scholarly journals Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei

2021 ◽  
Vol 15 (2) ◽  
pp. e0009132 ◽  
Author(s):  
Maria Lucia Sampaio Guther ◽  
Alan R. Prescott ◽  
Sabine Kuettel ◽  
Michele Tinti ◽  
Michael A. J. Ferguson
Keyword(s):  
Trypanosoma Brucei ◽  
Immunofluorescence Microscopy ◽  
Subcellular Fractionation ◽  
Bloodstream Form ◽  
Nucleotide Sugar ◽  
Procyclic Form ◽  
Nucleotide Sugars ◽  
Experimental Analyses ◽  
Nucleotide Sugar Biosynthesis

In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.

scholarly journals Regulation of Trypanosoma brucei Acetyl Coenzyme A Carboxylase by Environmental Lipids

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Sunayan S. Ray ◽  
Christina L. Wilkinson ◽  
Kimberly S. Paul
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Trypanosoma Brucei ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Bloodstream Form ◽  
Acetyl Coenzyme A ◽  
Content Type ◽  
Procyclic Form ◽  
High Lipid

ABSTRACT To satisfy its fatty acid needs, the extracellular eukaryotic parasite Trypanosoma brucei relies on two mechanisms: uptake of fatty acids from the host and de novo synthesis. We hypothesized that T. brucei modulates fatty acid synthesis in response to environmental lipid availability. The first committed step in fatty acid synthesis is catalyzed by acetyl coenzyme A (acetyl-CoA) carboxylase (ACC) and serves as a key regulatory point in other organisms. To test our hypothesis, T. brucei mammalian bloodstream and insect procyclic forms were grown in low-, normal-, or high-lipid media and the effect on T. brucei ACC (TbACC) mRNA, protein, and enzymatic activity was examined. In bloodstream form T. brucei, media lipids had no effect on TbACC expression or activity. In procyclic form T. brucei, we detected no change in TbACC mRNA levels but observed 2.7-fold-lower TbACC protein levels and 37% lower TbACC activity in high-lipid media than in low-lipid media. Supplementation of low-lipid media with the fatty acid stearate mimicked the effect of high lipid levels on TbACC activity. In procyclic forms, TbACC phosphorylation also increased 3.9-fold in high-lipid media compared to low-lipid media. Phosphatase treatment of TbACC increased activity, confirming that phosphorylation represented an inhibitory modification. Together, these results demonstrate a procyclic-form-specific environmental lipid response pathway that regulates TbACC posttranscriptionally, through changes in protein expression and phosphorylation. We propose that this environmental response pathway enables procyclic-form T. brucei to monitor the host lipid supply and downregulate fatty acid synthesis when host lipids are abundant and upregulate fatty acid synthesis when host lipids become scarce. IMPORTANCE Trypanosoma brucei is a eukaryotic parasite that causes African sleeping sickness. T. brucei is transmitted by the blood-sucking tsetse fly. In order to adapt to its two very different hosts, T. brucei must sense the host environment and alter its metabolism to maximize utilization of host resources and minimize expenditure of its own resources. One key nutrient class is represented by fatty acids, which the parasite can either take from the host or make themselves. Our work describes a novel environmental regulatory pathway for fatty acid synthesis where the parasite turns off fatty acid synthesis when environmental lipids are abundant and turns on synthesis when the lipid supply is scarce. This pathway was observed in the tsetse midgut form but not the mammalian bloodstream form. However, pharmacological activation of this pathway in the bloodstream form to turn fatty acid synthesis off may be a promising new avenue for sleeping sickness drug discovery.

scholarly journals Expression of normal levels of the Pumilio domain protein PUF3 is required for optimal growth of Trypanosoma brucei

2019 ◽  
Author(s):  
Kevin Kamanyi Marucha ◽  
Christine Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Specific Binding ◽  
Binding Protein ◽  
Ectopic Expression ◽  
Bloodstream Form ◽  
Fine Tuning ◽  
Growth Defect ◽  
Procyclic Form ◽  
Domain Protein

AbstractThe Trypanosoma brucei pumilio domain protein PUF3 is a cytosolic mRNA-binding protein that suppresses expression when tethered to a reporter mRNA. An induced reduction of PUF3 in bloodstream forms caused a slight growth defect and slightly delayed differentiation to the procyclic form, but the cells lost both defects upon prolonged cultivation. Both PUF3 genes could also be deleted in bloodstream-form and procyclic-form trypanosomes, suggesting that in vitro, at least, these life-cycle stages do not require PUF3. Procyclic forms without PUF3 grew somewhat slower than wild-type, but were able to transform to bloodstream forms after induced expression of the bloodstream-form RNA-binding protein RBP10. In contrast, ectopic expression of C-terminally tagged PUF3 in procyclic forms impaired viability. There was little evidence for specific binding of PUF3 to bloodstream-form mRNAs and RNAi had no significant effect on the transcriptome. Moreover, mass spectrometry revealed no PUF3 binding partners that might explain its suppressive activity. Since PUF3 is conserved in all Kinetoplastids, we suggest that it might be required within the invertebrate host, or perhaps implicated in fine-tuning gene expression.

scholarly journals Metabolic Engineering Optimizes Bioorthogonal Glycan Labeling in Living Cells

2021 ◽  
Author(s):  
Anna Cioce ◽  
Ganka Bineva-Todd ◽  
Anthony J. Agbay ◽  
Junwon Choi ◽  
Thomas M. Wood ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Protein Glycosylation ◽  
Side Reactions ◽  
Metabolic Fate ◽  
Nucleotide Sugar ◽  
Magnitude Comparison ◽  
Nucleotide Sugars ◽  
Salvage Pathways ◽  
Nucleotide Sugar Biosynthesis ◽  
Impact Probe

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac<sub>4</sub>GalNAlk and Ac<sub>4</sub>GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. Comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

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 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.

scholarly journals Changing Roles of Aurora-B Kinase in Two Life Cycle Stages of Trypanosoma brucei

2006 ◽  
Vol 5 (7) ◽  
pp. 1026-1035 ◽  
Author(s):  
Ziyin Li ◽  
C. C. Wang
Keyword(s):  
Dna Synthesis ◽  
Trypanosoma Brucei ◽  
Chromosome Segregation ◽  
Nuclear Dna ◽  
Developmental Stages ◽  
Bloodstream Form ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Spindle Formation ◽  
Procyclic Form

ABSTRACT Aurora-B kinase is a chromosomal passenger protein essential for chromosome segregation and cytokinesis. In the procyclic form of Trypanosoma brucei, depletion of an aurora-B kinase homologue TbAUK1 inhibited spindle formation, mitosis, cytokinesis, and organelle replication without altering cell morphology. In the present study, an RNA interference knockdown of TbAUK1 or overexpression of inactive mutant TbAUK1-K58R in the bloodstream form also resulted in defects in spindle formation, chromosome segregation, and cytokinesis but allowed multiple rounds of nuclear DNA synthesis, nucleolus multiplication, and continuous replication of kinetoplast, basal body, and flagellum. The typical trypanosome morphology was lost to an enlarged round shape filled with microtubules. It is thus apparent that there are distinctive mechanisms of action of TbAUK1 in regulating cell division between the two developmental stages of trypanosome. While it exerts a tight control on mitosis, organelle replication, and cytokinesis in the procyclic form, it regulates cytokinesis without rigid control over either nuclear DNA synthesis or organelle replication in the bloodstream form. The molecular basis underlining these discrepancies remains to be explored.

scholarly journals Distinct Roles of a Mitogen-Activated Protein Kinase in Cytokinesis between Different Life Cycle Forms of Trypanosoma brucei

2013 ◽  
Vol 13 (1) ◽  
pp. 110-118 ◽  
Author(s):  
Ying Wei ◽  
Ziyin Li
Keyword(s):  
Life Cycle ◽  
Protein Kinase ◽  
Map Kinase ◽  
Trypanosoma Brucei ◽  
Bloodstream Form ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Procyclic Form ◽  
Moderate Growth ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein kinase (MAPK) modules are evolutionarily conserved signaling cascades that function in response to the environment and play crucial roles in intracellular signal transduction in eukaryotes. The involvement of a MAP kinase in regulating cytokinesis in yeast, animals, and plants has been reported, but the requirement for a MAP kinase for cytokinesis in the early-branching protozoa is not documented. Here, we show that a MAP kinase homolog (TbMAPK6) from Trypanosoma brucei plays distinct roles in cytokinesis in two life cycle forms of T. brucei . TbMAPK6 is distributed throughout the cytosol in the procyclic form but is localized in both the cytosol and the nucleus in the bloodstream form. RNA interference (RNAi) of TbMAPK6 results in moderate growth inhibition in the procyclic form but severe growth defects and rapid cell death in the bloodstream form. Moreover, TbMAPK6 appears to be implicated in furrow ingression and cytokinesis completion in the procyclic form but is essential for cytokinesis initiation in the bloodstream form. Despite the distinct defects in cytokinesis in the two forms, RNAi of TbMAPK6 also caused defective basal body duplication/segregation in a small cell population in both life cycle forms. Altogether, our results demonstrate the involvement of the TbMAPK6-mediated pathway in regulating cytokinesis in trypanosomes and suggest distinct roles of TbMAPK6 in cytokinesis between different life cycle stages of T. brucei .

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