Positional Dynamics and Glycosomal Recruitment of Developmental Regulators during Trypanosome Differentiation

ABSTRACT Glycosomes are peroxisome-related organelles that compartmentalize the glycolytic enzymes in kinetoplastid parasites. These organelles are developmentally regulated in their number and composition, allowing metabolic adaptation to the parasite’s needs in the blood of mammalian hosts or within their arthropod vector. A protein phosphatase cascade regulates differentiation between parasite developmental forms, comprising a tyrosine phosphatase, Trypanosoma brucei PTP1 (TbPTP1), which dephosphorylates and inhibits a serine threonine phosphatase, TbPIP39, which promotes differentiation. When TbPTP1 is inactivated, TbPIP39 is activated and during differentiation becomes located in glycosomes. Here we have tracked TbPIP39 recruitment to glycosomes during differentiation from bloodstream “stumpy” forms to procyclic forms. Detailed microscopy and live-cell imaging during the synchronous transition between life cycle stages revealed that in stumpy forms, TbPIP39 is located at a periflagellar pocket site closely associated with TbVAP, which defines the flagellar pocket endoplasmic reticulum. TbPTP1 is also located at the same site in stumpy forms, as is REG9.1, a regulator of stumpy-enriched mRNAs. This site provides a molecular node for the interaction between TbPTP1 and TbPIP39. Within 30 min of the initiation of differentiation, TbPIP39 relocates to glycosomes, whereas TbPTP1 disperses to the cytosol. Overall, the study identifies a “stumpy regulatory nexus” (STuRN) that coordinates the molecular components of life cycle signaling and glycosomal development during transmission of Trypanosoma brucei. IMPORTANCE African trypanosomes are parasites of sub-Saharan Africa responsible for both human and animal disease. The parasites are transmitted by tsetse flies, and completion of their life cycle involves progression through several development steps. The initiation of differentiation between blood and tsetse fly forms is signaled by a phosphatase cascade, ultimately trafficked into peroxisome-related organelles called glycosomes that are unique to this group of organisms. Glycosomes undergo substantial remodeling of their composition and function during the differentiation step, but how this is regulated is not understood. Here we identify a cytological site where the signaling molecules controlling differentiation converge before the dispersal of one of them into glycosomes. In combination, the study provides the first insight into the spatial coordination of signaling pathway components in trypanosomes as they undergo cell-type differentiation.

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Positional dynamics and glycosomal recruitment of developmental regulators during trypanosome differentiation

10.1101/603258 ◽

2019 ◽

Author(s):

Balázs Szöőr ◽

Dorina V. Simon ◽

Federico Rojas ◽

Julie Young ◽

Derrick R. Robinson ◽

...

Keyword(s):

Life Cycle ◽

Tyrosine Phosphatase ◽

Sub Saharan Africa ◽

Metabolic Adaptation ◽

Tsetse Flies ◽

African Trypanosomes ◽

Signalling Molecules ◽

Life Cycle Stages ◽

Sub Saharan ◽

And Function

AbstractGlycosomes are peroxisome-related organelles that compartmentalise the glycolytic enzymes in kinetoplastid parasites. These organelles are developmentally regulated in their number and composition, allowing metabolic adaptation to the parasite’s needs in the blood of mammalian hosts or within their arthropod vector. A protein phosphatase cascade regulates differentiation between parasite developmental forms, comprising a tyrosine phosphatase, TbPTP1, that dephosphorylates and inhibits a serine threonine phosphatase TbPIP39 that promotes differentiation. When TbPTP1 is inactivated, TbPIP39 is activated and during differentiation becomes located in glycosomes. Here we have tracked TbPIP39 recruitment to glycosomes during differentiation from bloodstream stumpy forms to procyclic forms. Detailed microscopy and live cell imaging during the synchronous transition between life cycle stages revealed that in stumpy forms, TbPIP39 is located at a periflagellar pocket site closely associated with TbVAP, that defines the flagellar pocket endoplasmic reticulum. TbPTP1 is also located at the same site in stumpy forms, as is REG9.1, a regulator of stumpy-enriched mRNAs. This site provides a molecular node for the interaction between TbPTP1 and TbPIP39. Within 30 minutes of the initiation of differentiation TbPIP39 relocates to glycosomes whereas TbPTP1 disperses to the cytosol. Overall, the study identifies a ‘stumpy regulatory nexus’ (STuRN) that co-ordinates the molecular components of life cycle signalling and glycosomal development during transmission ofTrypanosoma brucei.ImportanceAfrican trypanosomes are parasites of sub-Saharan Africa responsible for both human and animal disease. The parasites are transmitted by tsetse flies and completion of their life cycle involves progression through several development steps. The initiation of differentiation between blood and tsetse forms is signalled by a phosphatase cascade, ultimately trafficked into peroxisome-related organelles called glycosomes that are unique to this group of organisms. Glycosomes undergo substantial remodelling of their composition and function during the differentiation step but how this is regulated is not understood. Here we identify a cytological site where the signalling molecules controlling differentiation converge before the dispersal of one of them into glycosomes. This coincides with a specialised ER site that may contribute to glycosome developmental biogenesis or regeneration. In combination, the study provides the first insight into the spatial co-ordination of signalling pathway components in trypanosomes as they undergo cell-type differentiation.

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Protein tyrosine phosphatase TbPTP1: a molecular switch controlling life cycle differentiation in trypanosomes

The Journal of Cell Biology ◽

10.1083/jcb.200605090 ◽

2006 ◽

Vol 175 (2) ◽

pp. 293-303 ◽

Cited By ~ 71

Author(s):

Balázs Szöőr ◽

Jude Wilson ◽

Helen McElhinney ◽

Lydia Tabernero ◽

Keith R. Matthews

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Tsetse Fly ◽

Mammalian Host ◽

African Trypanosomes ◽

Protein Tyrosine ◽

Ptp1b Inhibitors ◽

Stumpy Form

Differentiation in African trypanosomes (Trypanosoma brucei) entails passage between a mammalian host, where parasites exist as a proliferative slender form or a G0-arrested stumpy form, and the tsetse fly. Stumpy forms arise at the peak of each parasitaemia and are committed to differentiation to procyclic forms that inhabit the tsetse midgut. We have identified a protein tyrosine phosphatase (TbPTP1) that inhibits trypanosome differentiation. Consistent with a tyrosine phosphatase, recombinant TbPTP1 exhibits the anticipated substrate and inhibitor profile, and its activity is impaired by reversible oxidation. TbPTP1 inactivation in monomorphic bloodstream trypanosomes by RNA interference or pharmacological inhibition triggers spontaneous differentiation to procyclic forms in a subset of committed cells. Consistent with this observation, homogeneous populations of stumpy forms synchronously differentiate to procyclic forms when tyrosine phosphatase activity is inhibited. Our data invoke a new model for trypanosome development in which differentiation to procyclic forms is prevented in the bloodstream by tyrosine dephosphorylation. It may be possible to use PTP1B inhibitors to block trypanosomatid transmission.

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Colonization of the tsetse fly midgut with commensal Enterobacter inhibits trypanosome infection establishment

10.1101/472373 ◽

2018 ◽

Author(s):

Brian L. Weiss ◽

Michele A. Maltz ◽

Aurélien Vigneron ◽

Yineng Wu ◽

Katharine Walter ◽

...

Keyword(s):

Control Strategies ◽

Vector Competence ◽

Tsetse Fly ◽

Sub Saharan Africa ◽

Tsetse Flies ◽

Wild Type ◽

African Trypanosomes ◽

Control Programs ◽

Causative Agents ◽

Sub Saharan

AbstractTsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether an Enterobacter bacterium (Esp_Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Esp_Z established stable infections in tsetse’s gut, and exhibited no adverse effect on the survival of individuals from either group. Flies with established Esp_Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Esp_Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Esp_Z acidifies tsetse’s midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Esp_Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Esp_Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes.Author SummaryTsetse flies transmit pathogenic African trypanosomes, which are the causative agents of socio-economically devastating human and animal African trypanosomiases. These diseases are currently controlled in large part by reducing the population size of tsetse vectors through the use of insecticides, traps and sterile insect technique. However, logistic and monetary hurdles often preclude the prolonged application of procedures necessary to maintain these control programs. Thus, novel strategies, including those aimed at sustainably reducing the ability of tsetse to transmit trypanosomes, are presently under development. Herein we stably colonize tsetse flies with a bacterium (Enterobacter sp. Z, Esp_Z) that acidifies their midgut, thus rendering the environment inhospitable to infection with two distinct, epidemiologically important trypanosome strains as well as an entomopathogenic bacteria. In addition to inducing a trypanosome refractory phenotype, colonization of tsetse with Esp_Z exerts only a modest fitness cost on the fly. Taken together, these findings suggest that Esp_Z could be applied to enhance the effectiveness of currently employed tsetse control programs.

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Antioxidants promote establishment of trypanosome infections in tsetse

Parasitology ◽

10.1017/s0031182007002247 ◽

2007 ◽

Vol 134 (6) ◽

pp. 827-831 ◽

Cited By ~ 72

Author(s):

E. T. MacLEOD ◽

I. MAUDLIN ◽

A. C. DARBY ◽

S. C. WELBURN

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Trypanosoma Brucei ◽

Sub Saharan Africa ◽

Tsetse Flies ◽

Oxygen Species ◽

Infection Rates ◽

Trypanosoma Brucei Brucei ◽

Sub Saharan ◽

Cyclical Transmission

SUMMARYEfficient, cyclical transmission of trypanosomes through tsetse flies is central to maintenance of human sleeping sickness and nagana across sub-Saharan Africa. Infection rates in tsetse are normally very low as most parasites ingested with the fly bloodmeal die in the fly gut, displaying the characteristics of apoptotic cells. Here we show that a range of antioxidants (glutathione, cysteine, N-acetyl-cysteine, ascorbic acid and uric acid), when added to the insect bloodmeal, can dramatically inhibit cell death of Trypanosoma brucei brucei in tsetse. Both L- and D-cysteine invoked similar effects suggesting that inhibition of trypanosome death is not dependent on protein synthesis. The present work suggests that antioxidants reduce the midgut environment protecting trypanosomes from cell death induced by reactive oxygen species.

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Proteomics and theTrypanosoma bruceicytoskeleton: advances and opportunities

Parasitology ◽

10.1017/s0031182012000443 ◽

2012 ◽

Vol 139 (9) ◽

pp. 1168-1177 ◽

Cited By ~ 10

Author(s):

NEIL PORTMAN ◽

KEITH GULL

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

Cell Biology ◽

Molecular Composition ◽

Etiological Agent ◽

Sub Saharan Africa ◽

Parasitic Disease ◽

Cell Cycles ◽

Sub Saharan ◽

High Level

SUMMARYTrypanosoma bruceiis the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.

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Intraflagellar transport during the assembly of flagella of different length in Trypanosoma brucei isolated from tsetse flies

10.1101/2020.05.14.095216 ◽

2020 ◽

Author(s):

Eloïse Bertiaux ◽

Adeline Mallet ◽

Brice Rotureau ◽

Philippe Bastin

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

Live Cell Imaging ◽

Intraflagellar Transport ◽

Tsetse Fly ◽

Tsetse Flies ◽

Life Cycle Stages ◽

Summary Statement ◽

Cilia And Flagella ◽

Multicellular Organisms

AbstractMulticellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. This is the case of Trypanosoma brucei that assembles flagella of 3 to 30 µm during its development in the tsetse fly. It provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3-D electron microscopy and live cell imaging revealed that IFT was present in all life cycle stages. IFT proteins are concentrated at the base, IFT trains are located along doublets 3-4 & 7-8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed.Summary statementThis work investigated the assembly of flagella of different length during the development of Trypanosoma brucei in the tsetse fly, revealing a direct correlation between the amount of intraflagellar transport proteins and flagellum length.

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Unexpected plasticity in the life cycle of Trypanosoma brucei

eLife ◽

10.7554/elife.66028 ◽

2021 ◽

Vol 10 ◽

Author(s):

Sarah Schuster ◽

Jaime Lisack ◽

Ines Subota ◽

Henriette Zimmermann ◽

Christian Reuter ◽

...

Keyword(s):

Life Cycle ◽

Trypanosoma Brucei ◽

Parasite Load ◽

Tsetse Fly ◽

Productive Infection ◽

Complex Life Cycle ◽

Chronic Infections ◽

Population Densities ◽

African Trypanosomes ◽

Stage Transition

African trypanosomes cause sleeping sickness in humans and nagana in cattle. These unicellular parasites are transmitted by the bloodsucking tsetse fly. In the mammalian host's circulation, proliferating slender stage cells differentiate into cell cycle-arrested stumpy stage cells when they reach high population densities. This stage transition is thought to fulfil two main functions: first, it auto-regulates the parasite load in the host; second, the stumpy stage is regarded as the only stage capable of successful vector transmission. Here, we show that proliferating slender stage trypanosomes express the mRNA and protein of a known stumpy stage marker, complete the complex life cycle in the fly as successfully as the stumpy stage, and require only a single parasite for productive infection. These findings suggest a reassessment of the traditional view of the trypanosome life cycle. They may also provide a solution to a long-lasting paradox, namely the successful transmission of parasites in chronic infections, despite low parasitemia.

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Viviparity and habitat restrictions may influence the evolution of male reproductive genes in tsetse fly (Glossina) species

BMC Biology ◽

10.1186/s12915-021-01148-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Grazia Savini ◽

Francesca Scolari ◽

Lino Ometto ◽

Omar Rota-Stabelli ◽

Davide Carraretto ◽

...

Keyword(s):

Reproductive Behavior ◽

Selective Pressure ◽

Demographic History ◽

Reproductive Rate ◽

Tsetse Fly ◽

Sub Saharan Africa ◽

Comparative Genomic ◽

Tsetse Flies ◽

African Trypanosomes ◽

Reproductive Genes

Abstract Background Glossina species (tsetse flies), the sole vectors of African trypanosomes, maintained along their long evolutionary history a unique reproductive strategy, adenotrophic viviparity. Viviparity reduces their reproductive rate and, as such, imposes strong selective pressures on males for reproductive success. These species live in sub-Saharan Africa, where the distributions of the main sub-genera Fusca, Morsitans, and Palpalis are restricted to forest, savannah, and riverine habitats, respectively. Here we aim at identifying the evolutionary patterns of the male reproductive genes of six species belonging to these three main sub-genera. We then interpreted the different patterns we found across the species in the light of viviparity and the specific habitat restrictions, which are known to shape reproductive behavior. Results We used a comparative genomic approach to build consensus evolutionary trees that portray the selective pressure acting on the male reproductive genes in these lineages. Such trees reflect the long and divergent demographic history that led to an allopatric distribution of the Fusca, Morsitans, and Palpalis species groups. A dataset of over 1700 male reproductive genes remained conserved over the long evolutionary time scale (estimated at 26.7 million years) across the genomes of the six species. We suggest that this conservation may result from strong functional selective pressure on the male imposed by viviparity. It is noteworthy that more than half of these conserved genes are novel sequences that are unique to the Glossina genus and are candidates for selection in the different lineages. Conclusions Tsetse flies represent a model to interpret the evolution and differentiation of male reproductive biology under different, but complementary, perspectives. In the light of viviparity, we must take into account that these genes are constrained by a post-fertilization arena for genomic conflicts created by viviparity and absent in ovipositing species. This constraint implies a continuous antagonistic co-evolution between the parental genomes, thus accelerating inter-population post-zygotic isolation and, ultimately, favoring speciation. Ecological restrictions that affect reproductive behavior may further shape such antagonistic co-evolution.

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Mutualist-Provisioned Resources Impact Vector Competency

mBio ◽

10.1128/mbio.00018-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 2

Author(s):

Rita V. M. Rio ◽

Anna K. S. Jozwick ◽

Amy F. Savage ◽

Afsoon Sabet ◽

Aurelien Vigneron ◽

...

Keyword(s):

Life Cycle ◽

Disease Transmission ◽

Tsetse Fly ◽

Parasite Development ◽

Vitamin B ◽

Insect Vector ◽

Trypanosome Infection ◽

Content Type ◽

African Trypanosomes ◽

Vector Competency

ABSTRACT Many symbionts supplement their host’s diet with essential nutrients. However, whether these nutrients also enhance parasitism is unknown. In this study, we investigated whether folate (vitamin B9) production by the tsetse fly (Glossina spp.) essential mutualist, Wigglesworthia, aids auxotrophic African trypanosomes in completing their life cycle within this obligate vector. We show that the expression of Wigglesworthia folate biosynthesis genes changes with the progression of trypanosome infection within tsetse. The disruption of Wigglesworthia folate production caused a reduction in the percentage of flies that housed midgut (MG) trypanosome infections. However, decreased folate did not prevent MG trypanosomes from migrating to and establishing an infection in the fly’s salivary glands, thus suggesting that nutrient requirements vary throughout the trypanosome life cycle. We further substantiated that trypanosomes rely on symbiont-generated folate by feeding this vitamin to Glossina brevipalpis, which exhibits low trypanosome vector competency and houses Wigglesworthia incapable of producing folate. Folate-supplemented G. brevipalpis flies were significantly more susceptible to trypanosome infection, further demonstrating that this vitamin facilitates parasite infection establishment. Our cumulative results provide evidence that Wigglesworthia provides a key metabolite (folate) that is “hijacked” by trypanosomes to enhance their infectivity, thus indirectly impacting tsetse species vector competency. Parasite dependence on symbiont-derived micronutrients, which likely also occurs in other arthropod vectors, represents a relationship that may be exploited to reduce disease transmission. IMPORTANCE Parasites elicit several physiological changes in their host to enhance transmission. Little is known about the functional association between parasitism and microbiota-provisioned resources typically dedicated to animal hosts and how these goods may be rerouted to optimize parasite development. This study is the first to identify a specific symbiont-generated metabolite that impacts insect vector competence by facilitating parasite establishment and, thus, eventual transmission. Specifically, we demonstrate that the tsetse fly obligate mutualist Wigglesworthia provisions folate (vitamin B9) that pathogenic African trypanosomes exploit in an effort to successfully establish an infection in the vector’s MG. This process is essential for the parasite to complete its life cycle and be transmitted to a new vertebrate host. Disrupting metabolic contributions provided by the microbiota of arthropod disease vectors may fuel future innovative control strategies while also offering minimal nontarget effects.

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

mSphere ◽

10.1128/mspheredirect.00585-17 ◽

2018 ◽

Vol 3 (1) ◽

Cited By ~ 3

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.

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