Bloodstream form pre-adaptation to the tsetse fly in Trypanosoma brucei

African trypanosomes are sustained in the bloodstream of their mammalian hosts by their extreme capacity for antigenic variation. However, for life cycle progression, trypanosomes also must generate transmission stages called stumpy forms that are pre-adapted to survive when taken up during the bloodmeal of the disease vector, tsetse flies. These stumpy forms are rather different to the proliferative slender forms that maintain the bloodstream parasitaemia. Firstly, they are non proliferative and morphologically distinct, secondly, they show particular sensitivity to environmental cues that signal entry to the tsetse fly and, thirdly, they are relatively robust such that they survive the changes in temperature, pH and proteolytic environment encountered within the tsetse midgut. These characteristics require regulated changes in gene expression to pre-adapt the parasite and the use of environmental sensing mechanisms, both of which allow the rapid initiation of differentiation to tsetse midgut procyclic forms upon transmission. Interestingly, the generation of stumpy forms is also regulated and periodic in the mammalian blood, this being governed by a density-sensing mechanism whereby a parasite-derived signal drives cell cycle arrest and cellular development both to optimise transmission and to prevent uncontrolled parasite multiplication overwhelming the host.In this review we detail recent developments in our understanding of the molecular mechanisms that underpin the production of stumpy forms in the mammalian bloodstream and their signal perception pathways both in the mammalian bloodstream and upon entry into the tsetse fly. These discoveries are discussed in the context of conserved eukaryotic signalling and differentiation mechanisms. Further, their potential to act as targets for therapeutic strategies that disrupt parasite development either in the mammalian bloodstream or upon their transmission to tsetse flies is also discussed

Independent Pathways Can Transduce the Life-Cycle Differentiation Signal in Trypanosoma brucei

African trypanosomes cause disease in humans and livestock, generating significant health and welfare problems throughout sub-Saharan Africa. When ingested in a tsetse fly bloodmeal, trypanosomes must detect their new environment and initiate the developmental responses that ensure transmission. The best-established environmental signal is citrate/cis aconitate (CCA), this being transmitted through a protein phosphorylation cascade involving two phosphatases: one that inhibits differentiation (TbPTP1) and one that activates differentiation (TbPIP39). Other cues have been also proposed (mild acid, trypsin exposure, glucose depletion) but their physiological relevance and relationship to TbPTP1/TbPIP39 signalling is unknown. Here we demonstrate that mild acid and CCA operate through TbPIP39 phosphorylation, whereas trypsin attack of the parasite surface uses an alternative pathway that is dispensable in tsetse flies. Surprisingly, glucose depletion is not an important signal. Mechanistic analysis through biophysical methods suggests that citrate promotes differentiation by causing TbPTP1 and TbPIP39 to interact

Evolution, dynamics and specialized functions of glycosomes in metabolism and development of trypanosomatids

Kinetoplastea such as trypanosomatid parasites contain specialized peroxisomes that uniquely contain enzymes of the glycolytic pathway and other parts of intermediary metabolism and hence are called glycosomes. Their specific enzyme content can vary strongly, quantitatively and qualitatively, between different species and during the parasites' life cycle. The correct sequestering of enzymes has great importance for the regulation of the trypanosomatids' metabolism and can, dependent on environmental conditions, even be essential. Glycosomes also play a pivotal role in life-cycle regulation of Trypanosome brucei, as the translocation of a protein phosphatase from the cytosol forms part of a crucial developmental control switch. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative for unique forms of activity regulation, whereas many kinetic activity regulation mechanisms common for glycolytic enzymes are absent in these organisms. Glycosome turnover occurs by autophagic degradation of redundant organelles and assembly of new ones. This may provide the trypanosomatids with a manner to rapidly and efficiently adapt their metabolism to the sudden, major nutritional changes often encountered during the life cycle. This could also have helped facilitating successful adaptation of kinetoplastids, at multiple occasions during evolution, to their parasitic life style

A conserved trypanosomatid differentiation regulator controls substrate attachment and morphological development in Trypanosoma congolense

Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in that parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunoreactive proteins in experimental bovine infections, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission

Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

<p>Abstract</p> <p>Background</p> <p>Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β.</p> <p>Results</p> <p>URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that <it>Drosophila </it>Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a <it>uri </it>loss of function allele, and show that <it>uri </it>is essential for viability in <it>Drosophila</it>. <it>uri </it>mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei.</p> <p>Conclusion</p> <p>Uri is the first PP1α specific binding protein to be described in <it>Drosophila</it>. Uri protein plays a role in transcriptional regulation. Activity of <it>uri </it>is required to maintain DNA integrity and cell survival in normal development.</p

Functional dissection of T. brucei Protein Tyrosine Phosphatase 1 and investigation of its development as a therapeutic target

Trypanosoma brucei undergoes developmentally regulated morphological and biochemical changes during its life cycle, being transmitted between the mammalian host and the tsetse fly. It is generally recognized that cellular responses to environmental changes are mediated through signalling pathways, but our understanding of trypanosome signal transduction during differentiation is limited. Protein Tyrosine Phosphatase 1 (TbPTP1) is the one of the few factors identified to be responsible for differentiation from stumpy to procyclic form parasite, whereby TbPTP1 inhibition stimulates transition to insect-form cells (Szoor et al., 2006). In order to characterize the TbPTP1 signalling pathway, a substratetrapping approach was used, which identified a phosphatase TbPIP39 as substrate of TbPTP1. TbPIP39 interacts with, and is dephosphorylated by TbPTP1 in stumpy form cells. Additionally, it has been shown that upon citrate/cis-aconitate (CCA) treatment, phosphorylated TbPIP39 localizes to the parasite glycosomes, the organelles responsible for bloodstream forms metabolism, thereby promoting cellular differentiation to procyclic forms (Szoor et al., 2010). With the aim of further dissecting the TbPTP1 signalling pathway, the substrate-trapping approach was used, which identified one novel TbPTP1 substrate candidate, potentially involved in regulation of differentiation. In addition, the effect of other differentiation triggers, namely protease treatment or mild acid exposure, on the level of TbPIP39 phosphorylation was analyzed, to determine whether these stimuli operate via the same TbPIP39–dependent pathway as CCA signalling. Specifically, changes in the phosphorylation status of TbPIP39 were visualized and quantitated by the use of antibodies detecting either TbPIP39 or the Y278 phosphorylated form of TbPIP39 generated during CCA-dependent differentiation. Both protease treatment and mild acid exposure generated a different pattern of TbPIP39 phosphorylation, thus suggesting a different mechanism of action than CCA. Finally, the possibility of using piggyback strategies targeting TbPTP1 was investigated, as a means to decrease the number of the fly-transmissible stumpy form cells in the bloodstream, thereby controlling parasite transmission. For this purpose, natural and synthetic inhibitors of human PTP1B were tested against the parasite enzyme, since they are being developed by pharmaceutical companies for the treatment of diabetes and obesity. The compounds tested showed a moderate in vitro inhibitory activity against recombinant TbPTP1 and mainly a non-competitive type of inhibition, similarly to that observed for human PTP1B. However, none of the compounds showed in vivo specificity for TbPTP1, indicating that further studies will be needed to identify more specific inhibitors.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity-9

(PP1β9C) in this assay. NIPP1 shows no discrimination between the isozymes, and is shown as a control. PP1 isoforms were expressed as DNA binding domain fusions (bait), while Uri and NIPP1 were Activation domain fusions (prey). B. Uri is an specific inhibitor of PP1. The myelin basic protein phosphatase activity was measure in the presence of different concentrations of recombinant PP1α87B (triangles) or PP1β9C (squares). Phosphatase activity is shown as % of control in the absence of Uri. C. Uri immunoprecipitates ectopically expressed HA-PP1α87B more efficiently than HA-PP1β9C from fly extracts. Western blot showing similar levels of expression of HA-PP1α87B and HA-PP1β9C in total fly lysate, and proportion immuno-precipitated with the anti-Uri antibody. Normal guinea pig serum and Protein G sepharose only controls show no precipitation of the expressed proteins. D. Human PP1α (first lane) but not PP1β (second lane) co-immunoprecipitates with URI/RMP, and not URI/RMP-D2 (lanes 3 and 4) when co-expressed in COS7 cells (first panel). Expression controls are shown in the second and third panels.<p><b>Copyright information:</b></p><p>Taken from "Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity"</p><p>http://www.biomedcentral.com/1471-2199/9/36</p><p>BMC Molecular Biology 2008;9():36-36.</p><p>Published online 15 Apr 2008</p><p>PMCID:PMC2346476.</p><p></p