Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei.

Sarah Kabani,Katelyn Fenn,Keith Matthews,Terry K Smith,Peter Ghazal,Al Ivens,Alan Ross

doi:10.1186/1471-2164-10-427

Abstract

BackgroundTrypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level.ResultsIn this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry), thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation.ConclusionUsing stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition.

Highlights

Trypanosomes undergo extensive developmental changes during their complex life cycle
Stumpy forms were purified from host blood by DE52 chromatography [39] at 37°C and incubated for 1 h at 37°C in HMI-9 [40] to allow the cells to recover from the purification process, this providing the starting material for expression analyses during their differentiation to procyclic forms
Confirming earlier analyses, each subunit was up-regulated during differentiation to the procyclic form, this occurring rapidly after the initiation of the process. These analyses demonstrated that the mRNAs generated from parasites undergoing synchronous differentiation from stumpy to procyclic forms, matched expectation based on known bloodstream vs. procyclic expression profiles or previous studies of synchronous differentiation between bloodstream stumpy and procyclic forms

Summary

Introduction

Trypanosomes undergo extensive developmental changes during their complex life cycle Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. Among the most evolutionarily divergent eukaryotes for which there is significant molecular information [13], the contribution of RNA polymerase II promoter activity to regulated gene expression is unimportant [14] Instead, their genome is organised into long polycistronic transcription units in which genes are co-transcribed [15], primary transcripts being resolved into mRNAs by concerted trans-splicing and polyadenylation reactions [16]. Regulated mRNA stability is a major contributor to differential mRNA abundance, regulated protein synthesis, modification and turnover are clearly major additional contributors to regulated gene expression [19,20]

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