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Bloodstream Forms (bloodstream + form)
Selected AbstractsFunctional studies of an evolutionarily conserved, cytochrome b5 domain protein reveal a specific role in axonemal organisation and the general phenomenon of post-division axonemal growth in trypanosomesCYTOSKELETON, Issue 1 2009Helen Farr Abstract Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Several recent proteomic studies of cilia and flagella have been published, including a proteome of the flagellum of the protozoan parasite Trypanosoma brucei. Comparing proteomes reveals many novel proteins that appear to be widely conserved in evolution. Amongst these, we found a previously uncharacterised protein which localised to the axoneme in T. brucei, and therefore named it Trypanosome Axonemal protein (TAX)-2. Ablation of the protein using RNA interference in the procyclic form of the parasite has no effect on growth but causes a reduction in motility. Using transmission electron microscopy, various structural defects were seen in some axonemes, most frequently with microtubule doublets missing from the 9+2 arrangement. RNAi knockdown of TAX-2 expression in the bloodstream form of the parasite caused defects in growth and cytokinesis, a further example of the effects caused by loss of flagellar function in bloodstream form T. brucei. In procyclic cells we used a new set of vectors to ablate protein expression in cells expressing a GFP:TAX-2 fusion protein, which enabled us to easily quantify protein reduction and visualise axonemes made before and after RNAi induction. This establishes a useful generic technique but also revealed a specific observation that the new flagellum on the daughter trypanosome continues growth after cytokinesis. Our results provide evidence for TAX-2 function within the axoneme, where we suggest that it is involved in processes linking the outer doublet microtubules and the central pair. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] The essential neutral sphingomyelinase is involved in the trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma bruceiMOLECULAR MICROBIOLOGY, Issue 6 2010Simon A. Young Summary Sphingomyelin is the main sphingolipid in Trypanosoma brucei, the causative agent of African sleeping sickness. In vitro and in vivo characterization of the T. brucei neutral sphingomyelinase demonstrates that it is directly involved in sphingomyelin catabolism. Gene knockout studies in the bloodstream form of the parasite indicate that the neutral sphingomyelinase is essential for growth and survival, thus highlighting that the de novo biosynthesis of ceramide is unable to compensate for the loss of sphingomyelin catabolism. The phenotype of the conditional knockout has given new insights into the highly active endocytic and exocytic pathways in the bloodstream form of T. brucei. Hence, the formation of ceramide in the endoplasmic reticulum affects post-Golgi sorting and rate of deposition of newly synthesized GPI-anchored variant surface glycoprotein on the cell surface. This directly influences the corresponding rate of endocytosis, via the recycling endosomes, of pre-existing cell surface variant surface glycoprotein. The trypanosomes use this coupled endocytic and exocytic mechanism to maintain the cell density of its crucial variant surface glycoprotein protective coat. TbnSMase is therefore genetically validated as a drug target against African trypanosomes, and suggests that interfering with the endocytic transport of variant surface glycoprotein is a highly desirable strategy for drug development against African trypanosomasis. [source] Effect of the Disruption of RNA Pol II on the Transcription by RNA Pol l by Trypanosoma bruceiTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005S. DEVAUX Trypanosomes exist in two major distinct forms: the procyclic form in insects in which the surface molecule is procyclin and the bloodstream form in mammals in which the surface molecule is VSG. The promoter of these locus recruits RNA Pol I. Despite the use of this polymerase, the transcripts are spliced and polyadenylated exactly as the mRNAs synthesised by RNA Pol II. The fact that mRNA production from these sites must involve a concerted action between RNA Pol I and a "RNA factory" normally linked to RNA Pol II, led to the proposal that the control of these Pol I transcription units would involve a limiting component bridging RNA Pol I and an elongation/processing complex normally associated with RNA Pol II. To study the effects of the disruption of RNA Pol II on VSG expression level, we cloned the T. brucei homologue of the rpb9 subunit of RNA Pol II, which plays a role in RNA elongation in other eukaryotic cells. In PF, disruption of Tbrpb9 by conditional RNAi led to a strong transcriptional stimulation in the beginning of the ESs. This effect was linked to the inhibition of procyclin transcription. Thus, the disruption of RNA Pol II abolished the stage-specific regulation of the transcription units for the two major surface antigens, both of which recruit RNA Pol I. [source] Trypanosome Alternative Oxidase is Regulated Post-transcriptionally at the Level of RNA StabilityTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 4 2002MINU CHAUDHURI ABSTRACT In the bloodstream form of African trypanosomes, trypanosome alternative oxidase (TAO), the non-cytochrome ubiquinol:oxidoreductase, is the only terminal oxidase of the mitochondrial electron transport system. TAO is developmentally regulated during mitochondrial biogenesis in this parasite. During in vitro differentiation of Trypanosoma bmcei from the bloodstream to the procyclic form, the overall rate of oxygen consumption decreased about 80%. The mode of respiration changed over a 2- to 3-wk period from a cyanide-insensitive, SHAM-sensitive pathway to a predominantly cyanide-sensitive pathway. The TAO protein level gradually decreased to the level present in the procyclic forms during this 3-wk period. However, within the first week of differentiation, the TAO transcript level decreased about 90% and then in the following weeks it reached the level present in the established procyclic form, that is about 20% of that in bloodstream forms. Like other trypanosomatid genes TAO transcript synthesis remains unaltered in fully differentiated bloodstream and procyclic trypanosomes. The half-life of the TAO mRNA was about 3.2 h in the procyclic trypanosomes, whereas the TAO transcript level remained unaltered even after 4 h of incubation with actinomycin D in bloodstream forms. Inhibition of protein synthesis resulted in about a four-fold accumulation of the TAO transcript in the procyclic trypanosomes, comparable to the level present in the bloodstream forms. Thus, TAO is regulated at the level of mRNA stability and de novo protein synthesis is required for the reduction of the TAO mRNA pool in the procyclic form. [source] A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma bruceiMOLECULAR MICROBIOLOGY, Issue 1 2007Simon Urwyler Summary A ,two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3, flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins. [source] Trypanosome Alternative Oxidase is Regulated Post-transcriptionally at the Level of RNA StabilityTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 4 2002MINU CHAUDHURI ABSTRACT In the bloodstream form of African trypanosomes, trypanosome alternative oxidase (TAO), the non-cytochrome ubiquinol:oxidoreductase, is the only terminal oxidase of the mitochondrial electron transport system. TAO is developmentally regulated during mitochondrial biogenesis in this parasite. During in vitro differentiation of Trypanosoma bmcei from the bloodstream to the procyclic form, the overall rate of oxygen consumption decreased about 80%. The mode of respiration changed over a 2- to 3-wk period from a cyanide-insensitive, SHAM-sensitive pathway to a predominantly cyanide-sensitive pathway. The TAO protein level gradually decreased to the level present in the procyclic forms during this 3-wk period. However, within the first week of differentiation, the TAO transcript level decreased about 90% and then in the following weeks it reached the level present in the established procyclic form, that is about 20% of that in bloodstream forms. Like other trypanosomatid genes TAO transcript synthesis remains unaltered in fully differentiated bloodstream and procyclic trypanosomes. The half-life of the TAO mRNA was about 3.2 h in the procyclic trypanosomes, whereas the TAO transcript level remained unaltered even after 4 h of incubation with actinomycin D in bloodstream forms. Inhibition of protein synthesis resulted in about a four-fold accumulation of the TAO transcript in the procyclic trypanosomes, comparable to the level present in the bloodstream forms. Thus, TAO is regulated at the level of mRNA stability and de novo protein synthesis is required for the reduction of the TAO mRNA pool in the procyclic form. [source] Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Emmanuel Oluwadare Balogun In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His6 tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75,Å resolution indicated that the crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 63.84, b = 121.50, c = 154.59,Å. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (VM) of 2.5,Å3,Da,1, corresponding to 50% solvent content. [source] Azaanthraquinone inhibits respiration and in vitro growth of long slender bloodstream forms of Trypanosoma congolenseCELL BIOCHEMISTRY AND FUNCTION, Issue 3 2002Andrew Jonathan Nok Abstract An ethanolic extract of Mitracarpus scaber was found to possess in vitro and in vivo trypanocidal activity against Trypanosoma congolense. At a dosage of 50,mg kg,1 day,1 in normal saline for 5 days, the extract cured Balbc mice infected with T. congolense without any relapse. The isolated active component benz(g)isoquinoline 5,10 dione (Azaanthraquinone) (AQ) purified from the extract was found to inhibit glucose-dependent cellular respiration and glycerol-3-phosphate-dependent mitochondrial O2 assimilation of the long bloodstream forms of Trypanosoma congolense. On account of the pattern of inhibition, the target could be the mitochondrial electron transport system composed of glyceraldehyde 3-phosphate dehydrogenase (G3PDH). The azaanthraquinone specifically inhibited the reduced coenzyme Q1 -dependent O2 uptake of the mitochondria with respect to ubiquinone. The susceptible site could be due to ubiquinone redox system which links the two enzyme activities. Copyright © 2002 John Wiley & Sons, Ltd. [source] | ||||||||||