Project description:We analysed differentiation of the EATRO1125 strain of Trypanosoma brucei brucei, which was first isolated in 1966 from a bushbuck (Tragelaphus scriptus) in Uganda (origin stated (Bouteillea, 1995) without an original reference.<br>To analyse gene expression, we isolated at least 3 x 10e8 trypanosomes at different differentiation states, using two independent biological replicates. Bloodstream forms were harvested at a density of 2 x 10e5/ml (low density, logarithmic growth), and 2 x 10e6/ml (high density, logarithmic growth). Cells were also taken immediately upon attaining the density of 2 x 106/ml, treated with 3 mM cis-aconitate and moved to a room at 27M-0C. Samples were taken 30 min, 60 min, 12h and 24h after this. At 24 h the cells were centrifuged, resuspended (at 27M-0C) in MEM-Pros medium, which contains proline as the major energy source. Samples were taken again at 48h and 72h. A culture that had been maintained for several weeks after transformation was used as a source of established procyclic trypanosomes.
Project description:Cy3 and Cy5 direct labelled RNA from Bloodstream MiTat1.1 trypanosomes and Procyclic 427 Lister were hybridized onto JCVI Trypanosoma brucei oligoarrays (version2). Procyclic RNA were used as control for data analysis.
Project description:The first 4 samples belong to the RNA-IP using in situ TAP tagged ZC3H30 in procyclic (insect) form of the parasite T. brucei Lister 427, 2 samples are Elu or eluate, and 2 are FL or flowthrough (unbound) sample. The other 8 samples are also from procyclic cells. 4 samples belong to DKO(ZC3H30 gene double knockout), 2 are non-stressed and 2 are heat shocked samples; the rest 4 samples are DKO-ectopic (ZC3H30 double knockouts, expressing, ectopic copy of ZC3H30) 2 are non-stressed and 2 are heat shocked samples. Heat Shock experiment was done at 39 degree Celsius.
Project description:The life cycle of Trypanosoma brucei involves several cell differentiation transitions that allow transmission, survival and proliferation of these parasites. One of these transitions, the differentiation of growth-arrested stumpy forms in the mammalian blood into proliferating insect-stage procyclic forms, can be induced synchronously in vitro by addition of cis-aconitate (CA). Using single-cell analysis by flow-cytometry to follow differentiation, we show that this transition is an irreversible bistable switch where cells commit to differentiation after 1-3 hours of exposure to CA. This irreversibility implies the existence of positive feedback mechanisms that allow commitment to differentiation: i.e. the establishment of “memory” of exposure to the differentiation signal. Such mechanisms probably depend on post-translational modifications (e.g. phosphorylation) and/or synthesis of regulatory proteins. Using the reversible protein synthesis inhibitor cycloheximide, we find that protein synthesis is required for establishment of signal memory and normal commitment to differentiation. To characterize the ‘commitment proteome’, we performed SILAC phosphoproteomics to provide a detailed map of the protein expression and phosphorylation events during the early stages of differentiation in a synchronised parasite population. Using a rigorous candidate gene approach we have also demonstrated that the stumpy form enriched serine-throenine protein kinases TbNRKA/B stringently control the earliest events in differentiation identifying these kinases as major regulators of trypanosome development.
Project description:T bruce were heat shocked for 1 h at 41C then granules were prepared as per Fritz et al. 2015. After spinning down cytoskeletons with trapped contents and lysing them we sequenced the supernatant (trapped RNA) and pellet (granule) fractions. Input total RNA is included for comparison.
Project description:In this current study, we aimed to produce and develop an affinity matrix for enzymes involved in the folate metabolism of kinetoplastids, effectively the “folateome”folateome of these parasites. This information will then be used to determine the molecular targets of compounds targeting folate metabolism, and to correlate this to phenotypic responses. To achieve this, a small library of clinical and literature anti-folate compounds were immobilized onto resins and used in “pull-down” experiments with and without test compound present, to ascertain proteins binding specifically to particular resins.8,10-11 While the initial focus of this project was to establish the kinetoplastid folateome, the approach has the potential to be extended to other organisms.
Project description:Rats (four per isolate) were infected with Trypansooma rhodesiense taken from the CNS of two different patients from Lwala. Blood was taken at parasitaemias ranging from 3-20 E7. The results can be compared with those from human patient samples.
Project description:RBP10 was induced for 24 or 48 hours in procyclic cells derived from EATRO1125; this primes the procyclic cells to differentiate to bloodstream form. RNA-seq was performed to quantify mRNAs changes during the differentiation triggered by RBP10 overexpression.
Project description:SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using a chemo-proteomic approach. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. The diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field.
Project description:The enzyme N-myristoyltransferase (NMT) catalyses the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localisation of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labelling with an alkyne-tagged myristic acid analogue (“YnMyr”), enabling the capture of lipidated proteins in insect (PCF) and host (BSF) life stages of T. brucei. We further compare this with a longer chain palmitate analogue (“YnPal”) to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors (Cpds 1 and 2) and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites.