RNAi targeting ZC3H20 in differentiating EATRO1125 Trypanosoma brucei
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Trypanosoma brucei
SUBMITTER: Christine Clayton
PROVIDER: E-MTAB-7995 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2010-05-16 | E-GEOD-17026 | 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 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: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.
2010-01-01 | E-MEXP-2028 | biostudies-arrayexpress
Project description:Trypanosoma brucei were isolated from cattle, in Bunya, Uganda (Tb065BAPC) or Apuru, Uganda (Tb098AAPC). Parasites were stored as stabilities after a single mouse passage. After a further mouse passage the parasites were grown in rats to the parasitaemias indicated, isolated on DEAE cellulose and RNA was prepared. We also compared rRNA depletion with poly(A) selection.
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2017-02-10 | E-MTAB-4952 | biostudies-arrayexpress
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.
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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.
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Project description:PUF2 RNAi, V5-PUF2 and UBP1-myc pull-downs, all methods and protocols are described in detail in the publication
2018-05-21 | E-MTAB-5617 | biostudies-arrayexpress
Project description:During the bloodstream stage of the Trypanosoma brucei lifecycle, the parasite exists as the proliferative slender-form or the non-proliferative, transmissible, stumpy-form. The transition from the slender to stumpy-form is stimulated by a density-dependent mechanism and is important in infection dynamics, ordered antigenic variation and disease transmissibility. Here, we use a monomorphic reporter cell line in a whole-cell fluorescence-based assay to screen over 6000 small molecules from a kinase-focussed compound library for their ability to induce stumpy-like formation in a high-throughput screening programme. This identified one compound able to induce modest, yet specific, changes in gene expression indicative of a partial differentiation to stumpy forms. This not only provides a potential tool for the further understanding of stumpy formation, but also demonstrates the use of high throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes. Examination of gene expression in response to treatment with DDD00015314.
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