Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of C57BL/6J female mouse ovaries, hypothalamus, and pituitary gland tissue in animals infected with Trypanosoma brucei brucei at acute and chronic time points

ABSTRACT: Data from our group has shown that the protozoan parasite Trypanosoma brucei brucei can invade the mouse female reproductive tract (FRT) causing changes in tissue architecture and immune profile, as well as inducing reproductive cycle arrest. The purpose of this experiment was to identify transcriptional changes across the hypothalamic–pituitary–gonadal (HPG) axis during infection to link parasite-induced changes in hormonal regulation with the observed phenotype in the FRT. Female C57BL/6J mice were infected with 2x10^3 T. brucei brucei Antat 1.1E parasites via intraperitoneal injection. Mice were euthanised at 7 and 21-24 days post-infection, representing the acute and chronic stages of infection, respectively. Tissue was collected into RNAlater and stored at -80oC prior to RNA extraction. RNA was isolated using phenol-chloroform extraction. Libraries were prepared using the Illumina Stranded mRNA Prep. Ligation kit (Illumina, Inc.). Libraries were indexed and pooled for paired-end sequencing which was performed on an Illumina NovaSeq6000 instrument. The resulting data was de-multiplexed and BCL-to-Fastq conversion was performed using Illumina's bcl2fastq software (v2.20.0.4).

INSTRUMENT(S): N/A, Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Stefano Colombo

PROVIDER: E-MTAB-17174 | 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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RNA-seq of C57BL/6J female mouse ovaries, hypothalamus, and pituitary gland tissue in animals infected with Trypanosoma brucei brucei at acute and chronic time points

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