The trypanosome transcriptome is remodelled during differentiation but displays limited responsiveness within life stages

Abstract

BackgroundTrypanosomatids utilise polycistronic transcription for production of the vast majority of protein-coding mRNAs, which operates in the absence of gene-specific promoters. Resolution of nascent transcripts by polyadenylation and trans-splicing, together with specific rates of mRNA turnover, serve to generate steady state transcript levels that can differ in abundance across several orders of magnitude and can be developmentally regulated. We used a targeted oligonucleotide microarray, representing the strongly developmentally-regulated T. brucei membrane trafficking system and ~10% of the Trypanosoma brucei genome, to investigate both between-stage, or differentiation-dependent, transcriptome changes and within-stage flexibility in response to various challenges.Results6% of the gene cohort are developmentally regulated, including several small GTPases, SNAREs, vesicle coat factors and protein kinases both consistent with and extending previous data. Therefore substantial differentiation-dependent remodeling of the trypanosome transcriptome is associated with membrane transport. Both the microarray and qRT-PCR were then used to analyse transcriptome changes resulting from specific gene over-expression, knockdown, altered culture conditions and chemical stress. Firstly, manipulation of Rab5 expression results in co-ordinate changes to clathrin protein expression levels and endocytotic activity, but no detectable changes to steady-state mRNA levels, which indicates that the effect is mediated post-transcriptionally. Secondly, knockdown of clathrin or the variant surface glycoprotein failed to perturb transcription. Thirdly, exposure to dithiothreitol or tunicamycin revealed no evidence for a classical unfolded protein response, mediated in higher eukaryotes by transcriptional changes. Finally, altered serum levels invoked little transcriptome alteration beyond changes to expression of ESAG6/7, the transferrin receptor.ConclusionWhile trypanosomes regulate mRNA abundance to effect the major changes accompanying differentiation, a given differentiated state appears transcriptionally inflexible. The implications of the absence of a transcriptome response in trypanosomes for both virulence and models of life cycle progression are discussed.

Evolutionary reconstruction of the retromer complex and its function in Trypanosoma brucei

“…As an excavate, T. brucei is distantly related to mammals and yeast but possesses a well-characterised endomembrane system (Field and Carrington, 2009). T. brucei Vps5 mRNA expression (Tb09.211.4240) is strongly upregulated in the mammalian form (Koumandou et al, 2008) where endocytosis is also upregulated (Natesan et al, 2007); this is consistent with a role for T. brucei Vps5 in endocytic activity.…”

“…T. brucei has a complete, yet simplified, trafficking system, which has been extensively studied (Field and Carrington, 2009). T. brucei Vps5 is strongly upregulated in the mammalian infective form, compared with its expression in the insect proliferative stage, and thus could be important for pathogenesis (Koumandou et al, 2008). Indeed, qRT-PCR analysis confirmed that all components of retromer identified by comparative genomics in T. brucei are upregulated in the mammalian form; this might be related to the more active lysosomal delivery route in the procyclic stage, whereas in the bloodstream stage recycling is the major trafficking route in terms of flux (Field et al, 2007b).…”

“…RNA extraction and qRT-PCR analysis were as previously described (Koumandou et al, 2008). Primers were: TbVps5-RTF,5Ј-AGCGTTGGTCAAGTGCTTTC-3Ј; TbVps5-RTR, 5Ј-CCCTCCCATGCAGTCATATC-3Ј; TbVPS26-RTF, 5Ј-TG-TACGTTCTCCGTGTGGTC-3Ј; TbVPS26-RTR, TAGAGCGTCCG GCTGAGTAT-3Ј; TbVPS29-RTF, 5Ј-GTTTACACCTGGCCGCATAC-3Ј; TbVPS29-RTR, 5Ј-CGAGTCAAACTCACCCTTGG-3Ј; TbVPS35-RTF, 5Ј-GGTGATGGCAAGGGT-GTATT-3Ј; TbVPS35-RTR, 5Ј-CGTAGATCACCTCGGAAAGC-3Ј.…”

Rab28 function in trypanosomes: interactions with retromer and ESCRT pathways

“…RNAi is highly efficient in trypanosomes, and especially resistant to off-target effects (Koumandou et al, 2008). Moreover cosuppression requires high sequence similarity, and specific knockdown of trypanosomatid genes sharing over 60% identity has been achieved without off-target effects (Bastin et al, 2000); T. brucei Rab28 is 27% identical to its closest paralogue, Rab23.…”

Pseudogene-derived small interference RNAs regulate gene expression in African Trypanosoma brucei

“…To adapt to the disparate environments in mammalian and insect hosts, T. brucei must undergo complex differentiation during the life cycle. Various researchers have revealed that a diversity of transcripts is involved in the pathogen's differentiation across its life stages (12)(13)(14). However, the mechanisms that control expression shifts remain undetermined.…”