Tbrucei_H1_RNAi
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2000
ORGANISM(S): Trypanosoma brucei brucei
SUBMITTER: Helena Manso
PROVIDER: E-MTAB-1715 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Transcriptome responsiveness was further tested by attempts to invoke the unfolded protein repsonse (UPR), a classic ER-based pathway stimulated by the presence of increased levels of unfolded polypeptides. The UPR is mediated via transcriptional responses in both yeast and metazoan cells, and can be reliably activated by addition of dithiothreitol (DTT). Using DTT at concentrations that invoke a UPR in mammalian cells, Arabidopsis, yeast and other systems, we found that, in T. brucei, DTT exposure led to rapid cell death. We analysed the transcriptome at 1 mM DTT, under conditions where cells remained viable, as assessed by motility. <br><br>part 1: 3 biological replicates of SMB cells grown under normal conditions, and 3 replicates of SMB cells treated with 1mM DTT for 1hr, as well as dye swaps were used. <br><br>part 2: 3 biological replicates of SMB cells <br>grown under normal conditions, and 3 replicates of SMB cells treated <br>with 1mM DTT for 4hr, as well as dye swaps were used.<br><br>The UPR can also be activated by addition of tunicamycin. Using tunicamycin at concentrations that invoke a UPR in mammalian cells, Arabidopsis, yeast and other systems, we found that, in T. brucei, tunicamycin exposure efficiently inhibits trypanosome N-glycosylation and that it resulted in growth arrest over a period of up to 24 hours. We analysed the transcriptome at 5 ?g/ml tunicamycin under conditions where cells remained viable, as assessed by motility.<br><br>part 3: 3 biological replicates of SMB cells grown under normal conditions, and 3 replicates of SMB cells treated with 5 ?g/ml tunicamycin for 4hr, as well as dye swaps were used.<br><br>part 4: 3 biological replicates of SMB cells grown under normal conditions, and 3 replicates of SMB cells treated with 5 ?g/ml tunicamycin for 24hr, as well as dye swaps were used.
2008-06-26 | E-MEXP-1533 | biostudies-arrayexpress
Project description:Benzoxaboroles (BoBs) feature a boron-heterocyclic core and are an important recent innovation in the development of drugs against a range of pathogens and other pathologies. A broad spectrum of pharmacology is associated with chemically diverse BoB derivatives and includes multiple modes-of-action and targets. However, a consensus MoA for BoBs targeting evolutionarily diverse protozoan pathogens has emerged with the identification of CPSF3/CPSF73 in the CPSF complex in both apicomplexan and kinetoplastida parasites. We have detected a functional connection between protein sumoylation and the BoB boron-heterocyclic scaffold using comprehensive genetic screens in Trypanosoma brucei. Strikingly, as part of this sumoylation response, members of the CPSF complex are specifically and rapidly destabilised in a SUMO and proteosome-dependent manner. Here we deposit RNAseq data quantifying the effects of the aminomethyl-benzoxaborole AN3057 exposure on the transcriptome landscape in T. brucei. Specifically, T. brucei bloodstream-form cells in logarithmic growth phase were treated with 400 nm AN3057 (3 × EC50 determined after 24h) for 20 min (T20) and 60 min (T60), respectively. Nontreated control cells were prepared in parallel. All samples were in 2 biological replicates.
2024-03-08 | E-MTAB-13843 | biostudies-arrayexpress
Project description:As an additional strategy for investigating T. brucei transcriptome responsiveness, the mRNA of a highly important protein, the variant surface glycoprotein (VSG) the major surface protein in the bloodstream stage, was suppressed with RNAi. VSG RNAi results in arrest of cell cycle progression in the bloodstream stage. In addition, the mRNA of a highly important protein for endocytosis, the clathrin heavy chain (CLH), was suppressed with RNAi. CLH knockdown leads to a complete block to endocytosis. We hypothesized that if trypanosomes were able to sense alterations in trafficking and respond to these changes, then depletion of these ORFs by RNAi would be expected to elicit a response at the transcriptome level.<br> <br> part 1: 2 biological replicates of SMB cells transfected with the VSG- RNAi vector grown under normal conditions (non-induced), and 2 replicates of the same cells treated with 1 ug/ml tetracycline (induced) for 24hr, as well as dye swaps were used.<br> <br> part 2: 2 biological replicates of SMB cells transfected with the VSG- RNAi vector grown under normal conditions (non-induced), and 2 replicates of the same cells treated with 1 ug/ml tetracycline (induced) for 3 days, as well as dye swaps were used.<br> <br> part 3: 3 biological replicates of SMB cells transfected with the CLH- RNAi vector grown under normal conditions (non-induced), and 3 replicates of the same cells treated with 1 ug/ml tetracycline (induced), as well as dye swaps were used.
2008-06-26 | E-MEXP-1528 | biostudies-arrayexpress
Project description:In a phenotypic screening approach of novel molecules composed of a synergistic combination of phthalimide, benzimidazole, and triazole scaffolds we discovered compounds with potent anti-leishmanial activity. The resulting early-lead compound PHT-39, which contains a trifluoromethyl substitution, demonstrated the highest efficacy in a Leishmania infantum intramacrophage assay, with an EC50 of 1.2+/- 3.2 μM.Cytotoxicity testing of PHT-39 in Hep-G2 cells indicated high selectivity of over 90-fold. To investigate the mechanism of action we carried out experiments in Trypanosoma brucei, which is also sensitive to PHT-39. Here we used a genome-wide RNAi library approach (PMID: 22278056; PMID: 21363968) to detect sensitivity determinants. This high-throughput phenotyping approach identified sensitivity determinants for PHT-39, which included a P-type ATPase that is crucial for the uptake of miltefosine and amphotericin, strongly indicating a shared route for cellular entry.
2024-03-08 | E-MTAB-13844 | biostudies-arrayexpress
Project description:Bloodstream trypanosomes are sensitive to iron starvation, and upregulate ESAG6/7 transferrin receptor (TfR) mRNA and protein levels when placed in low transferrin or iron-depleted media, and adjust TfR expression to compensate for reduced endocytosis. Both observations suggest a specific transcriptional-level response resulting from an iron-sensing mechanism. The transcriptomes of BSFs cultured in different proportions of fetal bovine serum (FBS) were analysed. <br><br>part 1: 4 biological replicates of SMB cells grown with 10% FBS (normal conditions) and 4 replicates of SMB cells grown with 30% FBS, as well as dye swaps, were used. <br><br>part 2: 3 biological replicates of SMB cells grown with 10% FBS (normal conditions) and 3 replicates of SMB cells grown without FBS (but supplemented with 5 mg/ml BSA to simulate physiological concentrations of albumin in media with 10% FBS), as well as dye swaps were used. <br><br>part 3: 3 biological replicates of SMB cells grown with 10% FBS (normal conditions) and 3 replicates of SMB cells grown without FBS (but supplemented with 5 mg/ml BSA and 0.3 mg/ml bovine holo-transferrin to simulate physiological concentrations of albumin and transferrin in media with 10% FBS), as well as dye swaps were used.
2008-06-26 | E-MEXP-1530 | 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:The vast majority of molecular studies on T. brucei are carried out using in vitro cultures. Recently, whole-genome microarray analysis indicated significant differences between in vivo and in vitro cultures of the malaria parasite Plasmodium falciparum. Therefore, as an additional investigation into the influence of culturing conditions on intracellular transport we compared mRNA from bloodstream form MITat1.1 trypanosomes extracted from infected rats and from SMB cells cultured under standard conditions. 3 biological replicates of SMB cells grown under normal in vitro conditions, and 3 biological replicates of MITat1.1 cells grown in vivo in rats, as well as dye swaps were used.<br><br>The method for RNA isolation from cells grown in vivo contains steps carried out at 4oC. To eliminate the possibility that differences between the in vitro an in vivo samples arise from cold-shock during isolation of MITat1.1 trypomastigotes, we also extracted RNA from SMB cultures incubated at 4oC for 1hr before RNA extractions. 3 biological replicates of SMB cells grown under normal in vitro conditions, and 3 biological replicates of SMB cells grown under normal in vitro conditions but incubated at 4oC for 1 hr before RNA extraction, as well as dye swaps were used.
2008-06-26 | E-MEXP-1540 | biostudies-arrayexpress
Project description:Organization of the genome into compacted chromatin is a eukaryotic innovation facilitating increased sophistication in transcriptional regulation. In metazoa coiled-coil lamin proteins are major components of the chromatin organizer at the nuclear periphery and maintain nuclear integrity. While identifiable lamin homologues are restricted to metazoans, morphologically analogous structures maintaining nuclear organization in other eukaryotic lineages are known, but the molecular constituents remain undefined. Trypanosoma brucei NUP-1 is a large coiled-coil protein associated with fibrils at the inner face of the nuclear envelope. Using transcriptome analysis in combination with RNA interference and various imaging techniques, we demonstrate that NUP-1 forms a stable immobile cage around the nucleus, is required for viability and nuclear structural integrity, directs the positional organization of nuclear pore complexes, and serves to organize chromatin and specifically repress genes located at the nuclear periphery involved in immune evasion. Based on architectural similarity and functionality, we propose that NUP-1 is a novel, highly divergent lamin The effect of Nup-1 depletion on the transcriptome was examined in three independent experiments (A, B, & C). T. brucei cultures were either treated with RNAi (plus) or left untreated (minus) and RNA was extracted from each sample at the indicated time point (0h, 6h, 12h, 24h, or 48h). Two color microarrays were performed comparing treated and untreated samples at each time point. Dye swaps were performed and are indicated. Replicates of t=12h and t=24h for sample B were also included.
2011-12-15 | E-GEOD-26256 | biostudies-arrayexpress
Project description:The enzyme N-myristoyltransferase (NMT) catalyses the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localisation of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labelling with an alkyne-tagged myristic acid analogue (“YnMyr”), enabling the capture of lipidated proteins in insect (PCF) and host (BSF) life stages of T. brucei. We further compare this with a longer chain palmitate analogue (“YnPal”) to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors (Cpds 1 and 2) and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites.
2016-05-17 | PXD004053 | Pride
Project description:To look at differential expression of membrane-trafficking genes, <br>we extracted RNA from exponentially growing cultures of T. brucei <br>bloodstream (BSF) and procyclic (PCF) forms, labelled and competitively <br>hybridised the cDNA to the microarray. 8 arrays, representing <br>6 BSF and 5 PCF biological replicates, as well as dye swaps were used.
2008-06-26 | E-MEXP-1529 | biostudies-arrayexpress