Effect of in vitro culture on T. b. brucei MAK65 and MAK98
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PROVIDER: PRJEB44916 | ENA |
REPOSITORIES: ENA
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Project description:Fresh T. b. brucei isolates MAK65 and MAK98 (see E-MTAB-9320) were cultured in vitro for various times then the genomic DNA was sequenced.
2021-05-22 | E-MTAB-10457 | biostudies-arrayexpress
Project description:The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei. Keywords: Trypanosoma, VSG, antigenic switching, HDL-resistance
2007-07-04 | GSE8369 | GEO
Project description:The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei. Keywords: Trypanosoma, VSG, antigenic switching, HDL-resistance Bloodstream stages of the Lister strain 427 T. b. brucei (MiTat 1.2), expressing VSG221, were used in these studies. Cells were cultured in HMI-9 medium with the addition of heat inactivated fetal bovine serum (FBS) (10%) and Serum Plus (10%). T. b. brucei 427-221 is an antigenically stable line and contains a single copy of the vsg221 gene within the 221 expression site (221ES). At a cell density of approximately 1,000,000 cells/ml, T. b. brucei 427-221 were exposed to various amounts of human HDLs for 24 h in a 6 well plate. Surviving trypanosomes were counted using a hemocytometer then diluted into fresh HMI-9 medium and allowed to recover for 5-14 days. Once the cells had grown to a density of approximately 1,000,000 cells/ml, they were once again incubated with human HDLs. Each round of selection was performed with increasing concentrations of human HDLs and freezer stocks were prepared for each surviving population. Over nine months we conducted eight rounds of human HDL selection, resulting in a population of T. b. brucei that survived incubation with 800 μl of human HDLs (160 lytic U).
2007-07-04 | E-GEOD-8369 | biostudies-arrayexpress
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.
2021-01-22 | GSE165290 | GEO
Project description:In vitro generation of human high-density-lipoprotein-resistant Trypanosoma brucei brucei
| PRJNA101397 | ENA
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.
2012-05-30 | GSE37593 | GEO
Project description:Effect of CFB2 depletion in Trypanosoma brucei
| PRJEB40923 | ENA
Project description:Genomic shotgun sequences of Trypanosoma brucei brucei from Uganda with minimal laboratory passage.
2020-11-22 | E-MTAB-9759 | biostudies-arrayexpress
Project description:Purpose: Acoziborole is a recently developed benzoxaborole class compound, currently in clinical trials, for stage 1 and stage 2 treatment of Human African Trypanosomiasis. Recent studies have made significant progress in determining the molecular mode of action of acoziborole. However, less is known about the potential mechanisms leading to acoziborole resistance in trypanosomes. By characterising in vitro drug-resistance, this study aimed to gain a better understanding of the mechanisms involved in acoziborole resistance in the clinicaly relevant Trypanosoma brucei Methods: Drug resistance was generated in vitro through incremental dosage of acoziborole. RNA was isolated from axenic cultures of drug-resistant and parental drug sensitive cells and submitted for RNA-seq Results: Transcriptomics analysis revealed widepread downregulation of transcripts associated with mammalian-infective bloodstream-form parasites. Conversely, transcripts associated with insect-stage procyclic form parasites were increased, indicating that the resistant cells had undergone an unspecified "differentiation event", albeit on a transcriptomic level Conclusions: Trypanosoma brucei resistance to acoziborole can be generated under in vitro axenic conditions, and "transcriptional differentiation" is a mechanism of resistance. However, it is unknown whether this phenomenon is relevant to an in vivo setting
2021-03-07 | GSE168394 | GEO
Project description:In vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to be more biologically relevant, or to optimise growth, is traditionally a laborious task. Emerging metabolomics technology enables rapid evaluation of intra- and extra-cellular metabolites, and can be applied to the rational development of cell culture medium. In this study, untargeted semi-quantitative, and targeted quantitative, metabolomic analyses of fresh and spent media revealed the major nutritional requirements for growth of bloodstream-form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to more closely resemble concentrations normally found in blood. Our new minimal medium (CMM) supports in vitro growth equivalent to HMI11, and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (< 3-fold change; a = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole cell phenotypic screens, and in metabolomics mode of action assays. 400-fold decreases in IC50 were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomics studies and drug activity screening.
2015-05-14 | MTBLS49 | MetaboLights