RESUMEN
The AT-rich mitochondrial DNA (kDNA) of trypanosomatid parasites is a target of DNA minor groove binders. We report the synthesis, antiprotozoal screening, and SAR studies of three series of analogues of the known antiprotozoal kDNA binder 2-((4-(4-((4,5-dihydro-1H-imidazol-3-ium-2-yl)amino)benzamido)phenyl)amino)-4,5-dihydro-1H-imidazol-3-ium (1a). Bis(2-aminoimidazolines) (1) and bis(2-aminobenzimidazoles) (2) showed micromolar range activity against Trypanosoma brucei, whereas bisarylimidamides (3) were submicromolar inhibitors of T. brucei, Trypanosoma cruzi, and Leishmania donovani. None of the compounds showed relevant activity against the urogenital, nonkinetoplastid parasite Trichomonas vaginalis. We show that series 1 and 3 bind strongly and selectively to the minor groove of AT DNA, whereas series 2 also binds by intercalation. The measured pKa indicated different ionization states at pH 7.4, which correlated with the DNA binding affinities (ΔTm) for series 2 and 3. Compound 3a, which was active and selective against the three parasites and displayed adequate metabolic stability, is a fine candidate for in vivo studies.
Asunto(s)
Antiprotozoarios , Benzamidas , Leishmania donovani , Parásitos , Trypanosoma brucei brucei , Trypanosoma cruzi , Animales , Antiprotozoarios/química , ADN/metabolismo , ADN de Cinetoplasto/metabolismo , Imidazoles/química , Imidazoles/farmacología , Leishmania donovani/metabolismo , Parásitos/efectos de los fármacos , Parásitos/metabolismo , Benzamidas/química , Benzamidas/farmacologíaRESUMEN
Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.
Asunto(s)
ADN Mitocondrial , Trypanosoma brucei brucei , ADN Mitocondrial/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , ADN de Cinetoplasto/genética , ADN de Cinetoplasto/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Protozoarias/metabolismo , Replicación del ADNRESUMEN
Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme, remains enigmatic. De novo modeling of POLIB suggested a structure that is divergent from all other Family A polymerases, in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs RNA substrates and degrades single- and double-stranded DNA nonprocessively. Exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborates the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The enzymatic properties of POLIB suggest this paralog is not a replicative kDNA polymerase, and the noncanonical polymerase domain provides another example of exquisite diversity among DNA polymerases for specialized function.
Asunto(s)
Trypanosoma brucei brucei , ADN de Cinetoplasto/genética , ADN de Cinetoplasto/metabolismo , ADN Polimerasa gamma/metabolismo , Cartilla de ADN/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Exonucleasas/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismoRESUMEN
The protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only known TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Whereas the outer membrane region requires four essential subunits for proper TAC function, the inner membrane integral p166, via its interaction with TAC60 and TAC102, would theoretically suffice to bridge the distance between the OM and the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of additional TAC-associated proteins which loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC. However, binding of full length p166 to these TAC-associated proteins alone would not be sufficient to withstand the mechanical load imposed by the segregating basal bodies.
Asunto(s)
Genoma Mitocondrial , Trypanosoma brucei brucei , ADN de Cinetoplasto/genética , ADN de Cinetoplasto/metabolismo , Flagelos/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS). This process involves respiratory chain complexes and F1Fo-ATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly. IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought.
Asunto(s)
Parásitos , Trypanosoma brucei brucei , Trypanosoma , Tripanosomiasis Africana , Moscas Tse-Tse , Animales , Humanos , Trypanosoma brucei brucei/genética , Moscas Tse-Tse/parasitología , Parásitos/genética , ADN de Cinetoplasto/metabolismo , Fosforilación Oxidativa , Tripanosomiasis Africana/parasitología , Trypanosoma/metabolismo , Mamíferos/metabolismoRESUMEN
In the midst of numerous setbacks that beclouds the fight against leishmaniasis; a neglected tropical disease, the search for new chemotherapeutics against this disease is of utmost importance. Leishmaniasis is a disease closely associated with poverty and endemic in Africa, Asia, southern Europe and the Americas. It is caused by parasites of the genus Leishmania and transmitted by a sandfly vector. In this study, we evaluated the antileishmanial potency of eighteen pathogen box compounds and elucidated their biosafety and possible mechanisms of action against Leishmania donovani promastigotes and amastigotes in vitro. IC50s range of 0.12±0.15 to >6.25 µg/ml and 0.13±0.004 to >6.25µg/ml were observed for the promastigotes and amastigotes, respectively. We demonstrated the ability of some of the compounds to cause cytocidal effect on the parasites, induce increased production of reactive oxygen species (ROS), disrupt the normal parasite morphology and cause the accumulation of parasites at the DNA synthesis phase of the cell cycle. We recommend a further in vivo study on these compounds to validate the findings.
Asunto(s)
Antiprotozoarios/farmacología , Ciclo Celular , Leishmania donovani/citología , Ciclo Celular/efectos de los fármacos , ADN de Cinetoplasto/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , Leishmania donovani/efectos de los fármacos , Leishmania donovani/crecimiento & desarrollo , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosfatidilserinas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of the translocase of the mitochondrial inner membrane 50 (TbTim50). It has a pair of characteristic phosphatase signature motifs, DXDX(T/V). Here, we demonstrated that, besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner. Mutations of D242 and D244, but not of D345and D347, to alanine abolished these activities. In silico structural homology models identified the putative binding interfaces that may accommodate different phosphosubstrates. Interestingly, TbTim50 depletion in the bloodstream form (BF) of T. brucei reduced cardiolipin (CL) levels and decreased mitochondrial membrane potential (ΔΨ). TbTim50 knockdown (KD) also reduced the population of G2/M phase and increased that of G1 phase cells; inhibited segregation and caused overreplication of kinetoplast DNA (kDNA), and reduced BF cell growth. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed with upregulation of expression of a few stumpy marker genes. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice. Proteomics analysis showed reductions in levels of the translation factors, flagellar transport proteins, and many proteasomal subunits, including those of the mitochondrial heat shock locus ATPase (HslVU), which is known to play a role in regulation of kinetoplast DNA (kDNA) replication. Reduction of the level of HslV in TbTim50 KD cells was further validated by immunoblot analysis. Together, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and the cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis. IMPORTANCE African trypanosomiasis is a neglected tropical disease caused by the parasitic protozoan Trypanosoma brucei. During its digenetic life cycle, T. brucei undergoes multiple developmental changes to adapt in different environments. T. brucei BF parasites, dwelling in mammalian blood, produce ATP from glycolysis and hydrolyze ATP in mitochondria for generation of inner membrane potential. We found that TbTim50, a haloacid dehalogenase (HAD) family phosphatase, is critical for T. brucei BF survival in vitro and in vivo. Depletion of TbTim50 in BF reduced levels of CL and mitochondrial ΔΨ and caused a detrimental effect on many cellular functions. Cells accumulated in the G1 phase, and the kinetoplast was overreplicated, likely due to depletion of mitochondrial proteasome (mitochondrial heat shock locus ATPase [HslVU]), a master regulator of kDNA replication. Cell growth inhibition was accompanied by changes in morphology, AMPK phosphorylation, and upregulation of expression of a few stumpy-specific genes. TbTim50 is essential for T. brucei survival and is an important therapeutic target for African trypanosomiasis.
Asunto(s)
Ciclo Celular , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/parasitología , Animales , Línea Celular , ADN de Cinetoplasto/genética , ADN de Cinetoplasto/metabolismo , Ratones , Ratones Endogámicos BALB C , Mitocondrias/metabolismo , Fosforilación , Proteínas Protozoarias/genéticaRESUMEN
The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.
Asunto(s)
ADN de Cinetoplasto/metabolismo , Endorribonucleasas/metabolismo , Euglena/enzimología , Conformación de Ácido Nucleico , Proteínas Protozoarias/metabolismo , Procesamiento Postranscripcional del ARN , ARN Protozoario/metabolismo , Emparejamiento Base , Secuencia de Bases , ADN de Cinetoplasto/química , ADN de Cinetoplasto/genética , Endorribonucleasas/química , Endorribonucleasas/genética , Euglena/genética , Euglena/crecimiento & desarrollo , Kinetoplastida/enzimología , Kinetoplastida/genética , Kinetoplastida/crecimiento & desarrollo , Filogenia , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , ARN Protozoario/química , ARN Protozoario/genéticaRESUMEN
Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.
Asunto(s)
Bacterias/metabolismo , Replicación del ADN , ADN de Cinetoplasto/genética , ADN Protozoario/genética , Mitocondrias/genética , Proteínas Protozoarias/genética , Trypanosomatina/genética , División Celular , Núcleo Celular , ADN de Cinetoplasto/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , ADN Protozoario/metabolismo , Mitocondrias/metabolismo , Proteínas Protozoarias/metabolismo , Simbiosis , Trypanosomatina/metabolismo , Trypanosomatina/microbiologíaRESUMEN
The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.
Asunto(s)
Cuerpos Basales/fisiología , Quinasa de la Caseína I/metabolismo , ADN de Cinetoplasto/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Quinasa de la Caseína I/antagonistas & inhibidores , Quinasa de la Caseína I/genética , Citocinesis/fisiología , Citoplasma/metabolismo , División del ADN , Replicación del ADN , Mitocondrias/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Condensation and remodeling of nuclear genomes play an essential role in the regulation of gene expression and replication. Yet, our understanding of these processes and their regulatory role in other DNA-containing organelles, has been limited. This study focuses on the packaging of kinetoplast DNA (kDNA), the mitochondrial genome of kinetoplastids. Severe tropical diseases, affecting large human populations and livestock, are caused by pathogenic species of this group of protists. kDNA consists of several thousand DNA minicircles and several dozen DNA maxicircles that are linked topologically into a remarkable DNA network, which is condensed into a mitochondrial nucleoid. In vitro analyses implicated the replication protein UMSBP in the decondensation of kDNA, which enables the initiation of kDNA replication. Here, we monitored the condensation of kDNA, using fluorescence and atomic force microscopy. Analysis of condensation intermediates revealed that kDNA condensation proceeds via sequential hierarchical steps, where multiple interconnected local condensation foci are generated and further assemble into higher order condensation centers, leading to complete condensation of the network. This process is also affected by the maxicircles component of kDNA. The structure of condensing kDNA intermediates sheds light on the structural organization of the condensed kDNA network within the mitochondrial nucleoid.
Asunto(s)
Replicación del ADN/genética , ADN de Cinetoplasto/metabolismo , ADN Mitocondrial/genética , Núcleo Celular/metabolismo , Crithidia fasciculata/genética , ADN/metabolismo , ADN Circular/metabolismo , ADN de Cinetoplasto/genética , Proteínas de Unión al ADN/genética , Genoma Mitocondrial/genética , Mitocondrias/metabolismoRESUMEN
Trypanosoma brucei is a single celled eukaryotic parasite and the causative agent of human African trypanosomiasis and nagana in cattle. Aside from its medical relevance, T. brucei has also been key to the discovery of several general biological principles including GPI-anchoring, RNA-editing and trans-splicing. The parasite contains a single mitochondrion with a singular genome. Recent studies have identified several molecular components of the mitochondrial genome segregation machinery (tripartite attachment complex, TAC), which connects the basal body of the flagellum to the mitochondrial DNA of T. brucei. The TAC component in closest proximity to the mitochondrial DNA is TAC102. Here we apply and compare three different approaches (proximity labelling, immunoprecipitation and yeast two-hybrid) to identify novel interactors of TAC102 and subsequently verify their localisation. Furthermore, we establish the direct interaction of TAC102 and p166 in the unilateral filaments of the TAC.
Asunto(s)
ADN Mitocondrial/genética , Inmunoprecipitación , Proteínas Protozoarias/genética , Técnicas del Sistema de Dos Híbridos , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , ADN de Cinetoplasto/metabolismo , Flagelos/metabolismo , Genoma Mitocondrial/genética , Humanos , Mitocondrias/genética , Mitocondrias/fisiología , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/veterinariaRESUMEN
Conserved amongst all eukaryotes is a family of mitochondrial carrier proteins (SLC25A) responsible for the import of various solutes across the inner mitochondrial membrane. We previously reported that the human parasite Trypanosoma brucei possesses 26 SLC25A proteins (TbMCPs) amongst which two, TbMCP11 and TbMCP8, were predicted to function as phosphate importers. The transport of inorganic phosphate into the mitochondrion is a prerequisite to drive ATP synthesis by substrate level and oxidative phosphorylation and thus crucial for cell viability. In this paper we describe the functional characterization of TbMCP11. In procyclic form T. brucei, the RNAi of TbMCP11 blocked ATP synthesis on mitochondrial substrates, caused a drop of the mitochondrial oxygen consumption and drastically reduced cell viability. The functional complementation in yeast and mitochondrial swelling experiments suggested a role for TbMCP11 as inorganic phosphate carrier. Interestingly, procyclic form T. brucei cells in which TbMCP11 was depleted displayed an inability to either replicate or divide the kinetoplast DNA, which resulted in a severe cytokinesis defect.
Asunto(s)
Estadios del Ciclo de Vida/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas de Transporte de Fosfato/genética , Fosfatos/metabolismo , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Adenosina Trifosfato/biosíntesis , Supervivencia Celular , Citocinesis , ADN de Cinetoplasto/genética , ADN de Cinetoplasto/metabolismo , Prueba de Complementación Genética , Transporte Iónico , Mitocondrias/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Proteínas de Transporte de Fosfato/antagonistas & inhibidores , Proteínas de Transporte de Fosfato/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
Fluorescence microscopy enables the localization of proteins to specific structures within a cell which have either been fused to a fluorescence protein or detected by immunofluorescence. Here, we describe the various procedures that can be used to prepare both the procyclic form and bloodstream form of the human pathogen Trypanosoma brucei for fluorescence microscopy. The choice of procedure to be used is determined by various parameters, including protein characteristics and the scientific question being investigated.
Asunto(s)
Microscopía Intravital/métodos , Proteínas Luminiscentes/química , Parasitología/métodos , Trypanosoma brucei brucei/citología , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , ADN de Cinetoplasto/metabolismo , Estadios del Ciclo de Vida , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Trypanosoma brucei brucei/metabolismoRESUMEN
Base J, ß-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids.
Asunto(s)
ADN de Cinetoplasto/metabolismo , Proteínas Protozoarias/metabolismo , ARN Polimerasa II/metabolismo , Terminación de la Transcripción Genética , Trypanosoma brucei brucei/fisiología , Animales , ADN de Cinetoplasto/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Protozoarios , Glucósidos/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Histonas/genética , Histonas/metabolismo , Leishmania/fisiología , Mutación , Proteínas Protozoarias/genética , Interferencia de ARN , ARN Polimerasa II/genética , Timina/metabolismo , Uracilo/análogos & derivados , Uracilo/metabolismoRESUMEN
Trypanosomes have complex life cycles within which there are both proliferative and differentiation cell divisions. The coordination of the cell cycle to achieve these different divisions is critical for the parasite to infect both host and vector. From studying the regulation of the proliferative cell cycle of the Trypanosoma brucei procyclic life cycle stage, three subcycles emerge that control the duplication and segregation of (a) the nucleus, (b) the kinetoplast, and (c) a set of cytoskeletal structures. We discuss how the clear dependency relationships within these subcycles, and the potential for cross talk between them, are likely required for overall cell cycle coordination. Finally, we look at the implications this interdependence has for proliferative and differentiation divisions through the T. brucei life cycle and in related parasitic trypanosomatid species.
Asunto(s)
Ciclo Celular , Trypanosoma brucei brucei/crecimiento & desarrollo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , ADN de Cinetoplasto/metabolismo , ADN Protozoario/metabolismo , Regulación de la Expresión GénicaAsunto(s)
Chlamydomonas/enzimología , Chlamydomonas/genética , Dioxigenasas/genética , Dioxigenasas/metabolismo , Familia de Multigenes , Catálisis , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN de Cinetoplasto/genética , ADN de Cinetoplasto/metabolismo , Células Eucariotas/enzimología , Evolución Molecular , Interacciones Huésped-Patógeno/genética , Familia de Multigenes/fisiología , Especificidad por SustratoRESUMEN
Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis, the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38â¯C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95-235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization.
Asunto(s)
ADN de Cinetoplasto/metabolismo , ADN Mitocondrial/metabolismo , Leishmania/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/metabolismo , Telómero/metabolismo , Unión ProteicaRESUMEN
Trypanosoma (Herpetosoma) lewisi is a globally distributed rat trypanosome, currently considered as a zoonotic pathogen; however, a detailed understanding of the morphological events occurring during the cell cycle is lacking. This study aimed to investigate the cell cycle morphology and cleavage events of Trypanosoma lewisi (T. lewisi) during in vitro cultivation. By establishing in vitro cultivation of T. lewisi at 37°C, various cell morphologies and stages could be observed. We have provided a quantitative analysis of the morphological events during T. lewisi proliferation. We confirmed a generation time of 12.14 ± 0.79 hours, which is similar to that in vivo (12.21 ± 0.14 hours). We also found that there are two distinct cell cycles, with a two-way transformation connection in the developmental status of this parasite, which was contrasted with the previous model of multiple division patterns seen in T. lewisi. We quantified the timing of cell cycle phases (G1n, 0.56 U; Sn, 0.14 U; G2n, 0.16 U; M, 0.06 U; C, 0.08 U; G1k, 0.65 U; Sk, 0.10 U; G2k, 0.17 U; D, 0.03 U; A, 0.05 U) and their morphological characteristics, particularly with respect to the position of kinetoplast(s) and nucleus/nuclei. Interestingly, we found that both nuclear synthesis initiation and segregation in T. lewisi occurred prior to kinetoplast, different to the order of replication found in Trypanosoma brucei and Trypanosoma cruzi, implicating a distinct cell cycle control mechanism in T. lewisi. We characterized the morphological events during the T. lewisi cell cycle and presented evidence to support the existence of two distinct cell cycles with two-way transformation between them. These results provide insights into the differentiation and evolution of this parasite and its related species.
Asunto(s)
Trypanosoma lewisi/fisiología , Animales , Ciclo Celular , ADN de Cinetoplasto/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/parasitología , Microscopía Electrónica de Rastreo , Ratas , Ratas Sprague-Dawley , Imagen de Lapso de Tiempo , Trypanosoma lewisi/crecimiento & desarrolloRESUMEN
Trypanosoma brucei has a unique catenated mitochondrial DNA (mtDNA) network called kinetoplast DNA (kDNA). Replication of kDNA occurs once per cell cycle in near synchrony with nuclear S phase and requires the coordination of many proteins. Among these are three essential DNA polymerases (TbPOLIB, IC, and ID). Localization dynamics of these proteins with respect to kDNA replication stages and how they coordinate their functions during replication are not well understood. We previously demonstrated that TbPOLID undergoes dynamic localization changes that are coupled to kDNA replication events. Here, we report the localization of TbPOLIC, a second essential DNA polymerase, and demonstrate the accumulation of TbPOLIC foci at active kDNA replication sites (antipodal sites) during stage II of the kDNA duplication cycle. While TbPOLIC was undetectable by immunofluorescence during other cell cycle stages, steady-state protein levels measured by Western blot remained constant. TbPOLIC foci colocalized with the fraction of TbPOLID that localized to the antipodal sites. However, the partial colocalization of the two essential DNA polymerases suggests a highly dynamic environment at the antipodal sites to coordinate the trafficking of replication proteins during kDNA synthesis. These data indicate that cell cycle-dependent localization is a major regulatory mechanism for essential mtDNA polymerases during kDNA replication.