Unknown,Transcriptomics,Genomics,Proteomics

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Measurement of variant surface glycoprotein mRNA using RNAseq

ABSTRACT: Trypanosoma brucei Lister 427 bloodstream forms were cultured in HMI-11 medium. Total RNA was prepared using Qiagen RNAeasy kits for single sample RNAseq to estimate VSG mRNA abundance (and not to reconstruct the transcriptome). The cDNA libraries were prepared and sequenced at the Beijing Genomics Institute (Shenzhen, China). Polyadenylated RNA was purified from total RNA, converted to cDNA using random hexamer primers sheared and size selected for fragments ~200 bp in length using the Illumina TruSeq RNA Sample Preparation Kit v2. RNAseq of the resulting libraries was used for the determination of transcript abundances. Sequencing was performed on an Illumina Hiseq 2000 (Illumina, CA) platform and 90 base paired end reads obtained. Four samples were analysed: 1. Trypanosoma brucei Lister 427 expressing VSG2 2. Trypanosoma brucei Lister 427 expressing VSG6 3. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 28 days after electroporation 4. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 44 days after electroporation.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Trypanosoma brucei

SUBMITTER: Mark Carrington

PROVIDER: E-MTAB-9122 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 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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Measurement of variant surface glycoprotein mRNA using RNAseq

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