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 Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei

2015 ◽  
Vol 35 (23) ◽  
pp. 3945-3961 ◽  
Author(s):  
Suzanne M. McDermott ◽  
Jason Carnes ◽  
Kenneth Stuart
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Bloodstream Form ◽  
Single Amino Acid ◽  
Wild Type ◽  
Rnase Iii ◽  
Content Type ◽  
Life Cycle Stages

KREPB5 is an essential component of ∼20S editosomes inTrypanosoma bruceiwhich contains a degenerate, noncatalytic RNase III domain. To explore the function of this protein, we used a novel approach to make and screen numerous conditional nullT. bruceibloodstream form cell lines that express randomly mutagenized KREPB5 alleles. We identified nine single amino acid substitutions that could not complement the conditional loss of wild-type KREPB5. Seven of these were within the RNase III domain, and two were in the C-terminal region that has no homology to known motifs. Exclusive expression of these mutated KREPB5 alleles in the absence of wild-type allele expression resulted in growth inhibition, the loss of ∼20S editosomes, and inhibition of RNA editing in BF cells. Eight of these mutations were lethal in bloodstream form parasites but not in procyclic-form parasites, showing that multiple domains function in a life cycle-dependent manner. Amino acid changes at a substantial number of positions, including up to 7 per allele, allowed complementation and thus did not block KREPB5 function. Hence, the degenerate RNase III domain and a newly identified domain are critical for KREPB5 function and have differential effects between the life cycle stages ofT. bruceithat differentially edit mRNAs.

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 .

scholarly journals Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 683 ◽  
Author(s):  
Terry K. Smith ◽  
Frédéric Bringaud ◽  
Derek P. Nolan ◽  
Luisa M. Figueiredo
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Tsetse Fly ◽  
Metabolic Reprogramming ◽  
Mammalian Host ◽  
Insect Vector ◽  
Environmental Signals ◽  
Cellular Metabolic Activity

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

scholarly journals Glucose Signaling Is Important for Nutrient Adaptation during Differentiation of Pleomorphic African Trypanosomes

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Yijian Qiu ◽  
Jillian E. Milanes ◽  
Jessica A. Jones ◽  
Rooksana E. Noorai ◽  
Vijay Shankar ◽  
...  
Keyword(s):  
Life Cycle ◽  
Glucose Sensing ◽  
Negative Regulator ◽  
Bloodstream Form ◽  
Tsetse Flies ◽  
Insect Vector ◽  
Rapid Reduction ◽  
African Trypanosomes ◽  
Procyclic Form ◽  
African Trypanosome

ABSTRACT The African trypanosome has evolved mechanisms to adapt to changes in nutrient availability that occur during its life cycle. During transition from mammalian blood to insect vector gut, parasites experience a rapid reduction in environmental glucose. Here we describe how pleomorphic parasites respond to glucose depletion with a focus on parasite changes in energy metabolism and growth. Long slender bloodstream form parasites were rapidly killed as glucose concentrations fell, while short stumpy bloodstream form parasites persisted to differentiate into the insect-stage procyclic form parasite. The rate of differentiation was lower than that triggered by other cues but reached physiological rates when combined with cold shock. Both differentiation and growth of resulting procyclic form parasites were inhibited by glucose and nonmetabolizable glucose analogs, and these parasites were found to have upregulated amino acid metabolic pathway component gene expression. In summary, glucose transitions from the primary metabolite of the blood-stage infection to a negative regulator of cell development and growth in the insect vector, suggesting that the hexose is not only a key metabolic agent but also an important signaling molecule. IMPORTANCE As the African trypanosome Trypanosoma brucei completes its life cycle, it encounters many different environments. Adaptation to these environments includes modulation of metabolic pathways to parallel the availability of nutrients. Here, we describe how the blood-dwelling life cycle stages of the African trypanosome, which consume glucose to meet their nutritional needs, respond differently to culture in the near absence of glucose. The proliferative long slender parasites rapidly die, while the nondividing short stumpy parasite remains viable and undergoes differentiation to the next life cycle stage, the procyclic form parasite. Interestingly, a sugar analog that cannot be used as an energy source inhibited the process. Furthermore, the growth of procyclic form parasite that resulted from the event was inhibited by glucose, a behavior that is similar to that of parasites isolated from tsetse flies. Our findings suggest that glucose sensing serves as an important modulator of nutrient adaptation in the parasite.

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 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 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 Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

2011 ◽  
Vol 10 (6) ◽  
pp. 734-743 ◽  
Author(s):  
David F. Bruhn ◽  
Mark P. Sammartino ◽  
Michele M. Klingbeil
Keyword(s):  
Mitochondrial Dna ◽  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Dna Polymerases ◽  
Bloodstream Form ◽  
Complex Life Cycle ◽  
Replication Proteins ◽  
Kinetoplast Dna ◽  
Content Type ◽  
Parasite Viability

ABSTRACT Trypanosoma brucei , the causative agent of human African trypanosomiasis, has a complex life cycle that includes multiple life cycle stages and metabolic changes as the parasite switches between insect vector and mammalian host. The parasite's single mitochondrion contains a unique catenated mitochondrial DNA network called kinetoplast DNA (kDNA) that is composed of minicircles and maxicircles. Long-standing uncertainty about the requirement of kDNA in bloodstream form (BF) T. brucei has recently eroded, with reports of posttranscriptional editing and subsequent translation of kDNA-encoded transcripts as essential processes for BF parasites. These studies suggest that kDNA and its faithful replication are indispensable for this life cycle stage. Here we demonstrate that three kDNA replication proteins (mitochondrial DNA polymerases IB, IC, and ID) are required for BF parasite viability. Silencing of each polymerase was lethal, resulting in kDNA loss, persistence of prereplication DNA monomers, and collapse of the mitochondrial membrane potential. These data demonstrate that kDNA replication is indeed crucial for BF T. brucei . The contributions of mitochondrial DNA polymerases IB, IC, and ID to BF parasite viability suggest that these and other kDNA replication proteins warrant further investigation as a new class of targets for the development of antitrypanosomal drugs.

scholarly journals Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 683 ◽  
Author(s):  
Terry K. Smith ◽  
Frédéric Bringaud ◽  
Derek P. Nolan ◽  
Luisa M. Figueiredo
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Tsetse Fly ◽  
Metabolic Reprogramming ◽  
Mammalian Host ◽  
Insect Vector ◽  
Environmental Signals ◽  
Cellular Metabolic Activity

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

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