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1.
Open Biol ; 14(6): 240025, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38862021

RESUMO

Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. It remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.


Assuntos
Cinetocoros , Proteínas de Protozoários , Cinetocoros/metabolismo , Cinetocoros/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Modelos Moleculares , Sequência de Aminoácidos , Filogenia , Ligação Proteica , Cristalografia por Raios X , Segregação de Cromossomos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética
2.
PLoS One ; 19(4): e0298521, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38662801

RESUMO

In Trypanosoma brucei, gene expression is primarily regulated posttranscriptionally making RNA metabolism critical. T. brucei has an epitranscriptome containing modified RNA bases. Yet, the identity of the enzymes catalyzing modified RNA base addition and the functions of the enzymes and modifications remain unclear. Homology searches indicate the presence of numerous T. brucei cytosine RNA methyltransferase homologs. One such homolog, TbNop2 was studied in detail. TbNop2 contains the six highly conserved motifs found in cytosine RNA methyltransferases and is evolutionarily related to the Nop2 protein family required for rRNA modification and processing. RNAi experiments targeting TbNop2 resulted in reduced levels of TbNop2 RNA and protein, and a cessation of parasite growth. Next generation sequencing of bisulfite-treated RNA (BS-seq) detected the presence of two methylation sites in the large rRNA; yet TbNop2 RNAi did not result in a significant reduction of methylation. However, TbNop2 RNAi resulted in the retention of 28S internal transcribed spacer RNAs, indicating a role for TbNop2 in rRNA processing.


Assuntos
Processamento Pós-Transcricional do RNA , RNA Ribossômico , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/metabolismo , RNA Ribossômico/metabolismo , RNA Ribossômico/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , RNA de Protozoário/metabolismo , RNA de Protozoário/genética , Interferência de RNA , Metilação
3.
Mol Biol Rep ; 51(1): 578, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38668789

RESUMO

Mg2+-independent phosphatidic acid phosphatase (PAP2), diacylglycerol pyrophosphate phosphatase 1 (Dpp1) is a membrane-associated enzyme in Saccharomyces cerevisiae. The enzyme is responsible for inducing the breakdown of ß-phosphate from diacylglycerol pyrophosphate (DGPP) into phosphatidate (PA) and then removes the phosphate from PA to give diacylglycerol (DAG). In this study through RNAi suppression, we have demonstrated that Trypanosoma brucei diacylglycerol pyrophosphate phosphatase 1 (TbDpp1) procyclic form production is not required for parasite survival in culture. The steady-state levels of triacylglycerol (TAG), the number of lipid droplets, and the PA content are all maintained constant through the inducible down-regulation of TbDpp1. Furthermore, the localization of C-terminally tagged variants of TbDpp1 in the lysosome was demonstrated by immunofluorescence microscopy.


Assuntos
Glicerol/análogos & derivados , Lisossomos , Trypanosoma brucei brucei , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Lisossomos/metabolismo , Lisossomos/enzimologia , Triglicerídeos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Fosfatidato Fosfatase/metabolismo , Fosfatidato Fosfatase/genética , Interferência de RNA , Difosfatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Diglicerídeos/metabolismo , Ácidos Fosfatídicos/metabolismo
4.
Front Cell Infect Microbiol ; 14: 1381155, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38650737

RESUMO

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages.


Assuntos
Proteínas de Protozoários , Edição de RNA , Ribonuclease III , Trypanosoma brucei brucei , Substituição de Aminoácidos , Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Domínios Proteicos/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento
5.
J Cell Sci ; 137(10)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38572631

RESUMO

Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.


Assuntos
Proteínas de Protozoários , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sequência Conservada , Corpos Basais/metabolismo , Transporte Proteico , Fatores de Tempo , Flagelos/genética , Flagelos/metabolismo , Regulação da Expressão Gênica , Cílios/genética , Cílios/metabolismo
6.
Mol Biochem Parasitol ; 259: 111620, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38653348

RESUMO

Kinetoplastids, a group of flagellated protists that are often insect intestinal parasites, encounter various sources of oxidative stress. Such stressors include reactive oxygen species, both internally produced within the protist, and induced externally by host immune responses. This investigation focuses on the role of a highly conserved aspartate-based protein phosphatase, PTP-Interacting protein (PIP39) in managing oxidative stress. In addition to its well accepted role in a Trypanosoma brucei life stage transition, there is evidence of PIP39 participation in the T. brucei oxidative stress response. To examine whether this latter PIP39 role may exist more broadly, we aimed to elucidate PIP39's contribution to redox homeostasis in the monoxenous parasite Leptomonas seymouri. Utilizing CRISPR-Cas9-mediated elimination of PIP39 in conjunction with oxidative stress assays, we demonstrate that PIP39 is required for cellular tolerance to oxidative stress in L. seymouri, positing it as a putative regulatory node for adaptive stress responses. We propose that future analysis of L. seymouri PIP39 enzymatic activity, regulation, and potential localization to a specialized organelle termed a glycosome will contribute to a deeper understanding of the molecular mechanisms by which protozoan parasites adapt to oxidative environments. Our study also demonstrates success at using gene editing tools developed for Leishmania for the related L. seymouri.


Assuntos
Estresse Oxidativo , Proteínas de Protozoários , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Sistemas CRISPR-Cas , Kinetoplastida/genética , Kinetoplastida/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/genética , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/fisiologia
7.
Mol Microbiol ; 121(6): 1079-1094, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38558208

RESUMO

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Ribonucleoproteínas , Edição de Genes/métodos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Leishmania/genética , Leishmania/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma/genética , Trypanosoma/metabolismo , Transfecção
8.
Mol Microbiol ; 121(6): 1112-1126, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38622999

RESUMO

All mitochondria import >95% of their proteins from the cytosol. This process is mediated by protein translocases in the mitochondrial membranes, whose subunits are generally highly conserved. Most eukaryotes have two inner membrane protein translocases (TIMs) that are specialized to import either presequence-containing or mitochondrial carrier proteins. In contrast, the parasitic protozoan Trypanosoma brucei has a single TIM complex consisting of one conserved and five unique subunits. Here, we identify candidates for new subunits of the TIM or the presequence translocase-associated motor (PAM) using a protein-protein interaction network of previously characterized TIM and PAM subunits. This analysis reveals that the trypanosomal TIM complex contains an additional trypanosomatid-specific subunit, designated TbTim15. TbTim15 is associated with the TIM complex, lacks transmembrane domains, and localizes to the intermembrane space. TbTim15 is essential for procyclic and bloodstream forms of trypanosomes. It contains two twin CX9C motifs and mediates import of both presequence-containing and mitochondrial carrier proteins. While the precise function of TbTim15 in mitochondrial protein import is unknown, our results are consistent with the notion that it may function as an import receptor for the non-canonical trypanosomal TIM complex.


Assuntos
Mitocôndrias , Proteínas de Transporte da Membrana Mitocondrial , Membranas Mitocondriais , Transporte Proteico , Proteínas de Protozoários , Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/enzimologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Membranas Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Subunidades Proteicas/metabolismo
9.
Nat Commun ; 15(1): 2972, 2024 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-38582942

RESUMO

Adaptation to a change of environment is an essential process for survival, in particular for parasitic organisms exposed to a wide range of hosts. Such adaptations include rapid control of gene expression through the formation of membraneless organelles composed of poly-A RNA and proteins. The African trypanosome Trypanosoma brucei is exquisitely sensitive to well-defined environmental stimuli that trigger cellular adaptations through differentiation events that characterise its complex life cycle. The parasite has been shown to form stress granules in vitro, and it has been proposed that such a stress response could have been repurposed to enable differentiation and facilitate parasite transmission. Therefore, we explored the composition and positional dynamics of membraneless granules formed in response to starvation stress and during differentiation in the mammalian host between the replicative slender and transmission-adapted stumpy forms. We find that T. brucei differentiation does not reflect the default response to environmental stress. Instead, the developmental response of the parasites involves a specific and programmed hierarchy of membraneless granule assembly, with distinct components and regulation by protein kinases such as TbDYRK, that are required for the parasite to successfully progress through its life cycle development and prepare for transmission.


Assuntos
Trypanosoma brucei brucei , Trypanosoma , Animais , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Mamíferos
10.
Parasitol Int ; 101: 102874, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38417735

RESUMO

Trypanosoma brucei brucei is a parasitic protist that expresses cell surface proteins modified with complex-type N-linked glycan (NLG), like multicellular organisms. However, little is known about the role of complex-type NLG. In T. b. brucei, it has been shown that either one of the glycosyltransferases, TbGT11 or TbGT15, is sufficient to initiate the synthesis of complex-type NLG. To clarify the role of complex-type NLG, it is necessary to generate cells lacking both enzymes. Therefore, we deleted TbGT11 and TbGT15 from the genome of T. b. brucei for the phenotypic examination. The mutant strain grew in culture, with reduced maximum cell density; showed decreased susceptibility to normal human serum, which contains trypanolytic factors; and lacked uptake of the haptoglobin-hemoglobin complex. These data indicate that protein modification by complex-type NLG is not essential but is required for receptor function.


Assuntos
Polissacarídeos , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Soro
11.
Nat Commun ; 15(1): 1779, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38413606

RESUMO

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.


Assuntos
Trypanosoma brucei brucei , Trypanosoma cruzi , Tripanossomíase Africana , Animais , Humanos , Trypanosoma brucei brucei/genética , Interleucina-10/genética , Fatores de Virulência , Parasitemia/parasitologia , Tripanossomíase Africana/parasitologia
12.
BMC Genomics ; 25(1): 184, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365628

RESUMO

BACKGROUND: Almost all extant organisms use the same, so-called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non-canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypanosomatid flagellate that reassigned all three stop codons to encode amino acids. RESULTS: We in silico predicted the metabolism of B. nonstop and compared it with that of the well-studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes. CONCLUSIONS: In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop is extremely AT-rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.


Assuntos
Parasitos , Trypanosoma brucei brucei , Trypanosomatina , Animais , Códon de Terminação , Eucariotos/genética , Código Genético , Parasitos/genética , Trypanosoma brucei brucei/genética , Trypanosomatina/genética
13.
Sci Rep ; 14(1): 4158, 2024 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378867

RESUMO

Animal African trypanosomiasis (AAT) is a significant food security and economic burden in sub-Saharan Africa. Current AAT empirical and immunodiagnostic surveillance tools suffer from poor sensitivity and specificity, with blood sampling requiring animal restraint and trained personnel. Faecal sampling could increase sampling accessibility, scale, and species range. Therefore, this study assessed feasibility of detecting Trypanosoma DNA in the faeces of experimentally-infected cattle. Holstein-Friesian calves were inoculated with Trypanosoma brucei brucei AnTat 1.1 (n = 5) or T. congolense Savannah IL3000 (n = 6) in separate studies. Faecal and blood samples were collected concurrently over 10 weeks and screened using species-specific PCR and qPCR assays. T. brucei DNA was detected in 85% of post-inoculation (PI) faecal samples (n = 114/134) by qPCR and 50% by PCR between 4 and 66 days PI. However, T. congolense DNA was detected in just 3.4% (n = 5/145) of PI faecal samples by qPCR, and none by PCR. These results confirm the ability to consistently detect T. brucei DNA, but not T. congolense DNA, in infected cattle faeces. This disparity may derive from the differences in Trypanosoma species tissue distribution and/or extravasation. Therefore, whilst faeces are a promising substrate to screen for T. brucei infection, blood sampling is required to detect T. congolense in cattle.


Assuntos
Trypanosoma brucei brucei , Trypanosoma congolense , Trypanosoma , Tripanossomíase Africana , Humanos , Bovinos , Animais , Trypanosoma brucei brucei/genética , Trypanosoma congolense/genética , Tripanossomíase Africana/diagnóstico , Tripanossomíase Africana/veterinária , Tripanossomíase Africana/epidemiologia , Trypanosoma/genética , DNA , Fezes
14.
PLoS Negl Trop Dis ; 18(2): e0012007, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38394337

RESUMO

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.


Assuntos
Gluconeogênese , Trypanosoma brucei brucei , Animais , Humanos , Gluconeogênese/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Transaldolase/metabolismo , Glicerol/metabolismo , Glucose/metabolismo , Fosfofrutoquinases/metabolismo , Carbono/metabolismo , Trifosfato de Adenosina/metabolismo , Mamíferos
15.
Sci Rep ; 14(1): 2178, 2024 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-38272944

RESUMO

Recognition of the mRNA 5' end is a critical step needed for translation initiation. This step is performed by the cap binding protein eIF4E, which joins the larger eIF4G subunit to form the eIF4F complex. Trypanosomatids have a minimum of five different eIF4F-like complexes formed through specific but not well-defined interactions between four different eIF4E and five eIF4G homologues. The EIF4E6/EIF4G5 complex has been linked with the stage-specific translation of mRNAs encoding the major Trypanosoma brucei virulence factors. Here, to better define the molecular basis for the TbEIF4E6/TbEIF4G5 interaction, we describe the identification of the peptide interacting with TbEIF4E6 in the region comprising residues 79-166 of TbEIF4G5. The TbEIF4E6-TbEIF4G5_79-116 complex reconstituted with recombinant proteins is highly stable even in the absence of cap-4. The crystal structure of the complex was subsequently solved, revealing extensive interacting surfaces. Comparative analyses highlight the conservation of the overall structural arrangement of different eIF4E/eIF4G complexes. However, highly different interacting surfaces are formed with distinct binding contacts occurring both in the canonical and noncanonical elements within eIF4G and the respective eIF4E counterpart. These specific pairs of complementary interacting surfaces are likely responsible for the selective association needed for the formation of distinct eIF4F complexes in trypanosomatids.


Assuntos
Fator de Iniciação 4F em Eucariotos , Trypanosoma brucei brucei , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Trypanosoma brucei brucei/genética , Ligação Proteica , RNA Mensageiro/metabolismo
16.
J Cell Sci ; 137(3)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38205672

RESUMO

Tubulin polyglutamylation, catalysed by members of the tubulin tyrosine ligase-like (TTLL) protein family, is an evolutionarily highly conserved mechanism involved in the regulation of microtubule dynamics and function in eukaryotes. In the protozoan parasite Trypanosoma brucei, the microtubule cytoskeleton is essential for cell motility and maintaining cell shape. In a previous study, we showed that T. brucei TTLL6A and TTLL12B are required to regulate microtubule dynamics at the posterior cell pole. Here, using gene deletion, we show that the polyglutamylase TTLL1 is essential for the integrity of the highly organised microtubule structure at the cell pole, with a phenotype distinct from that observed in TTLL6A- and TTLL12B-depleted cells. Reduced polyglutamylation in TTLL1-deficient cells also leads to increased levels in tubulin tyrosination, providing new evidence for an interplay between the tubulin tyrosination and detyrosination cycle and polyglutamylation. We also show that TTLL1 acts differentially on specific microtubule doublets of the flagellar axoneme, although the absence of TTLL1 appears to have no measurable effect on cell motility.


Assuntos
Trypanosoma brucei brucei , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo
17.
BMC Mol Cell Biol ; 25(1): 3, 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38279116

RESUMO

BACKGROUND: Trypanosoma brucei is the causative agent for trypanosomiasis in humans and livestock, which presents a growing challenge due to drug resistance. While identifying novel drug targets is vital, the process is delayed due to a lack of functional information on many of the pathogen's proteins. Accordingly, this paper presents a computational framework for prioritizing drug targets within the editosome, a vital molecular machinery responsible for mitochondrial RNA processing in T. brucei. Importantly, this framework may eliminate the need for prior gene or protein characterization, potentially accelerating drug discovery efforts. RESULTS: By integrating protein-protein interaction (PPI) network analysis, PPI structural modeling, and residue interaction network (RIN) analysis, we quantitatively ranked and identified top hub editosome proteins, their key interaction interfaces, and hotspot residues. Our findings were cross-validated and further prioritized by incorporating them into gene set analysis and differential expression analysis of existing quantitative proteomics data across various life stages of T. brucei. In doing so, we highlighted PPIs such as KREL2-KREPA1, RESC2-RESC1, RESC12A-RESC13, and RESC10-RESC6 as top candidates for further investigation. This includes examining their interfaces and hotspot residues, which could guide drug candidate selection and functional studies. CONCLUSION: RNA editing offers promise for target-based drug discovery, particularly with proteins and interfaces that play central roles in the pathogen's life cycle. This study introduces an integrative drug target identification workflow combining information from the PPI network, PPI 3D structure, and reside-level information of their interface which can be applicable to diverse pathogens. In the case of T. brucei, via this pipeline, the present study suggested potential drug targets with residue-resolution from RNA editing machinery. However, experimental validation is needed to fully realize its potential in advancing urgently needed antiparasitic drug development.


Assuntos
Trypanosoma brucei brucei , Humanos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Citoplasma/metabolismo , Mitocôndrias/metabolismo
18.
Parasitol Int ; 99: 102831, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38048903

RESUMO

The biosynthesis of N-linked glycan precursors in the endoplasmic reticulum is important for many eukaryotes. In particular, the synthesis of Man5GlcNAc2-PP-dolichol (M5-DLO) at the cytoplasmic face of the endoplasmic reticulum is essential for maintaining cellular functions. In Trypanosoma brucei, the unicellular organism that causes African trypanosomiasis, homologs of the mannosyltransferases ALG2 and ALG11, which are involved in the biosynthesis of M5-DLO, are found, but the effects of their deletion on cells remain unknown. In this study, we generated conditional gene knockout strains of TbALG2 and TbALG11 in the bloodstream form T. brucei. Decreased N-linked glycosylation and cell death were observed in both strains under non-permissive conditions, with TbALG2 having a greater effect than TbALG11. Transcriptomic analysis of cells losing expression of TbALG11 showed decrease in mRNAs for enzymes involved in glucose metabolism and increase in mRNAs for procyclins and variant surface glycoproteins. These results indicate that the M5-DLO biosynthetic pathway is essential for the proliferation of the bloodstream form T. brucei. They also suggest that the failure of this pathway induces the transcriptomic change.


Assuntos
Trypanosoma brucei brucei , Animais , Trypanosoma brucei brucei/genética , RNA Mensageiro/metabolismo , Glicosilação , Retículo Endoplasmático/metabolismo , Morte Celular , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
19.
Mol Microbiol ; 121(1): 53-68, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38010644

RESUMO

Leishmania are flagellated eukaryotic parasites that cause leishmaniasis and are closely related to the other kinetoplastid parasites such as Trypanosoma brucei. In all these parasites there is a cell membrane invagination at the base of the flagellum called the flagellar pocket, which is tightly associated with and sculpted by cytoskeletal structures including the flagellum attachment zone (FAZ). The FAZ is a complex interconnected structure linking the flagellum to the cell body and has critical roles in cell morphogenesis, function and pathogenicity. However, this structure varies dramatically in size and organisation between these different parasites, suggesting changes in protein localisation and function. Here, we screened the localisation and function of the Leishmania orthologues of T. brucei FAZ proteins identified in the genome-wide protein tagging project TrypTag. We identified 27 FAZ proteins and our deletion analysis showed that deletion of two FAZ proteins in the flagellum, FAZ27 and FAZ34 resulted in a reduction in cell body size, and flagellum loss in some cells. Furthermore, after null mutant generation, we observed distinct and reproducible changes to cell shape, demonstrating the ability of the parasite to adapt to morphological perturbations resulting from gene deletion. This process of adaptation has important implications for the study of Leishmania mutants.


Assuntos
Leishmania , Leishmaniose , Trypanosoma brucei brucei , Humanos , Leishmania/genética , Leishmania/metabolismo , Flagelos/metabolismo , Citoesqueleto/metabolismo , Leishmaniose/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
20.
Biochem Pharmacol ; 219: 115937, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37995979

RESUMO

Mitochondrial uridine insertion/deletion RNA editing, catalyzed by a multiprotein complex (editosome), is essential for gene expression in trypanosomes and Leishmania parasites. As this process is absent in the human host, a drug targeting this mechanism promises high selectivity and reduced toxicity. Here, we successfully miniaturized our FRET-based full-round RNA editing assay, which replicates the complete RNA editing process, adapting it into a 1536-well format. Leveraging this assay, we screened over 100,000 compounds against purified editosomes derived from Trypanosoma brucei, identifying seven confirmed primary hits. We sourced and evaluated various analogs to enhance the inhibitory and parasiticidal effects of these primary hits. In combination with secondary assays, our compounds marked inhibition of essential catalytic activities, including the RNA editing ligase and interactions of editosome proteins. Although the primary hits did not exhibit any growth inhibitory effect on parasites, we describe eight analog compounds capable of effectively killing T. brucei and/or Leishmania donovani parasites within a low micromolar concentration. Whether parasite killing is - at least in part - due to inhibition of RNA editing in vivo remains to be assessed. Our findings introduce novel molecular scaffolds with the potential for broad antitrypanosomal effects.


Assuntos
Trypanosoma brucei brucei , Humanos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Ensaios de Triagem em Larga Escala , Edição de RNA , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA/metabolismo
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