RESUMEN

This study focuses on the biological impacts of deleting the telomerase RNA from Leishmania major (LeishTER), a parasite responsible for causing leishmaniases, for which no effective treatment or prevention is available. TER is a critical player in the telomerase ribonucleoprotein complex, containing the template sequence copied by the reverse transcriptase component during telomere elongation. The success of knocking out both LeishTER alleles was confirmed, and no off-targets were detected. LmTER-/- cells share similar characteristics with other TER-depleted eukaryotes, such as altered growth patterns and partial G0/G1 cell cycle arrest in early passages, telomere shortening, and elevated TERRA expression. They also exhibit increased γH2A phosphorylation, suggesting that the loss of LeishTER induces DNA damage signaling. Moreover, pro-survival autophagic signals and mitochondrion alterations were shown without any detectable plasma membrane modifications. LmTER-/- retained the ability to transform into metacyclics, but their infectivity capacity was compromised. Furthermore, the overexpression of LeishTER was also deleterious, inducing a dominant negative effect that led to telomere shortening and growth impairments. These findings highlight TER's vital role in parasite homeostasis, opening discussions about its potential as a drug target candidate against Leishmania.

Asunto(s)

Proliferación Celular , Leishmania major , ARN , Telomerasa , Leishmania major/genética , Leishmania major/patogenicidad , Telomerasa/genética , Telomerasa/metabolismo , ARN/genética , ARN/metabolismo , Animales , Técnicas de Inactivación de Genes , Telómero/metabolismo , Telómero/genética

RESUMEN

Maf1, originally described as a repressor of RNA polymerase III (RNAP III) transcription in yeast, participates in multiple functions across eukaryotes. However, the knowledge about Maf1 in protozoan parasites is scarce. To initiate the study of Maf1 in Leishmania major, we generated a cell line that overexpresses this protein. Overexpression of Maf1 led to a significant reduction in the abundance of tRNAs, 5S rRNA, and U4 snRNA, demonstrating that Maf1 regulates RNAP III activity in L. major. To further explore the roles played by Maf1 in this microorganism, global transcriptomic and proteomic changes due to Maf1 overexpression were determined using RNA-sequencing and label-free quantitative mass spectrometry. Compared to wild-type cells, differential expression was observed for 1082 transcripts (615 down-regulated and 467 up-regulated) and 205 proteins (132 down-regulated and 73 up-regulated) in the overexpressing cells. A correlation of 44% was found between transcriptomic and proteomic results. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the differentially expressed genes and proteins are mainly involved in transcription, cell cycle regulation, lipid metabolism and transport, ribosomal biogenesis, carbohydrate metabolism, autophagy, and cytoskeleton modification. Thus, our results suggest the involvement of Maf1 in the regulation of all these processes in L. major, as reported in other species, indicating that the functions performed by Maf1 were established early in eukaryotic evolution. Notably, our data also suggest the participation of L. major Maf1 in mRNA post-transcriptional control, a role that, to the best of our knowledge, has not been described in other organisms.

Asunto(s)

Leishmania major , Proteoma , Transcriptoma , Leishmania major/metabolismo , Leishmania major/genética , Proteoma/metabolismo , Humanos , ARN Polimerasa III/metabolismo , ARN Polimerasa III/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Regulación de la Expresión Génica

RESUMEN

In the protozoan parasite Leishmania, most genes encoding for ribosomal proteins (RPs) are present as two or more copies in the genome. However, their untranslated regions (UTRs) are predominantly divergent and might be associated with a distinct regulation of the expression of paralogous genes. Herein, we investigated the expression profiles of two RPs (S16 and L13a) encoded by duplicated genes in Leishmania major. The genes encoding for the S16 protein possess identical coding sequences (CDSs) and divergent UTRs, whereas the CDSs of L13a diverge by two amino acids and by their UTRs. Using CRISPR/Cas9 genome editing, we generated knockout (Δ) and endogenously tagged transfectants for each paralog of L13a and S16 genes. Combining tagged and Δ cell lines we found evidence of differential expression of both RPS16 and RPL13a isoforms throughout parasite development, with one isoform consistently more abundant than its respective copy. In addition, compensatory expression was observed for each paralog upon deletion of the corresponding isoform, suggesting functional conservation between these proteins. This differential expression pattern relates to post-translational processes, given compensation occurs at the level of the protein, with no alterations detected at transcript level. Ribosomal profiles for RPL13a indicate a standard behavior for these paralogues suggestive of interaction with heavy RNA-protein complexes, as already reported for other RPs in trypanosomatids. We identified paralog-specific bound to their 3'UTRs which may be influential in regulating paralog expression. In support, we identified conserved cis-elements within the 3'UTRs of RPS16 and RPL13a; cis-elements exclusive to the UTR of the more abundant paralog or to the less abundant ones were identified.

Asunto(s)

Leishmania major , Proteínas Protozoarias , Proteínas Ribosómicas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Leishmania major/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Sistemas CRISPR-Cas , Regulación de la Expresión Génica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo

RESUMEN

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

Asunto(s)

Antiprotozoarios , Leishmania major , Proteínas Protozoarias , Animales , Humanos , Antiprotozoarios/farmacología , Antiprotozoarios/química , Leishmania donovani/enzimología , Leishmania donovani/efectos de los fármacos , Leishmania donovani/genética , Leishmania major/enzimología , Leishmania major/efectos de los fármacos , Leishmania major/genética , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo

RESUMEN

Leishmaniasis is a neglected disease mainly affecting low-income populations. Conventional treatment involves several side effects, is expensive, and, in addition, protozoa can develop resistance. Photodynamic therapy (PDT) is a promising alternative in treating the disease. PDT involves applying light at a specific wavelength to activate a photosensitive compound (photosensitizer, PS), to produce reactive oxygen species (ROS). Curcumin and its photochemical characteristics make it a good candidate for photodynamic therapy. Studies evaluating gene expression can help to understand the molecular events involved in the cell death caused by PDT. In the present study, RNA was extracted from promastigotes from the control and treated groups after applying PDT. RT-qPCR was performed to verify the expression of the putative ATPase beta subunit (ATPS), ATP synthase subunit A (F0F1), argininosuccinate synthase 1 (ASS), ATP-binding cassette subfamily G member 2 (ABCG2), glycoprotein 63 (GP63), superoxide dismutase (FeSODA), and glucose-6-phosphate dehydrogenase (G6PDH) genes (QR). The results suggest that PDT altered the expression of genes that participate in oxidative stress and cell death pathways, such as ATPS, FeSODA, and G6PD. The ATP-F0F1, ASS, and GP63 genes did not have their expression altered. However, it is essential to highlight that other genes may be involved in the molecular mechanisms of oxidative stress and, consequently, in the death of parasites.

Asunto(s)

Curcumina , Leishmania major , Fotoquimioterapia , Curcumina/farmacología , Fotoquimioterapia/métodos , Leishmania major/genética , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéutico , Adenosina Trifosfato , Línea Celular Tumoral

RESUMEN

Sphingolipids (SLs) are essential components of all eukaryotic cellular membranes. In fungi, plants and many protozoa, the primary SL is inositol-phosphorylceramide (IPC). Trypanosoma cruzi is a protozoan parasite that causes Chagas disease (CD), a chronic illness for which no vaccines or effective treatments are available. IPC synthase (IPCS) has been considered an ideal target enzyme for drug development because phosphoinositol-containing SL is absent in mammalian cells and the enzyme activity has been described in all parasite forms of T. cruzi. Furthermore, IPCS is an integral membrane protein conserved amongst other kinetoplastids, including Leishmania major, for which specific inhibitors have been identified. Using a CRISPR-Cas9 protocol, we generated T. cruzi knockout (KO) mutants in which both alleles of the IPCS gene were disrupted. We demonstrated that the lack of IPCS activity does not affect epimastigote proliferation or its susceptibility to compounds that have been identified as inhibitors of the L. major IPCS. However, disruption of the T. cruzi IPCS gene negatively affected epimastigote differentiation into metacyclic trypomastigotes as well as proliferation of intracellular amastigotes and differentiation of amastigotes into tissue culture-derived trypomastigotes. In accordance with previous studies suggesting that IPC is a membrane component essential for parasite survival in the mammalian host, we showed that T. cruzi IPCS null mutants are unable to establish an infection in vivo, even in immune deficient mice.

Asunto(s)

Enfermedad de Chagas , Leishmania major , Trypanosoma cruzi , Ratones , Animales , Leishmania major/genética , Diferenciación Celular , Inositol/metabolismo , Inositol/farmacología , Mamíferos

RESUMEN

Leishmania major is the causative agent of cutaneous leishmaniasis. It is one of the most studied Leishmania species not only during vector interaction but also in the vertebrate host. Lipophosphoglycan (LPG) is the Leishmania multifunctional virulence factor during host-parasite interaction, whose polymorphisms are involved in the immunopathology of leishmaniasis. Although natural hybrids occur in nature, hybridization of L. major strains in the laboratory was successfully demonstrated. However, LPG expression in the hybrids remains unknown. LPGs from parental (Friedlin, Fn and Seidman, Sd) and hybrids (FnSd3, FnSd4A, FnSd4B, and FnSd6F) were extracted, purified, and their repeat units analyzed by immunoblotting and fluorophore-assisted carbohydrate electrophoresis. Parental strains have distinct profiles in LPG expression, and a mixed profile was observed for all hybrids. Variable levels of NO production by macrophages were detected after LPG exposure (parental and hybrids) and were strain specific.

Asunto(s)

Leishmania major , Leishmaniasis Cutánea , Glicoesfingolípidos/metabolismo , Humanos , Leishmania major/genética , Leishmania major/metabolismo , Leishmaniasis Cutánea/parasitología , Macrófagos/metabolismo

RESUMEN

Leishmania (Leishmania) major is an important agent of cutaneous leishmaniasis, having as a vector sandflies belonging to the genus Phlebotomus. Although this species has been described as restricted to the Old World, parasites similar to L. major have been isolated from South American patients who have never travelled abroad. These parasites were named "L. major-like", and several studies have been carried out to characterise them biochemically, molecularly, and biologically. However, the phylogenetic origin of these isolates is still unknown. In the present study we characterised three L. major-like isolates, named BH49, BH121 and BH129, using comparative genomics approaches. We evaluated the presence of gene and segmental duplications/deletions and the presence of aneuploidies that could explain the differences in infectivity observed in the BH49 and BH121 isolates. All isolates presented a pattern of mosaic aneuploidy and gene copy number variation, which are common in the genus Leishmania. Virulence factors such as phosphatases and peptidases were found to have increased gene copy numbers in the infective isolate, which could explain the difference in infectivity previously observed between BH121 and BH49. Phylogenetic analyses revealed that BH49, BH121 and BH129 L. major-like grouped with L. major isolates, and suggest they were imported from the Old World in at least two independent events. We suggest that new epidemiological inquiries should also evaluate L. major infections in South America, to assess the epidemiological importance of this species in the New World.

Asunto(s)

Leishmania major , Leishmaniasis Cutánea , Animales , Brasil , Variaciones en el Número de Copia de ADN , Genómica , Humanos , Leishmania major/genética , Leishmaniasis Cutánea/epidemiología , Filogenia

RESUMEN

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology.

Asunto(s)

Leishmania major/enzimología , Leishmaniasis Cutánea/patología , Neutrófilos/fisiología , Proteína Metiltransferasas/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Leishmania major/genética , Leishmania major/metabolismo , Leishmaniasis Cutánea/parasitología , Ratones , Proteína Metiltransferasas/genética

RESUMEN

Telomeres from different eukaryotes, including trypanosomatids, are transcribed into TERRA noncoding RNAs, crucial in regulating chromatin deposition and telomere length. TERRA is transcribed from the C-rich subtelomeric strand towards the 3'-ends of the telomeric array. Using bioinformatics, we confirmed the presence of subtelomeric splice acceptor sites at all L. major chromosome ends. Splice leader sequences positioned 5' upstream of L. major chromosomes subtelomeres were then mapped using SL-RNA-Seq libraries constructed from three independent parasite life stages and helped confirm TERRA expression from several chromosomes ends. Northern blots and RT-qPCR validated the results showing that L. major TERRA is processed by trans-splicing and polyadenylation coupled reactions. The number of transcripts varied with the parasite's life stage and continuous passages, being more abundant in the infective forms. However, no putative subtelomeric promoters involved in TERRA's transcriptional regulation were detected. In contrast, the observed changes in parasite's telomere length during development, suggest that differences in telomeric base J levels may control TERRA transcription in L. major. Also, TERRA-R loops' detection, mainly in the infective forms, was suggestive of TERRA's involvement in telomere protection. Therefore, Leishmania TERRA shares conserved features with other eukaryotes and advances new telomere specific functions in a Public Health-impacting parasite.

Asunto(s)

Clonación Molecular/métodos , Perfilación de la Expresión Génica/métodos , Leishmania major/crecimiento & desarrollo , Factores de Transcripción/genética , Bases de Datos Genéticas , Regulación del Desarrollo de la Expresión Génica , Leishmania major/genética , Leishmania major/metabolismo , Poliadenilación , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Empalme del ARN , Análisis de Secuencia de ARN , Telómero/genética , Factores de Transcripción/metabolismo

RESUMEN

Leishmania parasites are the causative agents of a group of neglected tropical diseases known as leishmaniasis. The molecular mechanisms employed by these parasites to adapt to the adverse conditions found in their hosts are not yet completely understood. DNA repair pathways can be used by Leishmania to enable survival in the interior of macrophages, where the parasite is constantly exposed to oxygen reactive species. In higher eukaryotes, DNA repair pathways are coordinated by the central protein kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR). The enzyme Exonuclease-1 (EXO1) plays important roles in DNA replication, repair, and recombination, and it can be regulated by ATM- and ATR-mediated signaling pathways. In this study, the DNA damage response pathways in promastigote forms of L. major were investigated using bioinformatics tools, exposure of lineages to oxidizing agents and radiation damage, treatment of cells with ATM and ATR inhibitors, and flow cytometry analysis. We demonstrated high structural and important residue conservation for the catalytic activity of the putative LmjEXO1. The overexpression of putative LmjEXO1 made L. major cells more susceptible to genotoxic damage, most likely due to the nuclease activity of this enzyme and the occurrence of hyper-resection of DNA strands. These cells could be rescued by the addition of caffeine or a selective ATM inhibitor. In contrast, ATR-specific inhibition made the control cells more susceptible to oxidative damage in an LmjEXO1 overexpression-like manner. We demonstrated that ATR-specific inhibition results in the formation of extended single-stranded DNA, most likely due to EXO1 nucleasic activity. Antagonistically, ATM inhibition prevented single-strand DNA formation, which could explain the survival phenotype of lineages overexpressing LmjEXO1. These results suggest that an ATM homolog in Leishmania could act to promote end resection by putative LmjEXO1, and an ATR homologue could prevent hyper-resection, ensuring adequate repair of the parasite DNA.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , ADN de Cadena Simple , Leishmania major , ADN Protozoario , Humanos , Leishmania major/efectos de los fármacos , Leishmania major/genética , Estrés Oxidativo , Fosforilación

RESUMEN

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR-Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.

Asunto(s)

Sistemas CRISPR-Cas , Endopeptidasa Clp/genética , Proteínas de Choque Térmico/genética , Leishmania braziliensis/genética , Proteínas Protozoarias/genética , Genética Inversa , Endopeptidasa Clp/metabolismo , Edición Génica , Marcación de Gen , Genes Protozoarios , Proteínas de Choque Térmico/metabolismo , Leishmania braziliensis/fisiología , Leishmania major/genética , Leishmania major/fisiología , Mutación , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/metabolismo , Termotolerancia

RESUMEN

Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.

Asunto(s)

Antihelmínticos/farmacología , Antiprotozoarios/farmacología , Brugia Malayi/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas del Helminto/antagonistas & inhibidores , Leishmania major/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antihelmínticos/química , Antiprotozoarios/química , Brugia Malayi/enzimología , Brugia Malayi/genética , Brugia Malayi/crecimiento & desarrollo , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Ensayos Analíticos de Alto Rendimiento , Leishmania major/enzimología , Leishmania major/genética , Leishmania major/crecimiento & desarrollo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Alineación de Secuencia

RESUMEN

Single-celled eukaryote genomes predominantly replicate through multiple origins. Although origin usage during the S-phase has been elucidated in some of these organisms, few studies have comparatively approached this dynamic. Here, we developed a user-friendly website able to calculate the length of the cell cycle phases for any organism. Next, using a formula developed by our group, we showed a comparative analysis among the minimum number of replication origins (MO) required to duplicate an entire chromosome within the S-phase duration in trypanosomatids (Trypanosoma cruzi, Leishmania major, and Trypanosoma brucei) and yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe). Using the data obtained by our analysis, it was possible to predict the MO required in a situation of replication stress. Also, our findings allow establishing a threshold for the number of origins, which serves as a parameter for genome approaches that map origins. Moreover, our data suggest that when compared to yeasts, trypanosomatids use much more origins than the minimum needed. This is the first time a comparative analysis of the minimum number of origins has been successfully applied. These data may provide new insight into the understanding of the replication mechanism and a new methodological framework for studying single-celled eukaryote genomes.

Asunto(s)

Cromosomas/genética , Leishmania major/genética , Origen de Réplica , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/genética , Ciclo Celular , Cromosomas/ultraestructura , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/ultraestructura , Replicación del ADN , ADN de Hongos/genética , ADN Protozoario/genética , Internet , Leishmania major/crecimiento & desarrollo , Especificidad de la Especie , Trypanosoma cruzi/crecimiento & desarrollo

RESUMEN

Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.

Asunto(s)

Leishmania major/genética , ARN Polimerasa III/genética , Factor de Transcripción TFIIIB/genética , Transcripción Genética , Simulación por Computador , Secuencia Conservada/genética , Regulación de la Expresión Génica/genética , Recombinación Homóloga/genética , Proteínas Mutantes/genética , Regiones Promotoras Genéticas , Dominios Proteicos/genética , Subunidades de Proteína/genética , ARN Ribosómico 5S/biosíntesis , ARN Nuclear Pequeño/biosíntesis , ARN de Transferencia/biosíntesis

RESUMEN

The major human pathogens Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major are collectively known as the Tritryps. The initial comparative analysis of their genomes has uncovered that Tritryps share a great number of genes, but repetitive DNA seems to be extremely variable between them. However, the in-depth characterization of repetitive DNA in these pathogens has been in part neglected, mainly due to the well-known technical challenges of studying repetitive sequences from de novo assemblies using short reads. Here, we compared the repetitive DNA repertories between the Tritryps genomes using genome-wide, low-coverage Illumina sequencing coupled to RepeatExplorer analysis. Our work demonstrates that this extensively implemented approach for studying higher eukaryote repeatomes is also useful for protozoan parasites like trypanosomatids, as we recovered previously observed differences in the presence and amount of repetitive DNA families. Additionally, our estimations of repetitive DNA abundance were comparable to those obtained from enhanced-quality assemblies using longer reads. Importantly, our methodology allowed us to describe a previously undescribed transposable element in Leishmania major (TATE element), highlighting its potential to accurately recover distinctive features from poorly characterized repeatomes. Together, our results support the application of this low-cost, low-coverage sequencing approach for the extensive characterization of repetitive DNA evolutionary dynamics in trypanosomatid and other protozoan genomes.

Asunto(s)

Evolución Molecular , Leishmania major/genética , Secuencias Repetitivas de Ácidos Nucleicos , Trypanosoma cruzi/genética

RESUMEN

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9-RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome-wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryotes.

Asunto(s)

Proteínas de Ciclo Celular/genética , Enzimas Reparadoras del ADN/genética , Endonucleasas/genética , Genoma de Protozoos , Leishmania major/genética , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Biología Computacional/métodos , Medios de Cultivo/química , Enzimas Reparadoras del ADN/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Endonucleasas/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Eliminación de Gen , Regulación de la Expresión Génica , Ingeniería Genética , Variación Genética , Inestabilidad Genómica , Histonas/genética , Histonas/metabolismo , Leishmania major/metabolismo , Secuenciación Completa del Genoma

RESUMEN

The N-end rule pathway leads to regulated proteolysis as an adaptive response to external stress and is ubiquitous from bacteria to mammals. In this study, we investigated a gene coding for a putative core enzyme of this post-translational regulatory pathway in Leishmania major, which may be crucial during cytodifferentiation and the environment adaptive responses of the parasite. Leucyl, phenylalanyl-tRNA protein transferase and arginyl-tRNA protein transferase are key components of this pathway in E. coli and eukaryotes, respectively. They catalyze the specific conjugation of leucine, phenylalanine or arginine to proteins containing exposed N-terminal amino acid residues, which are recognized by the machinery for the targeted proteolysis. Here, we characterized a conserved hypothetical protein coded by the LmjF.21.0725 gene in L. major. In silico analysis suggests that the LmjF.21.0725 protein is highly conserved among species of Leishmania and might belong to the Acyl CoA-N-acyltransferases (NAT) superfamily of proteins. Immunofluorescence cell imaging indicates that the cytosolic localization of the studied protein and the endogenous levels of the protein in promastigotes are barely detectable by western blotting assay. The knockout of the two alleles of LmjF.21.0725 by homologous recombination was only possible in the heterozygous transfectant expressing LmjF.21.0725 as a transgene from a plasmid. Moreover, the kinetics of loss of the plasmid in the absence of drug pressure suggests that maintenance of the gene is essential for promastigote survival. Here, evidence is provided that this putative aminoacyl tRNA-protein transferase is essential for parasite survival. The enzyme activity and corresponding post-translational regulatory pathway are yet to be investigated.

Asunto(s)

Aminoaciltransferasas , Leishmania major , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Protozoarias , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Simulación por Computador , Técnicas de Silenciamiento del Gen , Leishmania major/enzimología , Leishmania major/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo

RESUMEN

BACKGROUND: Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. METHODS: All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. RESULTS: Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n = 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n = 48, 53.93%) than L. tropica (n = 4, 4.49%) (Mann-Whitney U test: p < 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. CONCLUSION: L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.

Asunto(s)

Leishmania major/genética , Leishmania tropica/genética , Leishmaniasis Cutánea/parasitología , Animales , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Enfermedades Endémicas , Femenino , Haplotipos , Humanos , Irán , Leishmania major/aislamiento & purificación , Leishmania tropica/aislamiento & purificación , Leishmania tropica/ultraestructura , Leishmaniasis Cutánea/epidemiología , Leishmaniasis Cutánea/patología , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Población Rural

RESUMEN

ABSTRACT Background Leishmania major is a causative agent of zoonotic cutaneous leishmaniasis in the center of Iran, Abarkouh district. Molecular characterization and precise incrimination of Leishmania species was carried out to perform controlling measurements and to design treatment programs for zoonotic cutaneous leishmaniasis. Methods All smears isolated from ulcers of suspected patients were examined under a light microscope and graded for amastigotes frequency. Extraction of DNA, PCR, RFLP and sequencing of ITS-rDNA genotype were done to increase the efficacy of Leishmania parasites identification at their species-specific level and to detect any Leishmania infections within. Results Humans were found to be infected with L. major with high infection frequency and also Leishmania tropica was identified with low occurrence for the first time as non-native species using molecular analyses. The rates of infections was considerable with microscopic observation (n= 65, 73%) out of 89 smears prepared from suspected patients. Molecular analyses showed that the density of L. major was significantly higher (n= 48, 53.93%) than L. tropica (n= 4, 4.49%) (Mann-Whitney U test: p< 0.05) and two samples (2.25%) remained ambiguous after several sequencing. L. major did not have diversity with two common haplotypes but L. tropica were found to exhibit high diversity with three novel haplotypes. Conclusion L. major was considered the causative agent of leishmaniasis in the region, but the identification of a non-native L. tropica revealed the importance of further isolation of Leishmania parasites following molecular analyses and confirmation, and also revealed the importance of further isolation of Leishmania parasites from patients of the field areas who do not have easily access to health care centers for specialized treatment strategies.

Asunto(s)

Humanos , Animales , Masculino , Femenino , Leishmania tropica/genética , Leishmaniasis Cutánea/parasitología , Leishmania major/genética , Población Rural , Haplotipos , Polimorfismo de Longitud del Fragmento de Restricción , Leishmania tropica/aislamiento & purificación , Leishmania tropica/ultraestructura , Reacción en Cadena de la Polimerasa , ADN Protozoario/aislamiento & purificación , ADN Protozoario/genética , Leishmaniasis Cutánea/patología , Leishmaniasis Cutánea/epidemiología , Leishmania major/aislamiento & purificación , Enfermedades Endémicas , Irán