BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 ity of trypanosome mRNAs are unmodulated, suggesting that, for most messages, transcription and turnover are unaffected by signaling pathways. This implies that the trypanosome has a very limited capacity to respond to altered circumstances in vivo, except via pre-programmed differentiation pathways. The absence of transcriptome responsiveness to environ- mental cues has several consequences. Firstly, lack of a classical UPR indicates ER insensitivity to increased con- centrations of non-native polypeptides, and implies that changes in biosynthetic output similarly will not be sensed. This is consistent with extreme sensitivity to con- centrations of DTT that are well tolerated in higher eukaryotes. Whether the UPR is absent from additional excavates is unknown, but preliminary evidence suggests that Giardia also lacks the pathway [112]. Secondly, fail- ure to modify the transcriptome following gene knock- down or over-expression, but where a major impact on transport is found, suggests that modulation of traffick- ing, and potentially other systems, within a given life stage is restricted to proteomic changes [117]. Thirdly, and most significantly, the data indicate an inability of the trypanosome endomembrane system to adapt to altering conditions and, coupled with the possibility that such unresponsiveness extends to additional cellular functions, has profound implications. Limited transcriptional flexibility is clearly a restriction for the trypanosome, but is in agreement with a modest small GTPase signaling network [30], which may indicate a rather limited signal transduction capacity. As each life stage exists within a highly controlled environment the trypanosome's ability to modulate the trafficking system has likely been lost. The environment within the insect host is poorly understood, but the bloodstream pH range in H. sapiens is 7.35 – 7.45, blood glucose is at 4 – 8 mM and transferrin concentration is maintained at ~300 ± 50 μg/dl [129]; major alterations to these parameters are lethal, and hence there is little need for the parasite to retain mechanisms for responsiveness. By contrast, a new transcriptome would be triggered by appropriate signals when the trypanosome enters a new compartment or host. Hence the trypanosome life cycle may be viewed as a set of inflexible, sequential transcriptional profiles that serve to adapt the parasite to each new environment (Fig- ure 6). The in vitro cultured BSF is a convenient model for the mammalian bloodstream trypomastigote but, during infection, trypanosomes also invade tissue spaces, the lymphatic system and, in later disease stages, the cerebral spinal fluid [1]. Each environment differs greatly from the bloodstream in composition and hence transcriptome inflexibility suggests that a differentiation event may be required for adaptation. Further, the major transcriptome differences between cultured and animal-isolated trypanosomes underscores the potential for currently uncharted, but dramatic, adaptations associated with mammalian infection. A similar transcriptional rigidity is suggested for the intra- erythrocytic stages of the malaria parasite, Plasmodium fal- ciparum, possibly also related to the fact that Plasmodium enjoys a homeostatic host environment [130-132]. Trypanosomatids (Excavata) and Plasmodia (Chromalve- olata) are highly divergent and separated by many non- parasitic or free-living species, and thus transcriptome unresponsiveness most likely is the result of independent secondary loss accompanying adoption of the parasitic life style. Consistent with this view, environmental stress treatments do induce transcriptional responses in Euglena gracilis [133], a free-living protist closely related to kineto- plastids. Further, as Plasmodium utilises a more conven- tional promoter-mediated mechanism for transcription, this also rules out a trivial explanation that transcriptome inflexibility is simply the result of polycistronic transcrip- tion. Examination of transcriptome regulation in more free-living organisms related to both Plasmodium and trypanosomes would be highly informative. Finally, these data indicate that knockdown of a specific mRNA does not alter the transcript levels of additional gene products coordinated functionally with the RNAi tar- get. Therefore, in T. brucei RNAi is unlikely to suffer from off target effects as seen in higher eukaryotes [134-136], and hence is, in this regard, unusually clean. Secondary effects are likely mediated via proteome changes, and could explain the similar phenotypes observed for many knockdowns [61]. This is particularly prominent for cell cycle/cytokinesis defects that frequently emerge rather late following knockdown. Such effects may be the result of generic loss of the normal proteome, rather than absence of specific factors. Conclusion The life cycle of T. brucei includes a dramatic differentia- tion event as the parasite progresses from the insect to the mammalian host. Differentiation encompasses consider- able remodeling of the membrane trafficking system, which is also a vital component of the host-parasite inter- face. Using a subgenome microarray targeting the T. brucei trafficking systems together with qRT-PCR analysis, we find clear evidence for developmental alteration to ~6% of the trypanosome transport-associated transcriptome. This indicates that developmental remodeling of endomembrane transport is underpinned by alterations to mRNA abundance, and that such changes may be ana- lysed by microarray. Most protein-coding mRNAs in trypanosomatids are produced by polycistronic transcrip- tion, with specific rates of decay as the major mechanism controlling steady state transcript levels. Multiple stimuli, Page 20 of 28 (page number not for citation purposes)