BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 Background Trypanosoma brucei is the causative agent of sleeping sick- ness in humans and N'gana in cattle, and has a major eco- nomic and morbidity impact across much of Africa [1]. The principal mechanism of immune evasion in the mammalian host is antigenic variation, sequential expres- sion of immunologically distinct variant surface glycopro- teins (VSGs) at the cell surface [2], but additional mechanisms, including manipulation of the host immune system and antibody clearance from the surface also participate [3-5]. The full impact of these mecha- nisms on virulence is not understood at this time. The mammalian part of the life cycle is complex, involving penetration of multiple tissue spaces in the mammalian host, encompassing the bloodstream, lymphatic system and central nervous systems. Despite morphological sim- ilarities between mammalian forms, it is unknown if a developmental stimulus is required for tissue tropism or if this results from stochastic events. Further, differentiation to the procyclic insect form on entering the tsetse fly vec- tor is accompanied by massive cellular remodeling, including replacement of the VSG coat with procyclins, a family of acidic glycoproteins, activation of the mitochon- drion, changes to cellular and organelle morphology, altered cell cycle checkpoints and attenuation of endocytic activity [6-10]. Multiple stages have been described during fly infection, with clear changes in morphology and sur- face antigen expression [7,11,12]. How these alterations are controlled at the transcriptional level remains unclear; for example each form could represent a distinct develop- mental stage, implying stimulus-driven differentiation, or result from transcriptome flexibility, allowing modula- tion of levels of certain transcripts in response to altered conditions. Polycistronic transcription in trypanosomes [13] pre- cludes promoter-driven control of transcription, while specific degradation appears to be the major mechanism underpinning regulation of mRNA steady state levels. Microarray hybridization and real time (RT) PCR meth- ods can monitor steady-state RNA levels, and are inde- pendent of mechanisms controlling mRNA abundance. An earlier whole genome microarray study [14] identified ~2% of ORFs as developmentally regulated, but several developmentally regulated factors, including the clathrin heavy chain and Rab11 [10,15,16] were not detected in that analysis. Membrane trafficking is a defining characteristic of eukaryotic cells, playing major roles in nutrient uptake, turnover, signalling, immune defence, apoptosis and many other processes. Mechanisms regulating transport remain only partly understood in any organism, but tran- scriptional, post-transcriptional and post-translational processes are all implicated, with changes in transcription [17], complex kinase integration [18], and formation of multiple protein-protein interactions [19,20] contribut- ing to control. Further, copious evidence implicates small GTPases in regulating distinct aspects of transport, includ- ing Rabs in vesicle fusion [21], ARFs in regulation of membrane coat systems [22] and Rho-related proteins in cytoskeletal interactions [23,24]. Expression profiling for multiple human and mouse tissues indicates correlations between expression of certain Rabs and the SNAREs, adaptors, and other proteins with which they interact [17] and facilitates construction of potential interaction hubs and prediction of novel pathways. Understanding of membrane transport is comparatively advanced in trypanosomes [25,26], and various cellular changes are associated with life cycle progression [6], most notably an order of magnitude increase in endocytic activity in the mammalian infective bloodstream form (BSF) versus the insect procyclic (PCF) stages [10]. Increased endocytic activity in the BSF is likely related to immune evasion [4,26,27]. By contrast, knowledge of trypanosome signal transduction is poor [28]. Few Ras- like GTPases or their corresponding regulatory factors are present in trypanosomes, heterotrimeric GTPases are absent and there are no obvious receptor-type tyrosine kinases [29-31]. There is a complex predicted trypano- some kinome [31] but limited understanding as to how signaling is mediated via these factors [32-34]. There is evidence for phosphatidylinositol-mediated signaling, but this is comparatively unexplored [35], and a large novel family of receptor-coupled adenylate cyclases in kinetoplastids argues for significant environmental sens- ing via novel mechanisms [36,37]. We selected the trypanosome trafficking system as a clear example of a developmentally regulated process, where sufficient data are available for meaningful investigation of transcriptome changes. Using microarray analysis and quantitative RT-PCR, we assessed developmental regula- tion of trypanosome membrane transport and transcrip- tional flexibility by targeted genetic manipulation and altered environmental conditions designed to modulate trafficking pathways. From work in other organisms, we anticipated the involvement of protein kinases, small GTPases and, in the case of the unfolded protein response (UPR), a system that responds to increased concentrations of non-native polypeptides within the endoplasmic retic- ulum, clear evidence for a transcriptional mechanism. Our data indicates developmental remodeling of the tran- scriptome, but essentially no response to altered condi- tions. Page 2 of 28 (page number not for citation purposes)