RESUMEN
Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.
Asunto(s)
Parásitos , Plasmodium , Masculino , Animales , Axonema/metabolismo , Parásitos/metabolismo , Microtúbulos/metabolismo , Semillas , Proteínas/metabolismo , Flagelos/metabolismo , Eucariontes/metabolismo , Plasmodium/metabolismo , Dineínas/metabolismoRESUMEN
The ribosome is the universally conserved ribozyme that translates DNA coded instructions into proteins with the assistance of other RNA molecules, including transfer and messenger RNAs. Of particular interest is the segmentation phenomena, which is found in trypanosomatids and other protists. In these organisms, the large subunit ribosomal RNA is assembled from multiple smaller RNAs. This phenomenon posits several challenges to the folding and stabilization of such ribosomes to retain functionality and efficiency. In earlier studies, RNA/protein interactions were suggested to fully compensate for the fragmentation. Recently, several conserved RNA/RNA interaction regions were described in the cryo-EM structures of segmented ribosomes from trypanosomatids. These regions also seemed to aid in the folding and stabilization of such ribosomes, even before the ribosomal proteins start their association. In the present study, the existence of conserved RNA/RNA interaction regions shared between trypanosomatid and Euglena gracilis segmented ribosomes was confirmed, despite differences in segmentation patterns. Analysis of the crystallographic structures of unsegmented ribosomes from other Eukaryotes, Bacteria, and Archaea allowed us to estimate the relative age of highly conserved RNA/RNA interaction regions. These results strongly suggest that common interaction regions likely date far back into the ribosomes of the last common ancestor. Results also revealed that single hydrogen bonds are overwhelmingly facilitated by the 2'OH, a distinctive RNA feature. This supports the notion that RNA predates DNA and places some constraints on alternative nucleic acids proposals.
Asunto(s)
ARN , Ribosomas , ARN/metabolismo , Ribosomas/metabolismo , ARN Ribosómico/genética , Proteínas Ribosómicas/metabolismo , ARN Mensajero/metabolismoRESUMEN
Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.
Asunto(s)
División Celular , Microtúbulos , Simbiosis , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Microtúbulos/efectos de los fármacos , Trypanosomatina/genética , Trypanosomatina/metabolismo , Trypanosomatina/ultraestructura , Trypanosomatina/fisiología , Ácidos Hidroxámicos/farmacología , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Bacterias/metabolismo , Bacterias/genética , Acetilación , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasa 6/metabolismo , Histona Desacetilasa 6/genética , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructuraRESUMEN
Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.
RESUMEN
Trypanosomatids are obligatory parasites, some of which are responsible for important human and animal diseases, but the vast majority of trypanosomatids are restricted to invertebrate hosts. Isolation and in vitro cultivation of trypanosomatids from insect hosts enable their description, characterization, and subsequently genetic and genomic studies. However, exact nutritional requirements are still unknown for most trypanosomatids and thus very few defined media are available. This mini review provides information about the role of different ingredients, recommendations and advice on essential supplements and important physicochemical parameters of culture media with the aim of facilitating first attempts to cultivate insect-infesting trypanosomatids, with a focus on monoxenous trypanosomatids.
Asunto(s)
Trypanosomatina , Animales , Humanos , Trypanosomatina/genética , Insectos/parasitologíaRESUMEN
Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.
Asunto(s)
Amida Sintasas , Glutatión , NADH NADPH Oxidorreductasas , Trypanosoma , NADH NADPH Oxidorreductasas/metabolismo , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Humanos , Amida Sintasas/metabolismo , Amida Sintasas/antagonistas & inhibidores , Trypanosoma/efectos de los fármacos , Trypanosoma/metabolismo , Glutatión/metabolismo , Glutatión/análogos & derivados , Animales , Espermidina/análogos & derivados , Espermidina/metabolismo , Leishmania/efectos de los fármacos , Leishmania/metabolismo , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/metabolismo , Leishmaniasis/parasitología , Trypanosomatina/metabolismo , Trypanosomatina/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/metabolismoRESUMEN
BACKGROUND: Protists of the family Trypanosomatidae (phylum Euglenozoa) have gained notoriety as parasites affecting humans, domestic animals, and agricultural plants. However, the true extent of the group's diversity spreads far beyond the medically and veterinary relevant species. We address several knowledge gaps in trypanosomatid research by undertaking sequencing, assembly, and analysis of genomes from previously overlooked representatives of this protistan group. RESULTS: We assembled genomes for twenty-one trypanosomatid species, with a primary focus on insect parasites and Trypanosoma spp. parasitizing non-human hosts. The assemblies exhibit sizes consistent with previously sequenced trypanosomatid genomes, ranging from approximately 18 Mb for Obscuromonas modryi to 35 Mb for Crithidia brevicula and Zelonia costaricensis. Despite being the smallest, the genome of O. modryi has the highest content of repetitive elements, contributing nearly half of its total size. Conversely, the highest proportion of unique DNA is found in the genomes of Wallacemonas spp., with repeats accounting for less than 8% of the assembly length. The majority of examined species exhibit varying degrees of aneuploidy, with trisomy being the most frequently observed condition after disomy. CONCLUSIONS: The genome of Obscuromonas modryi represents a very unusual, if not unique, example of evolution driven by two antidromous forces: i) increasing dependence on the host leading to genomic shrinkage and ii) expansion of repeats causing genome enlargement. The observed variation in somy within and between trypanosomatid genera suggests that these flagellates are largely predisposed to aneuploidy and, apparently, exploit it to gain a fitness advantage. High heterogeneity in the genome size, repeat content, and variation in chromosome copy numbers in the newly-sequenced species highlight the remarkable genome plasticity exhibited by trypanosomatid flagellates. These new genome assemblies are a robust foundation for future research on the genetic basis of life cycle changes and adaptation to different hosts in the family Trypanosomatidae.
Asunto(s)
Trypanosomatina , Animales , Trypanosomatina/genética , Tamaño del Genoma , Aclimatación , Agricultura , AneuploidiaRESUMEN
Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as "lipoyl-relay", requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.
Asunto(s)
Redes y Vías Metabólicas , Ácido Tióctico , Trypanosoma brucei brucei , Bacillus subtilis/metabolismo , Ligasas/metabolismo , Redes y Vías Metabólicas/genética , Saccharomyces cerevisiae/metabolismo , Ácido Tióctico/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
G-quadruplexes (G4s) are nucleic acid secondary structures that have been linked to the functional regulation of eukaryotic organisms. G4s have been extensively characterised in humans and emerging evidence suggests that they might also be biologically relevant for human pathogens. This indicates that G4s might represent a novel class of therapeutic targets for tackling infectious diseases. Bioinformatic studies revealed a high prevalence of putative quadruplex-forming sequences (PQSs) in the genome of protozoans, which highlights their potential roles in regulating vital processes of these parasites, including DNA transcription and replication. In this work, we focus on the neglected trypanosomatid parasites, Trypanosoma and Leishmania spp., which cause debilitating and deadly diseases across the poorest populations worldwide. We review three examples where G4-formation might be key to modulate transcriptional activity in trypanosomatids, providing an overview of experimental approaches that can be used to exploit the regulatory roles and relevance of these structures to fight parasitic infections.
Asunto(s)
G-Cuádruplex , Parásitos , Trypanosoma , Animales , Humanos , Parásitos/genética , Trypanosoma/genética , ADN/química , GenomaRESUMEN
Trypanosomatids form a group of high prevalence protozoa that parasitise honey bees, with Lotmaria passim as the predominant species worldwide. However, the knowledge about the ecology of trypanosomatids in isolated areas is limited. The Portuguese archipelagos of Madeira and Azores provide an interesting setting to investigate these parasites because of their geographic isolation, and because they harbour honey bee populations devoid of two major enemies: Varroa destructor and Nosema ceranae. Hence, a total of 661 honey bee colonies from Madeira and the Azores were analysed using different molecular techniques, through which we found a high prevalence of trypanosomatids despite the isolation of these islands. L. passim was the predominant species and, in most colonies, was the only one found, even on islands free of V. destructor and/or N. ceranae with severe restrictions on colony movements to prevent the spread of them. However, islands with V. destructor had a significantly higher prevalence of L. passim and, conversely, islands with N. ceranae did not shown any significant correlation with the trypanosomatid. Crithidia bombi was detected in Madeira and on three islands of the Azores, almost always coincident with L. passim. By contrast, Crithidia mellificae was not detected in any sample. A high-throughput sequencing analysis distinguished two main haplotypes of L. passim, which accounted for 98% of the total sequence reads. This work suggests that L. passim and C. bombi are parasites that have been associated with honey bees predating the spread of V. destructor and N. ceranae.
Asunto(s)
Apicultura , Trypanosomatina , Animales , Abejas , Trypanosomatina/genética , Trypanosomatina/parasitología , Crithidia/genética , Crithidia/parasitología , Simbiosis , AzoresRESUMEN
A series of nine novel ether phospholipid-dinitroaniline hybrids were synthesized in an effort to deliver more potent antiparasitic agents with improved safety profile compared to miltefosine. The compounds were evaluated for their in vitro antiparasitic activity against L. infantum, L.donovani, L. amazonensis, L. major and L. tropica promastigotes, L. infantum and L. donovani intracellular amastigotes, Trypanosoma brucei brucei and against different developmental stages of Trypanosoma cruzi. The nature of the oligomethylene spacer between the dinitroaniline moiety and the phosphate group, the length of the side chain substituent on the dinitroaniline and the choline or homocholine head group were found to affect both the activity and toxicity of the hybrids. The early ADMET profile of the derivatives did not reveal major liabilities. Hybrid 3, bearing an 11-carbon oligomethylene spacer, a butyl side chain and a choline head group, was the most potent analogue of the series. It exhibited a broad spectrum antiparasitic profile against the promastigotes of New and Old World Leishmania spp., against intracellular amastigotes of two L. infantum strains and L. donovani, against T. brucei and against T. cruzi Y strain epimastigotes, intracellular amastigotes and trypomastigotes. The early toxicity studies revealed that hybrid 3 showed a safe toxicological profile while its cytotoxicity concentration (CC50) against THP-1 macrophages being >100 µM. Computational analysis of binding sites and docking indicated that the interaction of hybrid 3 with trypanosomatid α-tubulin may contribute to its mechanism of action. Furthermore, compound 3 was found to interfere with the cell cycle in T. cruzi epimastigotes, while ultrastructural studies using SEM and TEM in T. cruzi showed that compound 3 affects cellular processes that result in changes in the Golgi complex, the mitochondria and the parasite's plasma membrane. The snapshot pharmacokinetic studies showed low levels of 3 after 24 h following oral administration of 100 mg/Kg, while, its homocholine congener compound 9 presented a better pharmacokinetic profile.
Asunto(s)
Antiprotozoarios , Enfermedad de Chagas , Trypanosoma cruzi , Humanos , Antiparasitarios/farmacología , Antiprotozoarios/farmacología , Éteres Fosfolípidos/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Colina/uso terapéuticoRESUMEN
The endoplasmic reticulum (ER) presents unique properties to establishing bacterium symbiosis in eukaryotic cells since it synthesizes and glycosylates essential molecules like proteins and lipids. Tunicamycin (TM) is an antibiotic that inhibits the first step in the N-linked glycosylation in eukaryotes and has been used as an ER stress inducer to activate the Unfolded Protein Response (UPR). Mutualistic symbiosis in trypanosomatids is characterized by structural adaptations and intense metabolic exchanges, thus we investigated the effects of TM in the association between Angomonas deanei and its symbiotic bacterium, through ultrastructural and proteomic approaches. Cells treated with the inhibitor showed a decrease in proliferation, enlargement of the ER and Golgi cisternae and an increased distance between the symbiont and the ER. TM proved to be an important tool to better understand ER stress in trypanosomatids, since changes in protein composition were observed in the host protozoan, especially the expression of the Hsp90 chaperone. Furthermore, data obtained indicates the importance of the ER for the adaptation and maintenance of symbiotic associations between prokaryotes and eukaryotes, considering that this organelle has recognized importance in the biogenesis and division of cell structures.
Asunto(s)
Proteínas de Choque Térmico , Trypanosomatina , Bacterias , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Proteínas de Choque Térmico/metabolismo , Proteómica , Trypanosomatina/metabolismo , Trypanosomatina/microbiología , Tunicamicina/farmacologíaRESUMEN
Bee trypanosomatids have not been widely studied due to the original belief that these organisms were not pathogenic to honey bees. However, trypanosomatids have been linked to increased winter mortality in honey bee colonies in recent years and it has been shown that these pathogens can shorten a honey bee worker's lifespan in laboratory conditions. These studies found that this mortality corresponded to dose-dependent infection. Although Lotmaria passim is the most prevalent species worldwide, the natural load in colonies remains poorly investigated. Here we describe a new highly specific and sensitive qPCR method that allows the differentiation and quantification of the parasitic load of each of the three most common trypanosomatid species described to date in honey bee colonies: L. passim, Crithidia mellificae, and Crithidia bombi. We have used this new method to analyze honey bee colonies in central Spain and confirm that L. passim is the most common species and the one with higher parasitic loads in the colonies, which increased over the years, being higher in spring than in autumn. Crithidia mellificae was present along the study, with the highest prevalence in autumn 2019 and lately it was only found in non-quantifiable loads. Crithidia bombi was not detected in any of the colonies analyzed.
Asunto(s)
Crithidia , Trypanosomatina , Abejas , Animales , Crithidia/parasitología , España , Trypanosomatina/genética , Trypanosomatina/parasitologíaRESUMEN
Fatty acids have received growing interest in Leishmania biology with the characterization of the enzymes allowing the complete fatty acid synthesis of this trypanosomatid parasite. This review presents a comparative analysis of the fatty acid profiles of the major classes of lipids and phospholipids in different species of Leishmania with cutaneous or visceral tropism. Specificities relating to the parasite forms, resistance to antileishmanial drugs, and host/parasite interactions are described as well as comparisons with other trypanosomatids. Emphasis is placed on polyunsaturated fatty acids and their metabolic and functional specificities, in particular, their conversion into oxygenated metabolites that are inflammatory mediators able to modulate metacyclogenesis and parasite infectivity. The impact of lipid status on the development of leishmaniasis and the potential of fatty acids as therapeutic targets or candidates for nutritional interventions are discussed.
Asunto(s)
Leishmania , Leishmaniasis , Parásitos , Animales , Ácidos Grasos , BiomarcadoresRESUMEN
Trypanosoma and Leishmania parasites cause devastating tropical diseases resulting in serious global health consequences. These organisms have complex life cycles with mammalian hosts and insect vectors. The parasites must, therefore, survive in different environments, demanding rapid physiological and metabolic changes. These responses depend upon regulation of gene expression, which primarily occurs posttranscriptionally. Altering the composition or conformation of RNA through nucleotide modifications is one posttranscriptional mechanism of regulating RNA fate and function, and modifications including N6-methyladenosine (m6A), N1-methyladenosine (m1A), N5-methylcytidine (m5C), N4-acetylcytidine (ac4C), and pseudouridine (Ψ), dynamically regulate RNA stability and translation in diverse organisms. Little is known about RNA modifications and their machinery in Trypanosomatids, but we hypothesize that they regulate parasite gene expression and are vital for survival. Here, we identified Trypanosomatid homologs for writers of m1A, m5C, ac4C, and Ψ and analyze their evolutionary relationships. We systematically review the evidence for their functions and assess their potential use as therapeutic targets. This work provides new insights into the roles of these proteins in Trypanosomatid parasite biology and treatment of the diseases they cause and illustrates that Trypanosomatids provide an excellent model system to study RNA modifications, their molecular, cellular, and biological consequences, and their regulation and interplay.
Asunto(s)
Transcriptoma , Trypanosoma/genética , Tripanosomiasis/parasitología , Animales , Epigenómica , Humanos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Procesamiento Postranscripcional del ARN , ARN Protozoario/genética , ARN Protozoario/metabolismo , Trypanosoma/enzimología , Trypanosoma/metabolismoRESUMEN
Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. The survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here, we report the development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis. As proof of concept, we screened a diverse set of â¼14,000 small molecules and exemplify a validated hit as a novel kDNA-targeting compound.
Asunto(s)
Trypanosoma brucei brucei , Trypanosoma , ADN de Cinetoplasto/genética , ADN Mitocondrial/genética , Humanos , Mitocondrias/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Translation initiation is the first step in three essential processes leading to protein synthesis. It is carried out by proteins called translation initiation factors and ribosomes on the mRNA. One of the critical translation initiation factors in eukaryotes is eIF4G which is a scaffold protein that helps assemble translation initiation complexes that carry out translation initiation which ultimately leads to polypeptide synthesis. Trypanosomatids are a large family of kinetoplastids, some of which are protozoan parasites that cause diseases in humans through transmission by vectors. While the protein translation mechanisms in eukaryotes and prokaryotes are well understood, the protein translation factors and mechanisms in trypanosomatids are poorly understood necessitating further studies. Unlike other eukaryotes, trypanosomatids contain five eIF4G orthologues with diversity in length and sequences. Here, I have used bioinformatics tools to look at trypanosomatid keIF4G orthologue sequences and report that there are similarities and considerable differences in their domains/motifs organization and signature amino acid sequences that are required for different functions as compared to human eIF4G. My analysis suggests that there is likely to be considerable diversity and complexity in trypanosomatid keIF4G functions as compared to other eukaryotes.
Asunto(s)
Factor 4G Eucariótico de Iniciación , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Factor 4G Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Humanos , ARN Mensajero/metabolismoRESUMEN
Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.
Asunto(s)
Técnicas de Inactivación de Genes/métodos , Leishmania/genética , Sistemas CRISPR-Cas , Línea Celular , Edición Génica , Integrasas , Proteínas Proto-Oncogénicas c-crk/genética , Recombinación Genética , TransfecciónRESUMEN
Protein translation leading to polypeptide synthesis involves three distinct events, namely, initiation, elongation, and termination. Translation initiation is a multi-step process that is carried out by ribosomes on the mRNA with the assistance of a large number of proteins called translation initiation factors. Trypanosomatids are kinetoplastidas (flagellated protozoans), some of which cause acute disease syndromes in humans. Vector-borne transmission of protozoan parasites like Leishmania and Trypanosoma causes diseases that affect a large section of the world population and lead to significant morbidity and mortality. The mechanisms of translation initiation in higher eukaryotes are relatively well understood. However, structural and functional conservation of initiation factors in trypanosomatids are only beginning to be understood. Studies carried out so far suggests that at least in Leishmania and Trypanosoma eIF4E function may not be restricted to canonical translation initiation and some of the homologues may have alternate/non-canonical functions. Nonetheless, all of them bind the cap analogs, albeit with different efficiencies, indicating that this property may play an important role in the functionality of eIF4Es. Here, I give a brief background of trypanosomatid eIF4Es and revisit the cap-binding signatures of eIF4E orthologues in trypanosomatids, whose genome sequences are available, in detail, in comparison to human eIF4E1 and Trypanosoma cruzi eIF4E5, with an expanded list of members of this group in light of newer findings. The group 1 and 2 eIF4Es may use either a variation of heIF4E1 or T. cruzi eIF4E5 cap-4-binding signatures, while eIF4E5 and eIF4E6 use distinct amino acid contacts.
Asunto(s)
Factor 4E Eucariótico de Iniciación/clasificación , Factor 4E Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Trypanosomatina/metabolismo , Secuencia de Aminoácidos , Factor 4E Eucariótico de Iniciación/genética , Humanos , Unión Proteica , ARN Mensajero/genética , Alineación de Secuencia , Trypanosomatina/genéticaRESUMEN
Complex I (NADH dehydrogenase) is the first enzyme in the respiratory chain. It catalyses the electron transfer from NADH to ubiquinone that is associated with proton pumping out of the matrix. In this study, we characterized NADH dehydrogenase activity in seven monoxenous trypanosomatid species: Blechomonas ayalai, Herpetomonas tarakana, Kentomonas sorsogonicus, Leptomonas seymouri, Novymonas esmeraldas, Sergeia podlipaevi and Wallacemonas raviniae. We also investigated the subunit composition of the complex I in dixenous Phytomonas serpens, in which its presence and activity have been previously documented. In addition to P. serpens, the complex I is functionally active in N. esmeraldas and S. podlipaevi. We also identified 24-32 subunits of the complex I in individual species by using mass spectrometry. Among them, for the first time, we recognized several proteins of the mitochondrial DNA origin.