RESUMEN

The subcellular localisation of Rad1, a subunit of the Leishmania major 9-1-1 complex, remains unexplored. Herein, we reveal that Rad1 localises predominantly to the nucleus. Upon hydroxyurea treatment, the diffuse nuclear localisation of Rad1 becomes more punctate, suggesting that Rad1 is responsive to replication stress. Moreover, Rad1 localisation correlates with cell cycle progression. In the majority of G1 to early S-phase cells, Rad1 localises predominantly to the nucleus. As cells progress from late-S phase to mitosis, Rad1 relocalizes to both the nucleus and the cytoplasm in ∼90 % of cells. This pattern of distribution is different from Rad9 and Hus1, which remain nuclear throughout the cell cycle, suggesting Leishmania Rad1 may regulate 9-1-1 activities and/or perform relevant functions outside the 9-1-1 complex.

Asunto(s)

Proteínas de Ciclo Celular , Leishmania major , Proteínas de Ciclo Celular/genética , Leishmania major/metabolismo , Ciclo Celular , Daño del ADN

Asunto(s)

Leishmania , Leishmaniasis , Parásitos , Animales , Humanos , Leishmania/genética , Leishmaniasis/parasitología , Insectos Vectores/parasitología , Simbiosis

RESUMEN

Leishmania are kinetoplastid pathogens that cause leishmaniasis, a debilitating and potentially life-threatening infection if untreated. Unusually, Leishmania regulate their gene expression largely post-transcriptionally due to the arrangement of their coding genes into polycistronic transcription units that may contain 100s of functionally unrelated genes. Yet, Leishmania are capable of rapid and responsive changes in gene expression to challenging environments, often instead correlating with dynamic changes in their genome composition, ranging from chromosome and gene copy number variations to the generation of extrachromosomal DNA and the accumulation of point mutations. Typically, such events indicate genome instability in other eukaryotes, coinciding with genetic abnormalities, but for Leishmania, exploiting these products of genome instability can provide selectable substrates to catalyse necessary gene expression changes by modifying gene copy number. Unorthodox DNA replication, DNA repair, replication stress factors and DNA repeats are recognised in Leishmania as contributors to this intrinsic instability, but how Leishmania regulate genome plasticity to enhance fitness whilst limiting toxic under- or over-expression of co-amplified and co-transcribed genes is unclear. Herein, we focus on fresh, and detailed insights that improve our understanding of genome plasticity in Leishmania. Furthermore, we discuss emerging models and factors that potentially circumvent regulatory issues arising from polycistronic transcription. Lastly, we highlight key gaps in our understanding of Leishmania genome plasticity and discuss future studies to define, in higher resolution, these complex regulatory interactions.

Asunto(s)

Leishmania , Humanos , Leishmania/genética , Variaciones en el Número de Copia de ADN , Plásticos , Inestabilidad Genómica , Expresión Génica

RESUMEN

The subcellular localisation of Rad1, a subunit of the Leishmania major 9-1-1 complex, remains unexplored. Herein, we reveal that Rad1 localises predominantly to the nucleus. Upon hydroxyurea treatment, the diffuse nuclear localisation of Rad1 becomes more punctate, suggesting that Rad1 is responsive to replication stress. Moreover, Rad1 localisation correlates with cell cycle progression. In the majority of G1 to early S-phase cells, Rad1 localises predominantly to the nucleus. As cells progress from late-S phase to mitosis, Rad1 relocalizes to both the nucleus and the cytoplasm in ∼90 % of cells. This pattern of distribution is different from Rad9 and Hus1, which remain nuclear throughout the cell cycle, suggesting Leishmania Rad1 may regulate 9-1-1 activities and/or perform relevant functions outside the 9-1-1 complex.

RESUMEN

The current treatment of leishmaniasis is based on a few drugs that present several drawbacks, such as high toxicity, difficult administration route, and low efficacy. These disadvantages raise the necessity to develop novel antileishmanial compounds allied with a comprehensive understanding of their mechanisms of action. Here, we elucidate the probable mechanism of action of the antileishmanial binuclear cyclopalladated complex [Pd(dmba)(µ-N3)]2 (CP2) in Leishmania amazonensis. CP2 causes oxidative stress in the parasite, resulting in disruption of mitochondrial Ca2+ homeostasis, cell cycle arrest at the S-phase, increasing the reactive oxygen species (ROS) production and overexpression of stress-related and cell detoxification proteins, and collapsing the Leishmania mitochondrial membrane potential, and promotes apoptotic-like features in promastigotes, leading to necrosis, or directs programmed cell death (PCD)-committed cells toward necrotic-like destruction. Moreover, CP2 reduces the parasite load in both liver and spleen in Leishmania infantum-infected hamsters when treated for 15 days with 1.5 mg/kg body weight/day CP2, expanding its potential application in addition to the already known effectiveness on cutaneous leishmaniasis for the treatment of visceral leishmaniasis, showing the broad spectrum of action of this cyclopalladated complex. The data presented here bring new insights into the CP2 molecular mechanisms of action, assisting the promotion of its rational modification to improve both safety and efficacy.

Asunto(s)

Antiprotozoarios , Leishmania infantum , Leishmaniasis Cutánea , Animales , Antiprotozoarios/uso terapéutico , Calcio/metabolismo , Muerte Celular , Leishmaniasis Cutánea/tratamiento farmacológico , Macrófagos , Ratones , Ratones Endogámicos BALB C , Mitocondrias

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

Here we present the establishment of an inducible system based on the dimerizable Cre recombinase (DiCre) for controlled gene expression in the protozoan parasite Leishmania. Rapamycin-induced DiCre activation promoted efficient flipping and expression of gene products in a time and dose-dependent manner. The DiCre flipping activity induced the expression of target genes from both integrated and episomal contexts broadening the applicability of the system. We validated the system by inducing the expression of both full length and truncated forms of the checkpoint protein Rad9, which revealed that the highly divergent C-terminal domain of Rad9 is necessary for proper subcellular localization. Thus, by establishing the DiCre-based inducible system we have created and validated a robust new tool for assessing gene function in Leishmania.

Asunto(s)

Regulación de la Expresión Génica , Ingeniería Genética , Recombinación Homóloga , Integrasas/metabolismo , Leishmania major/genética , Leishmania major/metabolismo , Proteínas de Ciclo Celular/metabolismo , Orden Génico , Vectores Genéticos/genética

RESUMEN

The Rad9-Rad1-Hus1 (9-1-1) complex is a key component in the coordination of DNA damage sensing, cell cycle progression and DNA repair pathways in eukaryotic cells. This PCNA-related trimer is loaded onto RPA-coated single stranded DNA and interacts with ATR kinase to mediate effective checkpoint signaling to halt the cell cycle and to promote DNA repair. Beyond these core activities, mounting evidence suggests that a broader range of functions can be provided by 9-1-1 structural diversification. The protozoan parasite Leishmania is an early-branching eukaryote with a remarkably plastic genome, which hints at peculiar genome maintenance mechanisms. Here, we investigated the existence of homologs of the 9-1-1 complex subunits in L. major and found that LmRad9 and LmRad1 associate with chromatin in response to replication stress and form a complex in vivo with LmHus1. Similar to LmHus1, LmRad9 participates in telomere homeostasis and in the response to both replication stress and double strand breaks. However, LmRad9 and LmHus1-deficient cells present markedly opposite phenotypes, which suggest their functional compartmentalization. We show that some of the cellular pool of LmRad9 forms an alternative complex and that some of LmHus1 exists as a monomer. We propose that the diverse assembly of the Leishmania 9-1-1 subunits mediates functional compartmentalization, which has a direct impact on the response to genotoxic stress.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Daño del ADN/fisiología , Exonucleasas/metabolismo , Leishmania major/fisiología , Puntos de Control del Ciclo Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Leishmania major/genética , Leishmania major/metabolismo

RESUMEN

The ability of transposable elements to mobilize across genomes and affect the expression of genes makes them exceptional tools for genetic manipulation methodologies. Several transposon-based systems have been modified and incorporated into shuttle mutagenesis approaches in a variety of organisms. We have found that the Mos1 element, a DNA transposon from Drosophila mauritiana, is suitable and readily adaptable to a variety of strategies to the study of trypanosomatid parasitic protozoa. Trypanosomatids are the causative agents of a wide range of neglected diseases in underdeveloped regions of the globe. In this chapter we describe the basic elements and the available protocols for the in vitro use of Mos1 derivatives in the protozoan parasite Leishmania.

Asunto(s)

Drosophila/genética , Técnicas Genéticas , Leishmania/genética , Mutagénesis/genética , Animales , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Leishmania/parasitología , Transposasas/genética , Transposasas/aislamiento & purificación

RESUMEN

Genotoxic stress activates checkpoint-signalling pathways leading to cell cycle arrest and DNA repair. In many eukaryotes, the Rad9-Hus1-Rad1 (9-1-1) checkpoint complex participates in the early steps of the DNA damage response to replicative stress and is a pivotal contributor to genome homeostasis. The remarkable genome plasticity of the protozoan Leishmania hints at a peculiar DNA metabolism in these ancient eukaryotes. Therefore, we set out to investigate the existence of homologues of the 9-1-1 components in Leishmania major and found that LmHus1 and LmRad9 are phylogenetically related to the 9-1-1 complex subunits from other eukaryotes. Altered levels of LmHus1 and LmRad9 affected the parasite ability to manage genotoxic stress and LmHus1-defficent cells were defective in controlling cell cycle progression in response to genotoxic stress. Upon DNA damage, LmHus1 was recruited to the chromatin and colocalized with the single-stranded DNA-binding protein LmRpa1. Also, LmHus1 interacted with LmRad9 to form a DNA damage responsive complex in vivo. Altogether, our data strongly indicate the participation of LmHus1, LmRad9 and LmRpa1 in the L. major DNA damage response and suggest their involvement in genome maintenance mechanisms.

Asunto(s)

Daño del ADN , Proteínas de Unión al ADN/metabolismo , Genes Protozoarios , Leishmania major/genética , Leishmania major/metabolismo , Proteínas Protozoarias/metabolismo , Ciclo Celular , Línea Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Humanos , Modelos Moleculares , Filogenia , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Protozoarias/química

RESUMEN

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression.

Asunto(s)

Daño del ADN , Leishmania major/genética , Leishmania major/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Línea Celular , Daño del ADN/efectos de los fármacos , Humanos , Leishmania major/química , Leishmania major/efectos de los fármacos , Leishmaniasis Cutánea/parasitología , Datos de Secuencia Molecular , Mutágenos/toxicidad , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Alineación de Secuencia

RESUMEN

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

Asunto(s)

Genoma de Protozoos/genética , Péptido Hidrolasas/genética , Trypanosoma rangeli/enzimología , Trypanosoma rangeli/genética , Secuencia de Aminoácidos , Bases de Datos Genéticas , Regulación Enzimológica de la Expresión Génica , Genómica , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Alineación de Secuencia , Análisis de Secuencia

RESUMEN

Protozoan parasites affect millions of people around the world. Treatment and control of these diseases are complicated partly due to the intricate biology of these organisms. The interactions of species of Plasmodium, Leishmania and trypanosomes with their hosts are mediated by an unusual control of gene expression that is not fully understood. The availability of the genome sequence of these protozoa sets the stage for using more comprehensive, genome-wide strategies to study gene function. Transposons are effective tools for the systematic introduction of genetic alterations and different transposition systems have been adapted to study gene function in these human pathogens. A mariner transposon toolkit for use in vivo or in vitro in Leishmania parasites has been developed and can be used in a variety of applications. These modified mariner elements not only permit the inactivation of genes, but also mediate the rescue of translational gene fusions, bringing a major contribution to the investigation of Leishmania gene function. The piggyBac and Tn5 transposons have also been shown to mobilize across Plasmodium spp. genomes circumventing the current limitations in the genetic manipulation of these organisms.

Asunto(s)

Elementos Transponibles de ADN , Técnicas de Transferencia de Gen , Leishmania/fisiología , Mutagénesis , Animales , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Genoma de Protozoos , Humanos , Plasmodium/fisiología , Infecciones por Protozoos/parasitología , Transposasas/fisiología , Trypanosoma/fisiología

RESUMEN

In the present study, the molecular karyotypes of 12 KP1(+) and KP1(-) Trypanosoma rangeli strains were determined and 10 different molecular markers were hybridized to the chromosomes of the parasite, including seven obtained from T. rangeli [ubiquitin hydrolase (UH), a predicted serine/threonine protein kinase (STK), hexose transporter, hypothetical protein, three anonymous sequences] and three from Trypanosoma cruzi [ubiquitin-conjugating enzyme E2 (UBE2), ribosomal RNA methyltransferase (rRNAmtr), proteasome non-ATPase regulatory subunit 6 (PSMD6)]. Despite intraspecific variation, analysis of the karyotype profiles permitted the division of the T. rangeli strains into two groups coinciding with the KP1(+) and KP1(-) genotypes. Southern blot hybridization showed that, except for the hexose transporter probe, all other probes produced distinct patterns able to differentiate the KP1(+) and KP1(-) genotypes. The UH, STK and An-1A04 probes exclusively hybridized to the chromosomes of KP1(+) strains and can be used as markers of this group. In addition, the UBE2, rRNAmtr and PSMD6 markers, which are present in a conserved region in all trypanosomatid species sequenced so far, co-hybridized to the same T. rangeli chromosomal bands, suggesting the occurrence of gene synteny in these species. The finding of distinct molecular karyotypes in KP1(+) and KP1(-) strains of T. rangeli is noteworthy and might be used as a new approach to the study of genetic variability in this parasite. Together with the Southern blot hybridization results, these findings demonstrate that differences at the kDNA level might be associated with variations in nuclear DNA.

Asunto(s)

Genes Protozoarios , Variación Genética , Trypanosoma/clasificación , Trypanosoma/genética , Animales , Southern Blotting , Brasil , Colombia , Electroforesis en Gel de Campo Pulsado , Marcadores Genéticos , Cariotipificación , Proteínas Protozoarias/genética , Sintenía , Trypanosoma/aislamiento & purificación

RESUMEN

The use of transposable elements as a gene-trapping strategy is a powerful tool for gene discovery. Herein we describe the development of a transposable system, based on the bacterial Tn5 transposon, which has been used successfully in Leishmania braziliensis. The transposon carries the neomycin phosphotransferase gene, which is expressed only when inserted in-frame with a Leishmania gene present in the target DNA. Four cosmid clones from a L. braziliensis genomic library were used as targets in transposition reactions and four insertional libraries were constructed and transfected in L. braziliensis. Clones resistant to G418 were selected and analysed by immunofluorescence in order to identify the subcellular localisation of the protein coded by the trapped gene. A definitive subcellular localisation for neomycin phosphotransferase/targeted protein fusion was not obtained in any of the four Leishmania clones investigated. However, the constructed transposable element is highly efficient considering the frequency of insertion in large targets and is therefore a useful tool for functional genetic studies in Leishmania. Our data confirm the utility of the Tn5 transposon system for insertion of sequencing priming sites into target DNA. Furthermore, the high frequency of insertion and even distribution are important in studying genomic regions bearing long and polymorphic repetitive sequences.

Asunto(s)

Elementos Transponibles de ADN/genética , Leishmania braziliensis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biblioteca de Genes , Genómica/métodos , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Insercional , Transfección

RESUMEN

Resistance to antimonials is a major problem when treating visceral leishmaniasis in India and has already been described for New World parasites. Clinical response to meglumine antimoniate in patients infected with parasites of the Viannia sub-genus can be widely variable, suggesting the presence of mechanisms of drug resistance. In this work, we have compared L. major and L. braziliensis mutants selected in different drugs. The cross-resistance profiles of some cell lines resembled those of mutants bearing H locus amplicons. However, amplified episomal molecules were exclusively detected in L. major mutants. The analysis of the L. braziliensis H region revealed a strong conservation of gene synteny. The typical intergenic repeats that are believed to mediate the amplification of the H locus in species of the Leishmania sub-genus are partially conserved in the Viannia species. The conservation of these non-coding elements in equivalent positions in both species is indicative of their relevance within this locus. The absence of amplicons in L. braziliensis suggests that this species may not favour extra-chromosomal gene amplification as a source of phenotypic heterogeneity and fitness maintenance in changing environments.

Asunto(s)

Resistencia a Medicamentos/genética , Amplificación de Genes/genética , Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/genética , Proteínas Protozoarias/genética , Animales , Secuencia Conservada/genética , ADN Intergénico , Leishmania major/efectos de los fármacos , Leishmania major/genética , Mutación , Pruebas de Sensibilidad Parasitaria , Secuencias Repetitivas de Ácidos Nucleicos/genética

RESUMEN

Genetic manipulation of the protozoan Leishmania has led to a better understanding of the survival and development of these pathogens within their hosts. The association of the Leishmania genome sequencing information with the ability of transposons to introduce or destroy phenotypes allows a global perspective on the role and importance of genes in cellular pathways. Herein we report the construction and testing of mariner transposable elements carrying the neomycin phosphotransferase, green fluorescent protein, or beta-glucuronidase genes as reporters for translational fusion events. We demonstrate that the expression of the reporter genes will occur only when the genes are inserted in-frame within predicted genes. Our results not only add to the mariner toolkit for gene manipulation but also strengthen the evidence that the mariner system is a reliable means for the study of gene expression in Leishmania.

Asunto(s)

Elementos Transponibles de ADN/fisiología , Proteínas de Unión al ADN/genética , Leishmania/genética , Animales , Regulación de la Expresión Génica , Genes Reporteros/fisiología , Proteínas Fluorescentes Verdes/genética , Kanamicina Quinasa/genética , Mutagénesis Insercional , Especificidad de la Especie , Transfección , Transposasas

RESUMEN

Long-term nitric oxide (NO) blockade is known to induce a severe and progressive hypertension. The influence of the salt-intake on atrial natriuretic peptide (ANP) system in this hypertension model is unknown. The aim of this study was to evaluate ANP plasma levels, content and mRNA in atria of male Wistar rats chronically treated with oral Nomega-nitro-L-arginine methyl ester (L-NAME) after 4 weeks of high-salt diet. The high-salt diet induced an increase (P < 0.05) in ANP plasma levels in normotensive rats and no significant changes in hypertensive animals. We observed a significant increase in the ANP content in the left and right atria of hypertensive rats (P < 0.001) when compared to normotensive ones. However, no significant changes were observed during high-salt diet in normotensive and hypertensive animals. Northern blot analysis revealed that ANP gene expression is higher in the right and left atria of hypertensive rats when compared to normotensive rats. However, we found no significant changes in ANP mRNA of rats treated with high-salt diet in normotensive and hypertensive rats when compared to low-salt diet. The present observations indicate no interaction between salt-intake and activation of the ANP system during chronic nitric oxide synthase (NOS) inhibition.

Asunto(s)

Factor Natriurético Atrial/sangre , Hipertensión/sangre , Óxido Nítrico/antagonistas & inhibidores , Cloruro de Sodio Dietético/farmacología , Animales , Factor Natriurético Atrial/genética , Presión Sanguínea/efectos de los fármacos , Northern Blotting , Inhibidores Enzimáticos/farmacología , Atrios Cardíacos/efectos de los fármacos , Hipertensión/fisiopatología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , ARN Mensajero/genética , Ratas , Ratas Wistar

RESUMEN

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

Asunto(s)

Antiprotozoarios/farmacología , Elementos Transponibles de ADN/genética , Resistencia a Medicamentos/genética , Leishmania major/genética , Pentamidina/farmacología , Animales , ADN Protozoario/genética , Eliminación de Gen , Genes Protozoarios , Biblioteca Genómica , Leishmania major/efectos de los fármacos , Fenotipo , Mapeo Restrictivo

RESUMEN

A Pentamidina (PEN) é um composto alternativo para o tratamento de pacientes com leishmaniose que apresentam resistência ao antimônio, cujo alvo celular continua incerto. Uma abordagem para se identificar prováveis alvos seria a identificação e super-expressão de genes capazes de mediar resistência a PEN. A partir de uma genoteca construída com o DNA de Leishmania major em um vetor - cosmídio que se desenvolve tanto em bactérias como nas células do parasita, isolamos um locus que após transfecção é capaz de produzir resistência a PEN às células do parasita. Almejando o mapeamento desse locus de leishmania, o inserto clonado nesse cosmídio foi deletado através de duas digestões parciais sucessivas com enzimas de restrição, seguida de transfecção em células selvagens, super-expressão gênica, indução e testes funcionais na presença de PEN. Para determinar o gene de Leishmania relacionado com a resistência a PEN, o seqüenciamento de nucleotídeos foi executado após inserção de elementos transposicionais de Drosophila melanogaster no interior do inserto deletado para atuar como ilhas de iniciadores. Descrevemos aqui o mapeamento desse locus, após a inserção transposicional, que além de facilitar o seqüenciamento de nucleotídeos de grandes fragmentos de DNA, permite uma rápida identificação do gene relacionado com esse fenótipo. Experimentos posteriores revelaram neste locus a presença do gene de uma Glicoproteína-P de membrana, cujo papel no metabolismo na Leishmania está sendo analisado.

Asunto(s)

Animales , Antiprotozoarios , Elementos Transponibles de ADN , Resistencia a Medicamentos , Leishmania major , Pentamidina , ADN Protozoario , Eliminación de Gen , Genes Protozoarios , Biblioteca Genómica , Leishmania major , Fenotipo , Mapeo Restrictivo