Continuous culture of bloodstream form and procyclic form of Trypanosoma brucei
ABSTRACT: Trypanosomes are a globally important group of parasites which together kill and debilitate millions of people world-wide. In trypanosomes, genes do not have individual promoters, rather ~10000 genes share ~200 promoters and all gene expression is thus regulated post-transcriptionally. While effector proteins which modulate the expression of many genes have been described, the mechanisms by which trypanosomes sense changes in their environment and manifest changes in gene expression remain elusive. This study demonstrates that trypanosomes sense changes in their environment through temperature sensitive RNA secondary structure. We show that the majority of observed mRNA abundance changes which distinguish insect adapted and bloodstream adapted life cycle stages can be explained through change in temperature alone.
Project description:Trypanosomes are a globally important group of parasites which together kill and debilitate millions of people world-wide. In trypanosomes, genes do not have individual promoters, rather ~10000 genes share ~200 promoters and all gene expression is thus regulated post-transcriptionally. While effector proteins which modulate the expression of many genes have been described, the mechanisms by which trypanosomes sense changes in their environment and manifest changes in gene expression remain elusive. This study demonstrates that trypanosomes sense changes in their environment through temperature sensitive RNA secondary structure. We show that the majority of observed mRNA abundance changes which distinguish insect adapted and bloodstream adapted life cycle stages can be explained through change in temperature alone.
Project description:In trypanosomes, the apparent lack of regulation of RNA polymerase II-dependent transcription initiation poses a challenge to understand how these eukaryotes adjust gene expression in order to adapt to the contrasting environments they find during their life cycles. Evidence so far indicates that mRNA turnover and translation are the major control points in which regulation is exerted in trypanosomes. However, very little is known about which proteins are involved, and how do they regulate the abundance and translation of different mRNAs in different life stages. In this work, an RNA-binding protein, TbDRBD3, has been identified by affinity chromatography, and its function addressed using RNA interference, microarray analysis and immunoprecipitation of mRNA-protein complexes. The results obtained indicate that TbDRBD3 binds to a subset of developmentally regulated mRNAs encoding membrane proteins and intermediate metabolism enzymes, and that this association promotes the stabilization of the target transcripts. These observations raise the possibility that TbDRBD3-mRNA complexes act as a post-transcriptional operon, and provide a framework to interpret how trypanosomes regulate gene expression in the absence of transcriptional control. Keywords: Effect of depletion of an RNA-binding protein on the transcriptome Overall design: In order to analyze whether TbDRBD3 has a role in the regulation of mRNA turnover, the transcriptome of procyclic trypanosomes depleted of TbDRBD3 was compared to that of uninduced cells using hybridizations of genomic microarrays. RNA samples were isolated from cells harvested after 48 hours of induction with tetracycline. At this point, cell growth was barely affected and trypanosomes remained highly motile. 15 µg of RNA were reverse-transcribed in the presence of 100 ng of oligo(dT)12-18, 0.25 mM dATP, 0.25 mM dTTP, 0.25 mM dGTP, 0.05 mM dCTP, 0.05 mM Cy3- or Cy5-dCTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 40 U of RNaseOUT (Invitrogen) and 400 U of Superscript III reverse transcriptase (Invitrogen). Reactions were incubated for 3 hours at 50ºC, stopped by heating at 70ºC for 15 min and treated with 5 U of RNase H for 20 min at 37ºC. cDNA was purified using the MinElute kit (Qiagen), ethanol precipitated and resuspended in hybridization buffer. Genomic T. brucei microarray glass slides containing ca. 24,000 independent random genomic clones (Brems et al., 2005) were pre-hybridized and hybridized as described (Diehl et al., 2002). Four hybridizations (two biological replicates with a dye swap each) were analyzed.
Project description:T. brucei PF cells were treated with several chemical reagents and anti-trypanosomatid drugs. The effect of each chemical perturbation on the transcriptome of T. brucei was examined by transcript profiling of treated vs. control cells. The results indicated widespread changes, suggesting that the transcriptome of T. brucei is highly responsive to environmental factors that perturb its metabolic and biological pathways. 11 chemical perturbations, each co-hybridized with a common reference RNA from control non-treated cells. One array per treatment.
Project description:To address whether T. brucei has a circadian clock we probed its transcriptome by RNA-seq, searching for transcripts oscillating with a 24 hr period. For this we entrained/ synchronized parasites in vitro for three days using temperature and light as environmental stimuli and then collected parasite RNA every four hours for two consecutive days. Parasite RNA was subjected to RNA-seq analysis. We performed this protocol on two stages of the T. brucei life cycle (bloodstream and insect procyclic forms).
Project description:A procyclic form Trypansome brucei RNAi line (PTT parental line, transfected with pALC14 incorporating a TbNMD3 gene fragment) capable of inducing depletion of TbNMD3 was analysed for mRNA expression by RNAseq Cells were grown for 72 hours in culture; RNAi was induced in cells by the addition of 1 microgram/ml of tetracycline
Project description:Comparative phosphoproteomic analysis of SILAC labelled cultured bloodstream and procyclic form Trypansoma brucei. Phosphopeptide enrichement via SCX and TiO2, acquired on Oribtrap Velos using MSA. Triplicate biological replicate for each lifecycle stage (as unmixed, and two SILAC label swap expreiments), eight fraction injected in technical triplicate. For the SILAC labelled samples, the corresponding changes in the proteome were measured using unenriched peptides sperated by SCX. Data was processed using MaxQuant version 22.214.171.124 which incorporates the Andromeda search engine. Proteins were identified by searching a protein sequence database containingT. brucei brucei 927 annotated proteins (Version 4.0, downloaded from TriTrypDB http://www.tritrypdb.org/ supplemented with the VSG221 sequence and frequently observed contaminants (porcine trypsin, bovine serum albumins and mammalian keratins) that contains a total of 10,081 protein sequences. Search parameters specified an MS tolerance of 6 ppm, an MS/MS tolerance at 0.5 Da and full trypsin specificity, allowing for up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionines, N-terminal protein acetylation and N-pyroglutamate were allowed as variable modifications. Phosphoproteomic analysis included phosophorylation of serine, threonine and tyrosine as additional variable modifications. Peptides were required to be at least 7 amino acids in length and a MaxQuant score >5, with false discovery rates (FDRs) of 0.01 calculated at the levels of peptides, proteins and modification sites based on the number of hits against the reversed sequence database.
Project description:Procyclic trypanosomes (strain 427 lister) were grown in culture under standard conditions at 27ºC in SDM79 medium with 10% foetal bovine serume (Brun and Schnenberger, 1979), in a gazed incubator (5% CO2). Logarithmically growing procyclic cells (at about 5*10^6 cells/ml, at 27°C) were added to one volume medium that had been heated to 53°C and incubated at 41ºC for 60 minutes in a waterbath in a closed tube (41ºC sample). The control cells were added to one volume medium at 27ºC and also incubated for 60 minutes in a closed tube at 27°C. Cells were harvested and washed once in PBS. The harvesting was done within 8 minutes.
Project description:The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically ‘slender forms’ to transmissible ‘stumpy forms’ through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched in throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been identified to enrich transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream haves been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms. Examination of gene expression during life cycle stages.