scholarly journals Rab11 Function in Trypanosoma brucei: Identification of Conserved and Novel Interaction Partners

2011 ◽  
Vol 10 (8) ◽  
pp. 1082-1094 ◽  
Author(s):  
Carme Gabernet-Castello ◽  
Kelly N. DuBois ◽  
Camus Nimmo ◽  
Mark C. Field
Keyword(s):  
Trypanosoma Brucei ◽  
Intracellular Transport ◽  
Homo Sapiens ◽  
Coiled Coil ◽  
Membrane Composition ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Interaction Partners ◽  
Hybrid Screening

ABSTRACT The Ras-like GTPase Rab11 is implicated in multiple aspects of intracellular transport, including maintenance of plasma membrane composition and cytokinesis. In metazoans, these functions are mediated in part via coiled-coil Rab11-interacting proteins (FIPs) acting as Rab11 effectors. Additional interaction between Rab11 and the exocyst subunit Sec15 connects Rab11 with exocytosis. We find that FIPs are metazoan specific, suggesting that other factors mediate Rab11 functions in nonmetazoans. We examined Rab11 interactions in Trypanosoma brucei , where endocytosis is well studied and the role of Rab11 in recycling well documented. TbSec15 and TbRab11 interact, demonstrating evolutionary conservation. By yeast two-hybrid screening, we identified additional Rab11 interaction partners. Tb927.5.1640 (designated RBP74) interacted with both Rab11 and Rab5. RBP74 shares a coiled-coil architecture with metazoan FIPs but is unrelated by sequence and appears to play a role in coordinating endocytosis and recycling. A second coiled-coil protein, Tb09.211.4830 (TbAZI1), orthologous to AZI1 in Homo sapiens , interacts exclusively with Rab11. AZI1 is restricted to taxa with motile cilia/flagella. These data suggest that Rab11 functions are mediated by evolutionarily conserved (i.e., AZI1 and Sec15) and potentially lineage-specific (RBP74) interactions essential for the integration of the endomembrane system.

scholarly journals The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

2015 ◽  
Vol 14 (9) ◽  
pp. 898-907 ◽  
Author(s):  
Huiqing Hu ◽  
Yi Liu ◽  
Qing Zhou ◽  
Sara Siegel ◽  
Ziyin Li
Keyword(s):  
Cell Cycle ◽  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Basal Body ◽  
Essential Role ◽  
Microtubule Organizing Center ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Organizing Center

ABSTRACT The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei , an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei .

scholarly journals Novel Bilobe Components in Trypanosoma brucei Identified Using Proximity-Dependent Biotinylation

2012 ◽  
Vol 12 (2) ◽  
pp. 356-367 ◽  
Author(s):  
Brooke Morriswood ◽  
Katharina Havlicek ◽  
Lars Demmel ◽  
Sevil Yavuz ◽  
Marco Sealey-Cardona ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Protein Identification ◽  
Affinity Purification ◽  
Bioinformatic Analysis ◽  
Test Subject ◽  
Content Type ◽  
Interaction Partners ◽  
Nonionic Detergent ◽  
Protein Components ◽  
Attachment Zone

ABSTRACT The trypanosomes are a family of parasitic protists of which the African trypanosome, Trypanosoma brucei , is the best characterized. The complex and highly ordered cytoskeleton of T. brucei has been shown to play vital roles in its biology but remains difficult to study, in large part owing to the intractability of its constituent proteins. Existing methods of protein identification, such as bioinformatic analysis, generation of monoclonal antibody panels, proteomics, affinity purification, and yeast two-hybrid screens, all have drawbacks. Such deficiencies—troublesome proteins and technical limitations—are common not only to T. brucei but also to many other protists, many of which are even less well studied. Proximity-dependent biotin identification (BioID) is a recently developed technique that allows forward screens for interaction partners and near neighbors in a native environment with no requirement for solubility in nonionic detergent. As such, it is extremely well suited to the exploration of the cytoskeleton. In this project, BioID was adapted for use in T. brucei . The trypanosome bilobe, a discrete cytoskeletal structure with few known protein components, represented an excellent test subject. Use of the bilobe protein TbMORN1 as a probe resulted in the identification of seven new bilobe constituents and two new flagellum attachment zone proteins. This constitutes the first usage of BioID on a largely uncharacterized structure, and demonstrates its utility in identifying new components of such a structure. This remarkable success validates BioID as a new tool for the study of unicellular eukaryotes in particular and the eukaryotic cytoskeleton in general.

scholarly journals Acquisition and Role of Molybdate in Pseudomonas aeruginosa

2014 ◽  
Vol 80 (21) ◽  
pp. 6843-6852 ◽  
Author(s):  
Victoria G. Pederick ◽  
Bart A. Eijkelkamp ◽  
Miranda P. Ween ◽  
Stephanie L. Begg ◽  
James C. Paton ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fatty Acid Composition ◽  
Nitrate Reduction ◽  
Biofilm Formation ◽  
Energy Production ◽  
Anaerobic Growth ◽  
Membrane Composition ◽  
Content Type ◽  
High Affinity

ABSTRACTIn microaerophilic or anaerobic environments,Pseudomonas aeruginosautilizes nitrate reduction for energy production, a process dependent on the availability of the oxyanionic form of molybdenum, molybdate (MoO42−). Here, we show that molybdate acquisition inP. aeruginosaoccurs via a high-affinity ATP-binding cassette permease (ModABC). ModA is a cluster D-III solute binding protein capable of interacting with molybdate or tungstate oxyanions. Deletion of themodAgene reduces cellular molybdate concentrations and results in inhibition of anaerobic growth and nitrate reduction. Further, we show that conditions that permit nitrate reduction also cause inhibition of biofilm formation and an alteration in fatty acid composition ofP. aeruginosa. Collectively, these data highlight the importance of molybdate for anaerobic growth ofP. aeruginosaand reveal novel consequences of nitrate reduction on biofilm formation and cell membrane composition.

scholarly journals ThechbGGene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

2012 ◽  
Vol 194 (18) ◽  
pp. 4959-4971 ◽  
Author(s):  
Subhash Chandra Verma ◽  
Subramony Mahadevan
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Inflammatory Diseases ◽  
Regulatory Protein ◽  
Loss Of Function ◽  
Content Type ◽  
Reading Frame ◽  
E Coli ◽  
Evolutionarily Conserved

ABSTRACTThechboperon ofEscherichia coliis involved in the utilization of the β-glucosides chitobiose and cellobiose. The function ofchbG(ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show thatchbGencodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of thechbpromoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations inchbRconferring the ability to grow on both cellobiose and chitobiose are independent ofchbGfunction for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-β-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence ofchbG, suggesting an additional role ofchbGin the hydrolysis of chitooligosaccharides. The homologs ofchbGin metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function ofE. colichbGhas a broader significance.

scholarly journals A SAS-6-Like Protein Suggests that the Toxoplasma Conoid Complex Evolved from Flagellar Components

2013 ◽  
Vol 12 (7) ◽  
pp. 1009-1019 ◽  
Author(s):  
Jessica Cruz de Leon ◽  
Nicole Scheumann ◽  
Wandy Beatty ◽  
Josh R. Beck ◽  
Johnson Q. Tran ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Common Ancestor ◽  
Coiled Coil ◽  
Apicomplexan Parasite ◽  
Last Common Ancestor ◽  
Cell Cytoplasm ◽  
Content Type ◽  
Conserved Domain ◽  
Coiled Coil Domain ◽  
Order Structures

ABSTRACT SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii , SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma . The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.

scholarly journals Role of the Trypanosoma brucei HEN1 Family Methyltransferase in Small Interfering RNA Modification

2013 ◽  
Vol 13 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Huafang Shi ◽  
Rebecca L. Barnes ◽  
Nicholas Carriero ◽  
Vanessa D. Atayde ◽  
Christian Tschudi ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Small Interfering Rna ◽  
Leishmania Major ◽  
Rna Modification ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Content Type ◽  
Interfering Rna ◽  
Core Genes

ABSTRACT Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania ( Viannia ) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 ( Tb AGO1). The five genes are conserved in Leishmania ( Viannia ) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major . In T. brucei small interfering RNAs (siRNAs) are methylated at the 3′ end, whereas Leishmania ( Viannia ) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2′- O -methyltransferases, is required for methylation of siRNAs. Loss of Tb HEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1 −/− parasites are single stranded and associated with Tb AGO1, and a subset carry a nontemplated uridine at the 3′ end. These findings support a model wherein Tb HEN1 methylates siRNA 3′ ends after they are loaded into Tb AGO1 and this methylation protects siRNAs from uridylation and 3′ trimming. Moreover, expression of Tb HEN1 in Leishmania ( Viannia ) panamensis did not result in siRNA 3′ end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms.

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 Pyrophosphate Stimulates the Phosphate-Sodium Symporter ofTrypanosoma bruceiAcidocalcisomes andSaccharomyces cerevisiaeVacuoles

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Evgeniy Potapenko ◽  
Ciro D. Cordeiro ◽  
Guozhong Huang ◽  
Roberto Docampo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Potential ◽  
Trypanosoma Brucei ◽  
Wild Type ◽  
Inorganic Pyrophosphate ◽  
Content Type ◽  
Yeast Strains ◽  
Outward Currents ◽  
Sodium Dependent

ABSTRACTInorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPiand other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Piconductance inXenopusoocytes expressing aSaccharomyces cerevisiaeorTrypanosoma bruceiNa+/Pisymporter. PPiis not taken up byXenopusoocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPigenerated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type orpho91Δ yeast strains expressingTbPHO91but not from thepho91Δstrains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing PifromS. cerevisiaevacuoles andT. bruceiacidocalcisomes.IMPORTANCEAcidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities withS. cerevisiaevacuoles in their structure and composition. Both organelles share a Na+/Pisymporter involved in Pirelease to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in eitherXenopusoocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.

scholarly journals mSphere of Influence: Expanding the CRISPR Sphere with Single-Locus Proteomics

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Lucy Glover
Keyword(s):  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Strand Break ◽  
Single Locus ◽  
Repair Pathway ◽  
Genomic Locus ◽  
Content Type ◽  
Animal African Trypanosomiasis ◽  
Insight Into

ABSTRACT Lucy Glover’s research focuses on the role of DNA repair and recombination in antigenic variation in the parasite Trypanosoma brucei, the causative agent of both human and animal African trypanosomiasis. In this mSphere of Influence article, she reflects on how “A CRISPR-based approach for proteomic analysis of a single genomic locus” by Z. J. Waldrip, S. D. Byrum, A. J. Storey, J. Gao, et al. (Epigenetics 9:1207–1211, 2014, https://doi.org/10.4161/epi.29919) made an impact on her research by taking the precision of CRISPR-Cas9 and repurposing it to look at single-locus proteomics. By using this technology in trypanosomes, Dr. Glover and her colleagues could study the dynamic accumulation of repair proteins after specific damage and gain insight into how the location of a double-strand break (DSB) dictates repair pathway choice and how this may influence immune evasion in these parasites.

scholarly journals Yeast Viral Killer Toxin K1 Induces Specific Host Cell Adaptions via Intrinsic Selection Pressure

2019 ◽  
Vol 86 (4) ◽  
Author(s):  
Stefanie Gier ◽  
Martin Simon ◽  
Gilles Gasparoni ◽  
Salem Khalifa ◽  
Marcel H. Schulz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Host Cell ◽  
Selection Pressure ◽  
Biological Diversity ◽  
Killer Toxin ◽  
Content Type ◽  
Killer Yeast ◽  
Evolutionarily Conserved ◽  
K1 Toxin

ABSTRACT The killer phenomenon in yeast (Saccharomyces cerevisiae) not only provides the opportunity to study host-virus interactions in a eukaryotic model but also represents a powerful tool to analyze potential coadaptional events and the role of killer yeast in biological diversity. Although undoubtedly having a crucial impact on the abundance and expression of the killer phenotype in killer-yeast harboring communities, the influence of a particular toxin on its producing host cell has not been addressed sufficiently. In this study, we describe a model system of two K1 killer yeast strains with distinct phenotypical differences pointing to substantial selection pressure in response to the toxin secretion level. Transcriptome and lipidome analyses revealed specific and intrinsic host cell adaptions dependent on the amount of K1 toxin produced. High basal expression of genes coding for osmoprotectants and stress-responsive proteins in a killer yeast strain secreting larger amounts of active K1 toxin implies a generally increased stress tolerance. Moreover, the data suggest that immunity of the host cell against its own toxin is essential for the balanced virus-host interplay providing valuable hints to elucidate the molecular mechanisms underlying K1 immunity and implicating an evolutionarily conserved role for toxin immunity in natural yeast populations. IMPORTANCE The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two RNA viruses. In contrast to bacterial toxin producers, killer yeasts necessitate a specific immunity mechanism against their own toxin because they bear the same receptor populations as sensitive cells. Although the killer phenomenon is highly abundant and has a crucial impact on the structure of yeast communities, the influence of a particular toxin on its host cell has been barely addressed. In our study, we used two derivatives secreting different amount of the killer toxin K1 to analyze potential coadaptional events in this particular host/virus system. Our data underline the dependency of the host cell’s ability to cope with extracellular toxin molecules and intracellular K1 molecules provided by the virus. Therefore, this research significantly advances the current understanding of the evolutionarily conserved role of this molecular machinery as an intrinsic selection pressure in yeast populations.

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