Transcriptomics of suramin resistance in Trypanosoma brucei
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB20650 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR2234933.fastq.gz | Fastqsanger.gz | |||
| ERR2234934.fastq.gz | Fastqsanger.gz | |||
| ERR2234935.fastq.gz | Fastqsanger.gz | |||
| ERR2234936.fastq.gz | Fastqsanger.gz | |||
| ERR2234937.fastq.gz | Fastqsanger.gz |
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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).
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Project description:The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective Variant Surface Glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ~15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more ‘metacyclic’-like. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel architectural chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.
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Project description:Chromatin immunoprecipitation of genomic loci in Trypanosoma brucei where histone variant H3.V is deposited. This was achieved by deletion of one H3.V allele and N-terminal tagging of the second H3.V allele with a Ty1 tag. During the ChIP experiment, the DNA was digested with MNase to obtain mononucleosomes. Nucleosomes containing H3.V were pulled down by using a BB2 anti-Ty1 antibody. Cross links are reversed and mononucleosomal DNA is purified and prepared for Illumina sequencing.
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