scholarly journals Phosphatidylinositol synthesis is essential in bloodstream form Trypanosoma brucei

2006 ◽  
Vol 396 (2) ◽  
pp. 287-295 ◽  
Author(s):  
Kirstee L. Martin ◽  
Terry K. Smith
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Essential Gene ◽  
Recombinant Expression ◽  
Protozoan Parasite ◽  
High Specificity ◽  
Bloodstream Form ◽  
Head Group ◽  
Double Knockout

PI (phosphatidylinositol) is a ubiquitous eukaryotic phospholipid which serves as a precursor for messenger molecules and GPI (glycosylphosphatidylinositol) anchors. PI is synthesized either de novo or by head group exchange by a PIS (PI synthase). The synthesis of GPI anchors has previously been validated both genetically and chemically as a drug target in Trypanosoma brucei, the causative parasite of African sleeping sickness. However, nothing is known about the synthesis of PI in this organism. Database mining revealed a putative TbPIS gene in the T. brucei genome and by recombinant expression and characterization it was shown to encode a catalytically active PIS, with a high specificity for myo-inositol. Immunofluorescence revealed that in T. brucei, PIS is found in both the endoplasmic reticulum and Golgi. We created a conditional double knockout of TbPIS in the bloodstream form of T. brucei, which when grown under non-permissive conditions, clearly showed that TbPIS is an essential gene. In vivo labelling of these conditional double knockout cells confirmed this result, showing a decrease in the amount of PI formed by the cells when grown under non-permissive conditions. Furthermore, quantitative and qualitative analysis by GLC-MS and ESI-MS/MS (electrospray ionization MS/MS) respectively showed a significant decrease (70%) in cellular PI, which appears to affect all major PI species equally. A consequence of this fall in PI level is a knock-on reduction in GPI biosynthesis which is essential for the parasite's survival. The results presented here show that PI synthesis is essential for bloodstream form T. brucei, and to our knowledge this is the first report of the dependence on PI synthesis of a protozoan parasite by genetic validation.

The myo-inositol-1-phosphate synthase gene is essential in Trypanosoma brucei

2005 ◽  
Vol 33 (5) ◽  
pp. 983-985 ◽  
Author(s):  
K.L. Martin ◽  
T.K. Smith
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Essential Gene ◽  
Recombinant Expression ◽  
Conditional Knockout ◽  
Cellular Functions ◽  
Genome Database ◽  
Inositol 1 ◽  
Catalytically Active ◽  
Phosphate Synthase

The de novo synthesis of myo-inositol occurs via a two-step process: first, glucose 6-phosphate is converted into inositol 1-phosphate by an INO1 (myo-inositol-1-phosphate synthase; EC 5.5.1.4); then, it is dephosphorylated by an inositol monophosphatase. The myo-inositol can then be incorporated into PI (phosphatidylinositol), which is utilized in a variety of cellular functions, including the biosynthesis of GPI (glycosylphosphatidylinositol) anchors. A putative INO1 was identified in the Trypanosoma brucei genome database and, by recombinant expression in Escherichia coli, was shown to be a catalytically active INO1. To investigate the importance of INO1, we created a conditional knockout, which, under non-permissive conditions, showed that INO1 is an essential gene in bloodstream form T. brucei and that the de novo synthesized myo-inositol is used for the formation of PI and GPI anchors.

scholarly journals Indirubin Analogues Inhibit Trypanosoma brucei Glycogen Synthase Kinase 3 Short and T. brucei Growth

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Antonia Efstathiou ◽  
Nicolas Gaboriaud-Kolar ◽  
Vassilios Myrianthopoulos ◽  
Konstantina Vougogiannopoulou ◽  
Ines Subota ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Glycogen Synthase ◽  
Intracellular Localization ◽  
Protozoan Parasite ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Content Type ◽  
Antitrypanosomal Activity ◽  
Half Maximal Effective Concentration

ABSTRACT The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). The disease is fatal if it remains untreated, whereas most drug treatments are inadequate due to high toxicity, difficulties in administration, and low central nervous system penetration. T. brucei glycogen synthase kinase 3 short (TbGSK3s) is essential for parasite survival and thus represents a potential drug target that could be exploited for HAT treatment. Indirubins, effective leishmanicidals, provide a versatile scaffold for the development of potent GSK3 inhibitors. Herein, we report on the screening of 69 indirubin analogues against T. brucei bloodstream forms. Of these, 32 compounds had potent antitrypanosomal activity (half-maximal effective concentration = 0.050 to 3.2 μM) and good selectivity for the analogues over human HepG2 cells (range, 7.4- to over 641-fold). The majority of analogues were potent inhibitors of TbGSK3s, and correlation studies for an indirubin subset, namely, the 6-bromosubstituted 3′-oxime bearing an extra bulky substituent on the 3′ oxime [(6-BIO-3′-bulky)-substituted indirubins], revealed a positive correlation between kinase inhibition and antitrypanosomal activity. Insights into this indirubin-TbGSK3s interaction were provided by structure-activity relationship studies. Comparison between 6-BIO-3′-bulky-substituted indirubin-treated parasites and parasites silenced for TbGSK3s by RNA interference suggested that the above-described compounds may target TbGSK3s in vivo. To further understand the molecular basis of the growth arrest brought about by the inhibition or ablation of TbGSK3s, we investigated the intracellular localization of TbGSK3s. TbGSK3s was present in cytoskeletal structures, including the flagellum and basal body area. Overall, these results give insights into the mode of action of 6-BIO-3′-bulky-substituted indirubins that are promising hits for antitrypanosomal drug discovery.

scholarly journals Characterization of the Novel Mitochondrial Genome Segregation Factor TAP110 in Trypanosoma brucei

2020 ◽  
Author(s):  
Simona Amodeo ◽  
Ana Kalichava ◽  
Albert Fradera-Sola ◽  
Eloïse Bertiaux-Lequoy ◽  
Paul Guichard ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Eukaryotic Cell ◽  
The Novel ◽  
Detergent Treatment ◽  
Summary Statement ◽  
First Time

AbstractProper mitochondrial genome inheritance is key for eukaryotic cell survival, however little is known about the molecular mechanism controlling this process. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome aka kinetoplast DNA (kDNA). kDNA segregation requires anchoring of the genome to the basal body via the tripartite attachment complex (TAC). Several components of the TAC as well as their assembly have been described, it however remains elusive how the TAC connects to the kDNA. Here, we characterize the TAC associated protein TAP110 and for the first time use ultrastructure expansion microscopy in trypanosomes to reveal that TAP110 is the currently most proximal kDNA segregation factor. The kDNA proximal positioning is also supported by RNAi depletion of TAC102, which leads to loss of TAP110 at the TAC. Overexpression of TAP110 leads to expression level changes of several mitochondrial proteins and a delay in the separation of the replicated kDNA networks. In contrast to other kDNA segregation factors TAP110 remains only partially attached to the flagellum after DNAse and detergent treatment and can only be solubilized in dyskinetoplastic cells, suggesting that interaction with the kDNA might be important for stability of the TAC association. Furthermore, we demonstrate that the TAC, but not the kDNA, is required for correct TAP110 localization in vivo and suggest that TAP110 might interact with other proteins to form a >669 kDa complex.Summary StatementTAP110 is a novel mitochondrial genome segregation factor in Trypanosoma brucei that associates with the previously described TAC component TAC102. Ultrastructure expansion microscopy reveals its proximal position to the kDNA.

scholarly journals Adenosine Kinase of Trypanosoma brucei and Its Role in Susceptibility to Adenosine Antimetabolites

2007 ◽  
Vol 51 (11) ◽  
pp. 3895-3901 ◽  
Author(s):  
Alexandra Lüscher ◽  
Pinar Önal ◽  
Anne-Marie Schweingruber ◽  
Pascal Mäser
Keyword(s):  
Trypanosoma Brucei ◽  
De Novo ◽  
Growth Conditions ◽  
Adenosine Kinase ◽  
Bloodstream Form ◽  
Null Mutation ◽  
Transporter Gene ◽  
Purine Salvage ◽  
Kinase Gene ◽  
Pharmacological Interest

ABSTRACT Trypanosoma brucei cannot synthesize purines de novo and relies on purine salvage from its hosts to build nucleic acids. With adenosine being a preferred purine source of bloodstream-form trypanosomes, adenosine kinase (AK; EC 2.7.1.20) is likely to be a key player in purine salvage. Adenosine kinase is also of high pharmacological interest, since for many adenosine antimetabolites, phosphorylation is a prerequisite for activity. Here, we cloned and functionally characterized adenosine kinase from T. brucei (TbAK). TbAK is a tandem gene, expressed in both procyclic- and bloodstream-form trypanosomes, whose product localized to the cytosol of the parasites. The RNA interference-mediated silencing of TbAK suggested that the gene is nonessential under standard growth conditions. Inhibition or downregulation of TbAK rendered the trypanosomes resistant to cordycepin (3′-deoxyadenosine), demonstrating a role for TbAK in the activation of adenosine antimetabolites. The expression of TbAK in Saccharomyces cerevisiae complemented a null mutation in the adenosine kinase gene ado1. The concomitant expression of TbAK with the T. brucei adenosine transporter gene TbAT1 allowed S. cerevisiae ado1 ade2 double mutants to grow on adenosine as the sole purine source and, at the same time, sensitized them to adenosine antimetabolites. The coexpression of TbAK and TbAT1 in S. cerevisiae ado1 ade2 double mutants proved to be a convenient tool for testing nucleoside analogues for uptake and activation by T. brucei adenosine salvage enzymes.

scholarly journals RNase III Domain of KREPB9 and KREPB10 Association with Editosomes in Trypanosoma brucei

mSphere ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Jason Carnes ◽  
Suzanne M. McDermott ◽  
Kenneth Stuart
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Tsetse Fly ◽  
Accessory Proteins ◽  
Rnase Iii ◽  
Parasite Life Cycle ◽  
Null Cell ◽  
Content Type

ABSTRACT Editosomes are the multiprotein complexes that catalyze the insertion and deletion of uridines to create translatable mRNAs in the mitochondria of kinetoplastids. Recognition and cleavage of a broad diversity of RNA substrates in vivo require three functionally distinct RNase III-type endonucleases, as well as five additional editosome proteins that contain noncatalytic RNase III domains. RNase III domains have recently been identified in the editosome accessory proteins KREPB9 and KREPB10, suggesting a role related to editing endonuclease function. In this report, we definitively show that KREPB9 and KREPB10 are not essential in either bloodstream-form parasites (BF) or procyclic-form parasites (PF) by creating null or conditional null cell lines. While preedited and edited transcripts are largely unaffected by the loss of KREPB9 in both PF and BF, loss of KREPB10 produces distinct responses in BF and PF. BF cells lacking KREPB10 also lack edited CYb, while PF cells have increased edited A6, RPS12, ND3, and COII after loss of KREPB10. We also demonstrate that mutation of the RNase III domain of either KREPB9 or KREPB10 results in decreased association with ~20S editosomes. Editosome interactions with KREPB9 and KREPB10 are therefore mediated by the noncatalytic RNase III domain, consistent with a role in endonuclease specialization in Trypanosoma brucei. IMPORTANCE Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. U insertion/deletion RNA editing in T. brucei generates mature mitochondrial mRNAs. Editing is essential for survival in mammalian hosts and tsetse fly vectors and is differentially regulated during the parasite life cycle. Three multiprotein “editosomes,” typified by exclusive RNase III endonucleases that act at distinct sites, catalyze editing. Here, we show that editosome accessory proteins KREPB9 and KREPB10 are not essential for mammalian blood- or insect-form parasite survival but have specific and differential effects on edited RNA abundance in different stages. We also characterize KREPB9 and KREPB10 noncatalytic RNase III domains and show they are essential for editosome association, potentially via dimerization with RNase III domains in other editosome proteins. This work enhances the understanding of distinct editosome and accessory protein functions, and thus differential editing, during the parasite life cycle and highlights the importance of RNase III domain interactions to editosome architecture.

scholarly journals Golgi duplication in Trypanosoma brucei

2004 ◽  
Vol 165 (3) ◽  
pp. 313-321 ◽  
Author(s):  
Cynthia Y. He ◽  
Helen H. Ho ◽  
Joerg Malsam ◽  
Cecile Chalouni ◽  
Christopher M. West ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Time Course ◽  
Matrix Protein ◽  
De Novo ◽  
Protozoan Parasite ◽  
Er Export

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.

scholarly journals A UDP-GlcNAc : βGal β1-6 GlcNAc transferase involved in bloodstream form N-glycan and procyclic form GPI anchor elaboration in Trypanosoma brucei.

2021 ◽  
Author(s):  
Samuel Martin Duncan ◽  
Rupa Nagar ◽  
Manuela Damerow ◽  
Dmitry V. Yashunsky ◽  
Benedetta Buzzi ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Bloodstream Form ◽  
Wild Type ◽  
Dual Roles ◽  
Gpi Anchor ◽  
Procyclic Form ◽  
Life Cycle Stages ◽  
Glcnac Transferase

Trypanosoma brucei has large carbohydrate extensions on its N-linked glycans and glycosylphosphatidylinositol (GPI) anchors in its bloodstream form (BSF) and procyclic form (PCF), respectively. The parasites glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are unknown. Here, we probe the function(s) of a putative β3GT gene, TbGT10. The BSF null mutant is viable in vitro and in vivo and can differentiate into PCF, demonstrating non-essentiality. However, the absence of TbGT10 led to impaired elaboration of N-glycans and GPI anchor sidechains in BSF and PCF parasites, respectively. Glycosylation defects include reduced BSF glycoprotein binding to ricin and to monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope of lysosomal glycoprotein p67 that we show here, using synthetic glycans, consists of (-6Gal1-4GlcNAc1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase glycopeptides isolated from BSF wild-type and TbGT10 null parasites show a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. Together, these data suggest that TbGT10 encodes a UDP-GlcNAc : βGal β1-6 GlcNAc-transferase active in both BSF and PCF life-cycle stages elaborating complex N-glycans and GPI sidechains, respectively. The β1-6 specificity of this β3GT gene product and its dual roles in N-glycan and GPI glycan elaboration are notable.

scholarly journals An essential GDP-Fuc: β-D-Gal α-1,2-fucosyltransferase is located in the mitochondrion of Trypanosoma brucei

2019 ◽  
Author(s):  
Giulia Bandini ◽  
Sebastian Damerow ◽  
Maria Lucia Sampaio Güther ◽  
Angela Mehlert ◽  
Hongjie Guo ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Trypanosoma Brucei ◽  
Mitochondrial Function ◽  
Therapeutic Potential ◽  
Protozoan Parasite ◽  
Bloodstream Form ◽  
Insect Vector ◽  
Reaction Products ◽  
Growth And Survival ◽  
Animal African Trypanosomiasis

ABSTRACTThe biosynthesis of guanosine 5′-diphospho-β-L-fucose (GDP-Fuc), the activated donor for fucose, has been shown to be essential in the parasite Trypanosoma brucei. Fucose is a common constituent of eukaryotic glycan structures, but it has been rarely found in trypanosomatid glycoconjugates. A single putative T. brucei fucosyltransferase (TbFUT1) gene was identified in the trypanosome genome. The encoded TbFUT1 protein was enzymatically active when expressed in Escherichia coli. Structural characterization of its reaction products identified it as a GDP-Fuc: β-D-galactose α-1,2-fucosyltransferase, with a preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif among the substrates tested. Conditional null mutants of the TbFUT1 gene demonstrated that it is essential for growth of the mammalian-infective bloodstream form and insect vector dwelling procyclic form of the parasite. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be essential for mitochondrial function in bloodstream form trypanosomes, suggesting this kinetoplastid parasite possesses an unprecedented and essential mitochondrial fucosyltransferase activity.SIGNIFICANCEThe sugar fucose is a well-known component of cell-surface glycoproteins and glycolipids and typically plays roles in cell-cell adhesion. Fucose is generally incorporated into glycoproteins and glycolipids by fucosyltransferase enzymes that reside in the Golgi apparatus. Here we show that the single fucosyltransferase of the protozoan parasite Trypanosoma brucei, causative agent of human and animal African trypanosomiasis, resides in the mitochondrion and not the Golgi apparatus. While the exact role of fucosylation in the parasite mitochondrion remains to be determined, it is essential for mitochondrial function and for parasite growth and survival. The unusual nature of this parasite enzyme, and its orthologues in related parasite pathogens, suggests that selective inhibitors may have therapeutic potential across a family of parasites.

scholarly journals Functional Analyses of Cytokinesis Regulators in Bloodstream Stage Trypanosoma brucei Parasites Identify Functions and Regulations Specific to the Life Cycle Stage

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Xuan Zhang ◽  
Tai An ◽  
Kieu T. M. Pham ◽  
Zhao-Rong Lun ◽  
Ziyin Li
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Bloodstream Form ◽  
Signaling Cascade ◽  
Insect Vector ◽  
Content Type ◽  
Procyclic Form ◽  
Life Cycle Stages ◽  
Evolutionarily Conserved

ABSTRACT The early divergent protozoan parasite Trypanosoma brucei alternates between the insect vector and the mammalian hosts during its life cycle and proliferates through binary cell fission. The cell cycle control system in T. brucei differs substantially from that in its mammalian hosts and possesses distinct mitosis-cytokinesis checkpoint controls between two life cycle stages, the procyclic form and the bloodstream form. T. brucei undergoes an unusual mode of cytokinesis, which is controlled by a novel signaling cascade consisting of evolutionarily conserved protein kinases and trypanosome-specific regulatory proteins in the procyclic form. However, given the distinct mitosis-cytokinesis checkpoints between the two forms, it is unclear whether the cytokinesis regulatory pathway discovered in the procyclic form also operates in a similar manner in the bloodstream form. Here, we showed that the three regulators of cytokinesis initiation, cytokinesis initiation factor 1 (CIF1), CIF2, and CIF3, are interdependent for subcellular localization but not for protein stability as in the procyclic form. Further, we demonstrated that KLIF, a regulator of cytokinesis completion in the procyclic form, plays limited roles in cytokinesis in the bloodstream form. Finally, we showed that the cleavage furrow-localizing protein FRW1 is required for cytokinesis initiation in the bloodstream form but is nonessential for cytokinesis in the procyclic form. Together, these results identify conserved and life cycle-specific functions of cytokinesis regulators, highlighting the distinction in the regulation of cytokinesis between different life cycle stages of T. brucei. IMPORTANCE The early divergent protozoan parasite Trypanosoma brucei is the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. This parasite has a complex life cycle by alternating between the insect vector and the mammalian hosts and proliferates by binary cell fission. The control of cell division in trypanosomes appears to be distinct from that in its human host and differs substantially between two life cycle stages, the procyclic (insect) form and the bloodstream form. Cytokinesis, the final step of binary cell fission, is regulated by a novel signaling cascade consisting of two evolutionarily conserved protein kinases and a cohort of trypanosome-specific regulators in the procyclic form, but whether this signaling pathway operates in a similar manner in the bloodstream form is unclear. In this report, we performed a functional analysis of multiple cytokinesis regulators and discovered their distinct functions and regulations in the bloodstream form.

102Diagnosis of coronary plaque rupture, plaque erosion, and calcified nodule by using near-infrared spectroscopy intravascular ultrasound

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
K Terada ◽  
T Kubo ◽  
Y Matsuo ◽  
Y Ino ◽  
H Kitabata ◽  
...  
Keyword(s):  
Infrared Spectroscopy ◽  
Intravascular Ultrasound ◽  
Near Infrared Spectroscopy ◽  
Near Infrared ◽  
De Novo ◽  
Plaque Rupture ◽  
High Specificity ◽  
Plaque Erosion ◽  
Plaque Ulceration

Abstract Objectives This study sought to investigate the ability of near-infrared spectroscopy intravascular ultrasound (NIRS-IVUS) to differentiate among plaque rupture (PR), plaque erosion (PE), and calcified nodule (CN) in acute myocardial infarction (AMI) using an optical coherence tomography (OCT) diagnosis as a reference standard. Background In vivo, precise differentiation among PR, PE and CN is a major challenge for intravascular imaging. Methods The study enrolled 156 AMI patients who had a de novo culprit lesion in a native coronary artery. The culprit lesions were assessed by both NIRS-IVUS and OCT. Results OCT identified 112 PR, 29 PE, and 15 CN. IVUS-detected plaque ulceration showed a high specificity (100%) to identify OCT-PR although the sensitivity (62%) was intermediate. IVUS-detected convex calcium showed a high sensitivity (93%) and specificity (100%) to identify OCT-CN. In NIRS, the maximum lipid core burden index in 4 mm (maxLCBI4mm) was greatest in OCT-PR (values are median [interquartile range]) (671 [530 to 853]), followed by OCT-CN (355 [303 to 432]) and OCT-PE (283 [89 to 357]) (p<0.001). MaxLCBI4mm of <422 was the best cut-off to discriminate OCT-PE from OCT-PR and OCT-CN. The NIRS-IVUS classification algorithm using plaque ulceration, convex calcium, and maxLCBI4mm <422 showed a sensitivity and specificity of 96% and 95% for identifying OCT-PR, 93% and 95% for OCT-PE, and 93% and 100% for OCT-CN, respectively. NIRS-IVUS classification algorism Conclusion Lipid component assessed by NIRS-IVUS was different among OCT-PR, OCT-PE and OCT-CN. The NIRS-IVUS classification algorism was highly sensitive and specific for differentiating these unstable lesion types in AMI. Acknowledgement/Funding None

Sign in / Sign up

Export Citation Format

Share Document