APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins in Trypanosoma brucei

ABSTRACTTrypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into subdomains, each having specialized composition and function. Overall flagellum proteome has been well-studied, but a critical gap in knowledge is the protein composition of individual flagellum subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification of biotinylated proteins yields a DRC1 “proximity proteome” showing good overlap with proteomes obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily purified.IMPORTANCESleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (aka cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and has allowed identification of proximity proteomes for different subdomains. This capacity opens the possibility to study the composition and function of other compartments. We further expect that this approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilia function is impaired.

Download Full-text

APEX2 Proximity Proteomics Resolves Flagellum Subdomains and Identifies Flagellum Tip-Specific Proteins in Trypanosoma brucei

mSphere ◽

10.1128/msphere.01090-20 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Daniel E. Vélez-Ramírez ◽

Michelle M. Shimogawa ◽

Sunayan S. Ray ◽

Andrew Lopez ◽

Shima Rayatpisheh ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Model Organism ◽

Protein Composition ◽

Principal Component ◽

Protozoan Parasite ◽

Sleeping Sickness ◽

Content Type ◽

Critical Knowledge ◽

Regulatory Complex ◽

And Function

ABSTRACT Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum (cilium) that plays critical roles in transmission and pathogenesis. An emerging concept is that the flagellum is organized into subdomains, each having specialized composition and function. The overall flagellum proteome has been well studied, but a critical knowledge gap is the protein composition of individual subdomains. We have tested whether APEX-based proximity proteomics could be used to examine the protein composition of T. brucei flagellum subdomains. As APEX-based labeling has not previously been described in T. brucei, we first fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, a well-characterized axonemal complex. We found that DRC1-APEX2 directs flagellum-specific biotinylation, and purification of biotinylated proteins yields a DRC1 “proximity proteome” having good overlap with published proteomes obtained from purified axonemes. Having validated the use of APEX2 in T. brucei, we next attempted to distinguish flagellar subdomains by fusing APEX2 to a flagellar membrane protein that is restricted to the flagellum tip, AC1, and another one that is excluded from the tip, FS179. Fluorescence microscopy demonstrated subdomain-specific biotinylation, and principal-component analysis showed distinct profiles between AC1-APEX2 and FS179-APEX2. Comparing these two profiles allowed us to identify an AC1 proximity proteome that is enriched for tip proteins, including proteins involved in signaling. Our results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve the proteome composition of flagellum subdomains that cannot themselves be readily purified. IMPORTANCE Sleeping sickness is a neglected tropical disease caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need for approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei and has allowed identification of proximity proteomes for different flagellar subdomains that cannot be purified. This capacity opens the possibility to study the composition and function of other compartments. We expect this approach may be extended to other eukaryotic pathogens and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilium function is impaired.

Download Full-text

Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽

10.12688/f1000research.10342.2 ◽

2017 ◽

Vol 6 ◽

pp. 683 ◽

Cited By ~ 31

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.

Download Full-text

Human African trypanosomiasis

Oxford Textbook of Medicine ◽

10.1093/med/9780199204854.003.070810_update_001 ◽

2010 ◽

pp. 1119-1127

Author(s):

August Stich

Keyword(s):

West Africa ◽

East Africa ◽

Trypanosoma Brucei ◽

Human African Trypanosomiasis ◽

Protozoan Parasite ◽

Sleeping Sickness ◽

Tsetse Flies ◽

African Trypanosomiasis ◽

Tropical Africa

Human African trypanosomiasis (HAT, sleeping sickness) is caused by two subspecies of the protozoan parasite Trypanosoma brucei: T. b. rhodesiense is prevalent in East Africa among many wild and domestic mammals; T. b. gambiense causes an anthroponosis in Central and West Africa. The disease is restricted to tropical Africa where it is transmitted by the bite of infected tsetse flies (...

Download Full-text

Diversification of Function by Different Isoforms of Conventionally Shared RNA Polymerase Subunits

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0841 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1293-1301 ◽

Cited By ~ 27

Author(s):

Sara Devaux ◽

Steven Kelly ◽

Laurence Lecordier ◽

Bill Wickstead ◽

David Perez-Morga ◽

...

Keyword(s):

Rna Interference ◽

Rna Polymerase ◽

Trypanosoma Brucei ◽

Model Organism ◽

Protozoan Parasite ◽

Higher Plant ◽

The Core ◽

Form Part ◽

Rna Polymerase Subunits

Eukaryotic nuclei contain three classes of multisubunit DNA-directed RNA polymerase. At the core of each complex is a set of 12 highly conserved subunits of which five—RPB5, RPB6, RPB8, RPB10, and RPB12—are thought to be common to all three polymerase classes. Here, we show that four distantly related eukaryotic lineages (the higher plant and three protistan) have independently expanded their repertoire of RPB5 and RPB6 subunits. Using the protozoan parasite Trypanosoma brucei as a model organism, we demonstrate that these distinct RPB5 and RPB6 subunits localize to discrete subnuclear compartments and form part of different polymerase complexes. We further show that RNA interference-mediated depletion of these discrete subunits abolishes class-specific transcription and hence demonstrates complex specialization and diversification of function by conventionally shared subunit groups.

Download Full-text

A previously unrecognized membrane protein in the Rhodobacter sphaeroides LH1-RC photocomplex

Nature Communications ◽

10.1038/s41467-021-26561-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kazutoshi Tani ◽

Kenji V. P. Nagashima ◽

Ryo Kanno ◽

Saki Kawamura ◽

Riku Kikuchi ◽

...

Keyword(s):

Membrane Protein ◽

Ring Opening ◽

Model Organism ◽

Hypothetical Protein ◽

Integral Membrane Protein ◽

Bacterial Photosynthesis ◽

Core Complex ◽

Rc Structures ◽

Complex Forms ◽

And Function

AbstractRhodobacter (Rba.) sphaeroides is the most widely used model organism in bacterial photosynthesis. The light-harvesting-reaction center (LH1-RC) core complex of this purple phototroph is characterized by the co-existence of monomeric and dimeric forms, the presence of the protein PufX, and approximately two carotenoids per LH1 αβ-polypeptides. Despite many efforts, structures of the Rba. sphaeroides LH1-RC have not been obtained at high resolutions. Here we report a cryo-EM structure of the monomeric LH1-RC from Rba. sphaeroides strain IL106 at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αβ-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its channels. Our findings offer an exciting new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

Download Full-text

Oversized flagellar membrane protein in paralyzed mutants of Chlamydomonas reinhardrii.

The Journal of Cell Biology ◽

10.1083/jcb.85.2.258 ◽

1980 ◽

Vol 85 (2) ◽

pp. 258-272 ◽

Cited By ~ 37

Author(s):

J W Jarvik ◽

J L Rosenbaum

Keyword(s):

Membrane Protein ◽

Genetic Analysis ◽

Chlamydomonas Reinhardtii ◽

Mutant Strain ◽

Protein Composition ◽

Temperature Sensitive ◽

Membrane Biogenesis ◽

Radial Spoke ◽

Flagellar Membrane ◽

Flagellar Assembly

A mutant strain of Chlamydomonas reinhardtii is shown to possess an oversized flagellar membrane protein. The mutant has paralyzed flagella, is temperature sensitive for flagellar assembly, and has an abnormal axonemal protein composition. All phenotypes appear to derive from a single Mendelian mutation, and genetic analysis suggests that the mutation, which call ts222, is in the gene pfl. Because pf1 mutants are known to have radial-spoke defects (Piperno et al., 1977, Proc. Natl. Acad. Sci. U. S. A. 74:1600-1604; and Witman et al., 1978, J. Cell Biol. 76:729-797), a relation as yet undefined appears to exist between radial-spoke and flagellar membrane biogenesis.

Download Full-text

The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00879-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6151-6160 ◽

Cited By ~ 16

Author(s):

Luis Carvalho ◽

Marta Martínez-García ◽

Ignacio Pérez-Victoria ◽

José Ignacio Manzano ◽

Vanessa Yardley ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Antimalarial Drug ◽

Protozoan Parasite ◽

Sleeping Sickness ◽

Phase Iii ◽

Content Type ◽

Clinical Phase ◽

Intracellular Ca ◽

Cytoplasmic Content

ABSTRACTThe protozoan parasiteTrypanosoma bruceicauses human African trypanosomiasis, or sleeping sickness, a neglected tropical disease that requires new, safer, and more effective treatments. Repurposing oral drugs could reduce both the time and cost involved in sleeping sickness drug discovery. Tafenoquine (TFQ) is an oral antimalarial drug belonging to the 8-aminoquinoline family which is currently in clinical phase III. We show here that TFQ efficiently kills differentT. bruceispp. in the submicromolar concentration range. Our results suggest that TFQ accumulates into acidic compartments and induces a necrotic process involving cell membrane disintegration and loss of cytoplasmic content, leading to parasite death. Cell lysis is preceded by a wide and multitarget drug action, affecting the lysosome, mitochondria, and acidocalcisomes and inducing a depolarization of the mitochondrial membrane potential, elevation of intracellular Ca2+, and production of reactive oxygen species. This is the first report of an 8-aminoquinoline demonstrating significantin vitroactivity againstT. brucei.

Download Full-text

Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells

Cell Discovery ◽

10.1038/s41421-021-00281-2 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Jiayan Zhang ◽

Hui Wang ◽

Simon Imhof ◽

Xueting Zhou ◽

Shiqing Liao ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Disease Transmission ◽

Electron Tomography ◽

Signal Transmission ◽

Protozoan Parasite ◽

Empty Space ◽

Eukaryotic Cells ◽

Critical Gap ◽

Proximal Zone ◽

Paracrystalline Array

AbstractEukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of accessory filaments and their axoneme interface are mostly unknown, presenting a critical gap in understanding structural foundations of eukaryotic flagella. In the flagellum of the protozoan parasite Trypanosoma brucei (T. brucei), the axoneme is accompanied by a paraflagellar rod (PFR) that supports non-planar motility and signaling necessary for disease transmission and pathogenesis. Here, we employed cryogenic electron tomography (cryoET) with sub-tomographic averaging, to obtain structures of the PFR, PFR-axoneme connectors (PACs), and the axonemal central pair complex (CPC). The structures resolve how the 8 nm repeat of the axonemal tubulin dimer interfaces with the 54 nm repeat of the PFR, which consist of proximal, intermediate, and distal zones. In the distal zone, stacked “density scissors” connect with one another to form a “scissors stack network (SSN)” plane oriented 45° to the axoneme axis; and ~370 parallel SSN planes are connected by helix-rich wires into a paracrystalline array with ~90% empty space. Connections from these wires to the intermediate zone, then to overlapping layers of the proximal zone and to the PACs, and ultimately to the CPC, point to a contiguous pathway for signal transmission. Together, our findings provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology, to engineering principles in bionics.

Download Full-text