Project description:Trypanosoma congolense IL3000 parasites were grown in adult MF1 mice with parasites harvested on day 5 post infection ('ascend') or on day 6/7 post infection ('peak').
Project description:Transcriptome sequencng of Trypanosoma congolense for future protein-protein interaction studiesThis 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:Purpose: The is a major paucity of knowledge regarding the biology of Trypanosoma congolense, a protozoan parasite primarily responsible for Animal African Trypanosomiasis. In contrast, the closely related species T. brucei, is far better understood. To characterise core metabolism in T. congolense, comparative RNAseq analysis was undertaken to assess similarities and differences in transcript levels of genes associated with metabolism Methods: Samples from both in vitro culture and ex vivo (isolated from murine infections) bloodstream-form T. brucei and T. congolense were RNA-sequenced. Data was analyzed using a pipeline that allows for inter-species comparison Results: T. congolense exhibits increased transcript abundance in genes associated with the glycosomal succinate shunt, as well as mitochondrial metabolism, in particular the catabolism of pyruvate to acetate, compared to T. brucei. These differences occur both in vitro and ex vivo. Furthermore there are differences in nucleotide metabolism, and transcript levels of genes involved in fatty acid synthesis are reduced in T. congolense compared to T. brucei. Conclusions: Comparative RNAseq between two closely related species provided a detailed overview of similarities and differences in core metabolism. This carries significant implications for adaptation to in vitro culture, and drug efficacy, mode of action and mode of resistance.
Project description:Isometamidium Chloride (ISM) is one of the principal drugs used to counteract Trypanosoma congolense infection in livestock, both as a prophylactic as well as a curative treatment. However, numerous cases of ISM resistance have been reported in different African regions, representing a significant constraint in the battle against Animal African Trypanosomiasis. In order to identify genetic signatures associated with ISM resistance in T. congolense, the sensitive strain MSOROM7 was selected for induction of ISM resistance in a murine host. Administered ISM concentrations in immune-suppressed mice were gradually increased from 0.001 mg/kg to 1 mg/kg, the maximal dose used in livestock. As a result, three independent MSOROM7 lines acquired full resistance to this concentration after five months of induction, and retained this full resistant phenotype following a six months period without drug pressure. In contrast, parasites did not acquire ISM resistance in immune-competent animals, even after more than two years under ISM pressure, suggesting that the development of full ISM resistance is strongly enhanced when the host immune response is compromised. Genomic analyses comparing the ISM resistant lines with the parental sensitive line identified shifts in read depth at heterozygous loci in genes coding for different transporters and transmembrane products, and several of these shifts were also found within natural ISM resistant isolates. These findings suggested that the transport and accumulation of ISM inside the resistant parasites may be modified, which was confirmed by flow cytometry and ex vivo ISM uptake assays that showed a decrease in the accumulation of ISM in the resistant parasites.
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.