur mainly in the subcutis, that we further validated at the protein level by flow cytometry. In silico cell-cell communication analyses between adipocytes and T cells suggests that adipocytes trigger T cell activation locally via Il6, Il10, and Tnfsf18 signalling, amongst others. Thirdly, mice deficient of Vg6 gdT cells show extensive inflammation, increased IFNg-producing CD4+ T cells, and tissue parasite burden compared to naïve controls, indicating that Vg6 gdT cells are important to limit skin inflammation and parasite replication. Based on these observations, we proposed a model whereby interstitial preadipocytes (and potentially adipocytes) as well as Vg6 gdT cells act concertedly in the subcutis to limit tissue damage and parasite load, thus imposing an immunological barrier for transmission. These studies shed light into the mechanisms of gdT cells-mediated immunity in the skin in the context of African trypanosomes infection, as well as a potential role of immature and mature adipocytes as homeostatic regulators on the skin during chronic infection.
Project description:Since the initial publication of the trypanosomatid genomes, curation has been ongoing. Here we apply the technique of ribosome profiling to Trypanosoma brucei, identifying 223 new coding regions by virtue of ribosome occupancy in the corresponding transcripts. A small number of these putative genes correspond to extra copies of previously annotated genes but 85% are novel. The median size of these novels CDSs is small (74 aa) indicating that past annotation work has excelled at detecting large CDSs. Of the unique CDSs discovered here, over half have candidate orthologues in other trypanosomatid genomes, most of which were not yet annotated as genes. Still, approximately one-third of the new CDSs were found only in T. brucei subspecies. When combined with RNA-seq and spliced leader mapping, we were able to definitively revise the start sites for 430 CDSs as compared to the current gene models. Such data also allowed us to use a structured approach to eliminate 701 putative genes as protein-coding. Finally, the data pointed to several regions of the genome that had sequence errors that altered coding region boundaries.
Project description:Human African Trypanosomiasis (HAT) is a disease of major economic importance in Sub-Saharan Africa. In eastern and southern Africa. Here we analysed clinical isolates of T brucei rhodensiense, resistant to suramin by shotgun proteomics . And identified parasite proteins whose expression is associated with resistance to suramin.
Project description:African trypanosomiasis is still one of the infectious diseases threatening the health of human beings and animals, and severely impeding the continental economic development. Currently, the disease control programs are mainly relying on a limited number of parasiticides which are either expensive or with severe side-effect, and insecticides. In this study, we investigated the anti-trypanosome effect of two noble metal nanoclusters (NM-NCs), Ag2S-NC@MPA and AuNC@GSH. Both types of NCs can be efficiently up-taken by Trypanosoma brucei via a clathrin-dependent endocytosis pathway, and displayed dose-dependent anti-parasite effect by inducing apoptosis-associated organelle pathological alterations. The data not only revealed the clathrin-dependent endocytosis pathway in African trypanosome, but also proposed a new avenue for NM-NC-based drug development for trypanosomiasis.