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1.
Mikrobiyol Bul ; 53(4): 408-418, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709938

RESUMO

Leishmaniasis is a parasitic disease that is transmitted to humans by the bites of infected female phlebotomine sandflies. In the diagnosis of cutaneous leishmaniasis (CL), in the smear samples, the demonstration of the parasite by microscope remains a gold standard method. However, it becomes difficult to diagnose the parasite since the number of amastigotes in chronic cases with a lesion of one year or longer is very low. Due to many factor such as patients primarily do not to take any notice these lesions in their bodies, do not apply to health institutions or late applied, receive wrong treatment; the diagnosis and treatment are delayed. In addition, it is been worse prognosis by add secondary infection to lesions and wounds become chronic. For this reason, molecular methods are used in addition to microscopic examination in chronic suspected CL cases. It was aimed to reveal of the molecular diagnostic value in chronic suspected CL cases by polymerase chain reaction (PCR) in the smear belonging to Turkish patients that reported to be evaluated clinically because it can not be seen Leishmania amastigotes in microscopic examination. Smear of 50 Turkish patients who were clinically reported of the evaluation of chronic CL were selected. These samples were smears belonging to suspected CL patients that applied Hatay Mustafa Kemal University, Faculty of Medicine, Parasitology laboratory from different polyclinics and were decided to be evaluated clinically as a result of microscopic examination because they came from endemic regions (such as Hassa, Altinözü, Yayladagi). DNA was isolated from selected samples and PCR was performed using 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. The samples found positive by PCR were typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using LITSR and L5.8S primers targeting internal transcribed spacer (ITS-1) region. Of the 50 smear samples, 17 (34%) were determined positive with 13A, 13B primers targeting the kinetoplastid DNA (kDNA) region. Positive samples were also found to be positive with LITSR and L5.8S primers targeting ITS-1 region. The PCR products obtained from PCR with ITS-1 gene region were digested with the restriction endonucleases BsuRI (HaeIII). As a result of PCR-RFLP analysis, it was determined that 11 of Leishmania tropica, one of Leishmania major and five of Leishmania infantum/donovani out of 17 samples. Chronic CL can be confused with skin diseases such as sarcoidosis, tuberculosis, malignant tumors. In particular, chronic CL cases can be escaped the attention for many reasons such as failure to diagnose correctly, insufficient microscope experience, fail to see due to low number of parasites. For this reason, it was concluded that PCR, which is a molecular method, should be used in chronic suspected CL samples which are negative for the parasite by microscopic examination.


Assuntos
DNA de Protozoário , Leishmania , Leishmaniose Cutânea , Reação em Cadeia da Polimerase , DNA de Protozoário/genética , Feminino , Humanos , Leishmania/genética , Leishmaniose Cutânea/diagnóstico , Microscopia , Polimorfismo de Fragmento de Restrição , Reprodutibilidade dos Testes , Turquia
2.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 31(4): 418-422, 2019 Jul 24.
Artigo em Chinês | MEDLINE | ID: mdl-31612679

RESUMO

OBJECTIVE: To understand Leishmania infections among employees of China Petroleum First Construction Corporation returning from Uzbekistan, and take timely actions to prevent the spread of the epidemic. METHODS: Questionnaire survey was conducted to collect screening subjects'information. Palpation of the liver, spleen and superficial lymph nodes was performed by a physician, and the lesions on the frequently exposed skin were detected by a dermatologist. In addition, the liver and spleen sizes were measured using B-mode ultrasonography, and serum samples were collected to be subjected to an rK39-based rapid diagnostic test for detection of visceral leishmaniasis. Leishmania was detected using microscopy in the specimens sampled from the lesioned skin, and the parasites species was identified using molecular assays in parasitologically positive specimens. RESULTS: Among the 181 employees screened, enlarged cervical lymph nodes were palpable in 6 subjects, and skin lesions were found in 12 cases. B-mode ultrasonography displayed hepatosplenomegaly in 5 cases, and rK39 test were positive in 3 serum samples. Two classical lesioned skin specimens were sampled, and Leishmania was detected in one specimen. The promastigote DNA was extracted and two fragments of 120 bp and 350 bp in sizes were amplified using PCR assay with K13A/K13B and L5.8S/LITSR primers specific to Leishmania. The two amplification products were 90% and 98% homologous to the corresponding sequences of L. major (GenBank accession numbers: EU370906.1 and FN677342.1). CONCLUSIONS: Six patients with cutaneous leishmaniasis were screened, including 2 uncured cases. One uncured case was diagnosed as imported cutaneous leishmaniasis caused by L. major infection.


Assuntos
Doenças Transmissíveis Importadas , Leishmania , Leishmaniose Cutânea , China/epidemiologia , Doenças Transmissíveis Importadas/diagnóstico , Doenças Transmissíveis Importadas/epidemiologia , Doenças Transmissíveis Importadas/parasitologia , DNA de Protozoário , Epidemias , Humanos , Leishmania/genética , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , Fígado/diagnóstico por imagem , Linfonodos/patologia , Reação em Cadeia da Polimerase , Baço/diagnóstico por imagem , Inquéritos e Questionários , Ultrassonografia , Uzbequistão
3.
Braz J Med Biol Res ; 52(9): e8224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482975

RESUMO

Leishmaniasis is a neglected disease that affects a large part of the world population. Knowing the sand fly fauna of a region is of fundamental importance for guiding health surveillance actions related to the prevention and control of leishmaniasis. A total of 86 specimens of sand flies (60 females and 26 males) were collected. Using the classification proposed by Galati (2003), the following species were identified: Lutzomyia longipalpis (Lutz & Neiva, 1912), Migonemyia migonei (França, 1920), Evandromyia cortelezzi (Brethes, 1923), Ev. sallesi (Galvão & Coutinho, 1939), Nyssomyia whitmani (Atunes & Coutinho, 1939), Psathyromyia lutziana (Costa Lima, 1932), Ev. lenti (Mangabeira, 1938), Brumptomyia sp. (França and Parrot, 1921), and Pressatia sp. (Mangabeira, 1942). Using PCR with internal transcribed spacer target to identify infected sand flies, five Lu. longipalpis females were infected with Leishmania spp. Despite the small number of specimens collected, considerable species diversity was found in the study area.


Assuntos
Insetos Vetores/classificação , Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Psychodidae/classificação , Psychodidae/parasitologia , Animais , Brasil , DNA Espaçador Ribossômico/genética , Feminino , Leishmania/genética , Leishmaniose/transmissão , Masculino , Reação em Cadeia da Polimerase , RNA de Protozoário/genética
4.
Parasit Vectors ; 12(1): 348, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300064

RESUMO

BACKGROUND: In the last decade, resistance to antimonials has become a serious problem due to the emergence of drug-resistant strains. Therefore, understanding the mechanisms used by Leishmania parasites to survive under drug pressure is essential, particularly for species of medical-veterinary importance such as L. amazonensis. METHODS: Here, we used RNA-seq technology to analyse transcriptome profiles and identify global changes in gene expression between antimony-resistant and -sensitive L. amazonensis promastigotes. RESULTS: A total of 723 differentially expressed genes were identified between resistant and sensitive lines. Comparative transcriptomic analysis revealed that genes encoding proteins involved in metabolism (fatty acids) and stress response, as well as those associated with antimony resistance in other Leishmania species, were upregulated in the antimony-resistant line. Most importantly, we observed upregulation of genes encoding autophagy proteins, suggesting that in the presence of trivalent stibogluconate (SbIII) L. amazonensis can activate these genes either as a survival strategy or to induce cell death, as has been observed in other parasites. CONCLUSIONS: This work identified global transcriptomic changes in an in vitro-adapted strain in response to SbIII. Our results provide relevant information to continue understanding the mechanism used by parasites of the subgenus Leishmania (L. amazonensis) to generate an antimony-resistant phenotype.


Assuntos
Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/farmacologia , Resistência a Medicamentos/genética , Leishmania/efeitos dos fármacos , Leishmania/genética , Transcriptoma , DNA de Protozoário/genética , Perfilação da Expressão Gênica , Ontologia Genética , Fenótipo , Análise de Sequência de RNA , Regulação para Cima
5.
Parasit Vectors ; 12(1): 303, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202271

RESUMO

Parasites comprise diverse and complex organisms, which substantially impact human and animal health. Most parasites have complex life-cycles, and by virtue of co-evolution have developed multifaceted, often life-cycle stage-specific relationships with the immune system of their hosts. The complexity in the biology of many parasites often limits our knowledge of parasite-specific immune responses, to in vitro studies only. The relatively recent development of methods to stably manipulate the genetic make-up of many parasites has allowed a better understanding of host-parasite interactions, particularly in vivo. In this regard, the use of transgenic parasites can facilitate the study of immunomodulatory mechanisms under in vivo conditions. Therefore, in this review, we specifically highlighted the current developments in the use of transgenic parasites to unravel the host's immune response to different life-cycle stages of some key parasite species such as Leishmania, Schistosoma, Toxoplasma, Plasmodium and Trypanosome and to some degree, the use of transgenic nematode parasites is also briefly discussed.


Assuntos
Técnicas de Transferência de Genes , Interações Hospedeiro-Parasita/imunologia , Parasitos/genética , Parasitos/imunologia , Animais , Interações Hospedeiro-Parasita/genética , Humanos , Leishmania/genética , Leishmania/imunologia , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/imunologia , Camundongos , Plasmodium/genética , Plasmodium/imunologia , Toxoplasma/genética , Toxoplasma/imunologia
6.
PLoS Negl Trop Dis ; 13(6): e0007496, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31220120

RESUMO

To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru.


Assuntos
Citocromos b/genética , Leishmania/classificação , Leishmania/genética , Leishmaniose Cutânea/epidemiologia , Manose-6-Fosfato Isomerase/genética , Filogeografia , Proteínas de Protozoários/genética , Humanos , Leishmania/isolamento & purificação , Peru/epidemiologia
7.
PLoS Negl Trop Dis ; 13(5): e0007288, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31071080

RESUMO

BACKGROUND: Leishmania development in the sand fly gut leads to highly infective forms called metacyclic promastigotes. This process can be routinely mimicked in culture. Gene expression-profiling studies by transcriptome analysis have been performed with the aim of studying promastigote forms in the sand fly gut, as well as differences between sand fly-and culture-derived promastigotes. FINDINGS: Transcriptome analysis has revealed the crucial role of the microenvironment in parasite development within the sand fly gut because substantial differences and moderate correlation between the transcriptomes of cultured and sand fly-derived promastigotes have been found. Sand fly-derived metacyclics are more infective than metacyclics in culture. Therefore, some caution should be exercised when using cultured promastigotes, depending on the experimental design. The most remarkable examples are the hydrophilic acidic surface protein/small endoplasmic reticulum protein (HASP/SHERP) cluster, the glycoprotein 63 (gp63), and autophagy genes, which are up-regulated in sand fly-derived promastigotes compared with cultured promastigotes. Because HASP/SHERP genes are up-regulated in nectomonad and metacyclic promastigotes in the sand fly, the encoded proteins are not metacyclic specific. Metacyclic promastigotes are distinguished by morphology and high infectivity. Isolating them from the sand fly gut is not exempt from technical difficulty, because other promastigote forms remain in the gut even 15 days after infection. Leishmania major procyclic promastigotes within the sand fly gut up-regulate genes involved in cell cycle regulation and glucose catabolism, whereas metacyclics increase transcript levels of fatty acid biosynthesis and ATP-coupled proton transport genes. Most parasite's signal transduction pathways remain uncharacterized. Future elucidation may improve understanding of parasite development, particularly signaling molecule-encoding genes in sand fly versus culture and between promastigote forms in the sand fly gut. CONCLUSIONS: Transcriptome analysis has been demonstrated to be technically efficacious to study differential gene expression in sand fly gut promastigote forms. Transcript and protein levels are not well correlated in these organisms (approximately 25% quantitative coincidences), especially under stress situations and at differentiation processes. However, transcript and protein levels behave similarly in approximately 60% of cases from a qualitative point of view (increase, decrease, or no variation). Changes in translational efficiency observed in other trypanosomatids strongly suggest that the differences are due to translational regulation and regulation of the steady-state protein levels. The lack of low-input sample strategies does not allow translatome and proteome analysis of sand fly-derived promastigotes so far.


Assuntos
Leishmania/crescimento & desenvolvimento , Leishmania/genética , Proteínas de Protozoários/genética , Psychodidae/parasitologia , Animais , Trato Gastrointestinal/parasitologia , Genômica , Leishmania/classificação , Leishmania/isolamento & purificação , Proteínas de Protozoários/metabolismo , Transcriptoma
8.
Parasite ; 26: 30, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31120019

RESUMO

Tegumentary Leishmaniasis (TL) in the Brazilian Amazon region is associated with several Leishmania species. In this report, we describe two cases of TL related to Leishmania lindenbergi occurring in different locations of Rondônia state. After clinical diagnosis, lesion samples were collected for parasitological diagnoses via direct microscopic visualization, parasite isolation, and PCR. PCR reactions were positive in both clinical samples. Parasite isolation was possible for both patients, and isolates were submitted to species identification by isoenzyme electrophoresis and DNA sequencing. This report is the first to describe human infections caused by L. lindenbergi since the initial description and record of human infection by this species in 2002.


Assuntos
Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Pele/patologia , Adulto , Brasil , Feminino , Humanos , Leishmania/classificação , Leishmania/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Pele/parasitologia
9.
Braz J Infect Dis ; 23(2): 111-120, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31054271

RESUMO

Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.


Assuntos
DNA de Protozoário/urina , Leishmania/genética , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/urina , DNA de Protozoário/isolamento & purificação , Humanos , Leishmania/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
10.
PLoS Negl Trop Dis ; 13(5): e0007403, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31059516

RESUMO

PCR-Restriction Fragment Length Polymorphism (RFLP) analyses targeting multiple nuclear genes were established for the simple and practical identification of Leishmania species without using expensive equipment. This method was applied to 92 clinical samples collected at 33 sites in 14 provinces of Ecuador, which have been identified at the species level by the kinetoplast cytochrome b (cyt b) gene sequence analysis, and the results obtained by the two analyses were compared. Although most results corresponded between the two analyses, PCR-RFLP analyses revealed distribution of hybrid strains between Leishmania (Viannia) guyanensis and L. (V.) braziliensis and between L. (V.) guyanensis and L. (V.) panamensis, of which the latter was firstly identified in Ecuador. Moreover, unexpected parasite strains having the kinetoplast cyt b gene of L. (V.) braziliensis and nuclear genes of L. (V.) guyanensis, L. (V.) panamensis, or a hybrid between L. (V.) guyanensis and L. (V.) panamensis were identified. This is the first report of the distribution of a protozoan parasite having mismatches between kinetoplast and nuclear genes, known as mito-nuclear discordance. The result demonstrated that genetically complex Leishmania strains are present in Ecuador. Since genetic exchanges such as hybrid formation were suggested to cause higher pathogenicity in Leishmania and may be transmitted by more species of sand flies, further country-wide epidemiological studies on clinical symptoms, as well as transmissible vectors, will be necessary.


Assuntos
Núcleo Celular/genética , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Mucocutânea/parasitologia , Mitocôndrias/genética , Animais , Pareamento Incorreto de Bases , DNA de Cinetoplasto , Equador , Humanos , Leishmania/isolamento & purificação , Leishmania/fisiologia , Leishmaniose Cutânea/transmissão , Leishmaniose Mucocutânea/transmissão , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/genética , Psychodidae/parasitologia , Psychodidae/fisiologia
11.
Methods Mol Biol ; 1971: 95-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980299

RESUMO

High-throughput sequencing of cDNA copies of mRNA (RNA-seq) provides a digital readout of mRNA levels over several orders of magnitude, as well as mapping the transcripts to the nucleotide level. Here we describe two different RNA-seq approaches, including one that exploits the 39-nucleotide mini-exon or spliced leader (SL) sequence found at the 5' end of all Leishmania (and other trypanosomatid) mRNAs.


Assuntos
Éxons , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leishmania/genética , RNA Mensageiro/genética , RNA de Protozoário/genética , Análise de Sequência de RNA/métodos
12.
Acta Trop ; 195: 23-27, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30998901

RESUMO

Leishmania is an intracellular parasite, which is transmitted by the bite of infected female Phlebotominae sand flies. Turkey is a crossroad between Europe and Asia that makes it important in terms of epidemiology. In the present study, we aimed to evaluate Leishmania infection among non-autochthonous patients admitted to Health Sciences University, Dr. Sadi Konuk Research and Training hospital between 2014-2018. Slides were prepared by sampling the edge of the lesions for each patient. Microscopical examination was performed after staining procedures. After microscopical examination slides were washed and DNA extraction was performed. ITS-1 real-time PCR was performed to identify the species of the causative agents. Demographic data were recorded for each patient. Also number, type and location of the lesions were recorded. Totally 13 patients were included in this. Majority (12/13) of them were found to be infected with L. tropica, while one patient was infected with L. infantum. Two of the lesions were wet type and 11 of them were dry type lesions. Several papers were published recently about leishmaniasis in Turkey but to best of our knowledge, this is the first study reporting refugee leishmaniasis in Istanbul.


Assuntos
Leishmaniose/epidemiologia , Refugiados , Adolescente , Adulto , Animais , Criança , Feminino , Humanos , Leishmania/genética , Masculino , Turquia/epidemiologia , Adulto Jovem
13.
PLoS Negl Trop Dis ; 13(4): e0007264, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31017892

RESUMO

Trypanosomatids are flagellated protozoan parasites that are very unusual in terms of cytoskeleton organization but also in terms of cell death. Most of the Trypanosomatid cytoskeleton consists of microtubules, forming different substructures including a subpellicular corset. Oddly, the actin network appears structurally and functionally different from other eukaryotic actins. And Trypanosomatids have an apoptotic phenotype under cell death conditions, but the pathways involved are devoid of key mammal proteins such as caspases or death receptors, and the triggers involved in apoptotic induction remain unknown. In this article, we have studied the role of the post-translational modifications, deglutamylation and polyglutamylation, in Leishmania. We have shown that Leishmania apoptosis was linked to polyglutamylation and hypothesized that the cell survival process autophagy was linked to deglutamylation. A balance seems to be established between polyglutamylation and deglutamylation, with imbalance inducing microtubule or other protein modifications characterizing either cell death if polyglutamylation was prioritized, or the cell survival process of autophagy if deglutamylation was prioritized. This emphasizes the role of post-translational modifications in cell biology, inducing cell death or cell survival of infectious agents.


Assuntos
Apoptose/efeitos dos fármacos , Leishmania/citologia , Microtúbulos/fisiologia , Processamento de Proteína Pós-Traducional , Actinas/metabolismo , Sobrevivência Celular , Curcumina/farmacologia , Citoesqueleto/fisiologia , Imunofluorescência , Leishmania/efeitos dos fármacos , Leishmania/genética , Peptídeo Sintases/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia
14.
Methods Mol Biol ; 1971: 9-68, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980297

RESUMO

Phylogenetics is an important component of the systems biology approach. Knowledge about evolution of the genus Leishmania is essential to understand various aspects of basic biology of these parasites, such as parasite-host or parasite-vector relationships, biogeography, or epidemiology. Here, we present a comprehensive guideline for performing phylogenetic studies based on DNA sequence data, but with principles that can be adapted to protein sequences or other molecular markers. It is presented as a compilation of the most commonly used genetic targets for phylogenetic studies of Leishmania, including their respective primers for amplification and references, as well as details of PCR assays. Guidelines are, then, presented to choose the best targets in relation to the types of samples under study. Finally, and importantly, instructions are given to obtain optimal sequences, alignments, and datasets for the subsequent data analysis and phylogenetic inference. Different bioinformatics methods and software for phylogenetic inference are presented and explained. This chapter aims to provide a compilation of methods and generic guidelines to conduct phylogenetics of Leishmania for nonspecialists.


Assuntos
Biologia Computacional , DNA de Protozoário/genética , Leishmania/genética , Filogenia , Análise de Sequência de DNA , Software
15.
Methods Mol Biol ; 1971: 69-94, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980298

RESUMO

Next generation sequencing (NGS) technology transformed Leishmania genome studies and became an indispensable tool for Leishmania researchers. Recent Leishmania genomics analyses facilitated the discovery of various genetic diversities including single nucleotide polymorphisms (SNPs), copy number variations (CNVs), somy variations, and structural variations in detail and provided valuable insights into the complexity of the genome and gene regulation. Many aspects of Leishmania NGS analyses are similar to those of related pathogens like trypanosomes. However, the analyses of Leishmania genomes face a unique challenge because of the presence of frequent aneuploidy. This makes characterization and interpretation of read depth and somy a key part of Leishmania NGS analyses because read depth affects the accuracy of detection of all genetic variations. However, there are no general guidelines on how to explore and interpret the impact of aneuploidy, and this has made it difficult for biologists and bioinformaticians, especially for beginners, to perform their own analyses and interpret results across different analyses. In this guide we discuss a wide range of topics essential for Leishmania NGS analyses, ranging from how to set up a computational environment for genome analyses, to how to characterize genetic variations among Leishmania samples, and we will particularly focus on chromosomal copy number variation and its impact on genome analyses.


Assuntos
Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leishmania/genética , Polimorfismo de Nucleotídeo Único
16.
Methods Mol Biol ; 1971: 123-140, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980301

RESUMO

Cosmid libraries can represent an entire genome in a library of circular DNA molecules, allowing for the faithful amplification, cloning and isolation of large genomic DNA fragments. Moreover, using the so-called shuttle cosmid vectors, genomic DNA may be propagated in bacteria and in eukaryotic cells, which is a prerequisite for classic functional cloning and for the newly described Cos-Seq strategies.


Assuntos
Clonagem Molecular , Cosmídeos/genética , Biblioteca Gênica , Leishmania/genética
17.
Methods Mol Biol ; 1971: 141-167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980302

RESUMO

Leishmania is still a major cause of mortality and morbidity worldwide. Few efficient drugs are available, and resistance threatens actual treatments. In order to improve knowledge about the mode of action of current drugs and those in development, as well as to understand the mechanisms pertaining to their resistance, we recently described a sensitive and high-throughput method termed Cos-Seq. Here we provide a detailed protocol for every step of the procedure, from library construction to drug selection, cosmid extraction, and next-generation sequencing of extracted cosmids. A section on the bioinformatics of Cos-Seq is also included. Cos-Seq facilitates the identification of gain-of-function resistance mechanisms and drug targets and is a useful tool in resistance and drug development studies.


Assuntos
Antiprotozoários , Resistência a Medicamentos/genética , Mutação com Ganho de Função , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Leishmania/genética
18.
Methods Mol Biol ; 1971: 169-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980303

RESUMO

While homologous recombination-based gene replacement is about to be supplanted by more modern approaches, it is still retaining usefulness for genes that prove to be poor targets for CRISPR/cas-based approaches. Homologous recombination has proven to be relatively robust to minor sequence mismatches between GOI-flanking sequences and the gene replacement constructs, and the faithfulness of recombination events is easily verified by whole-genome sequencing. Moreover, the availability of custom synthetic gene production by numerous service providers should allow for a relatively quick generation of null mutants without the need to introduce additional protein-coding genes beyond the selection markers.


Assuntos
Sistemas CRISPR-Cas , Marcação de Genes , Recombinação Homóloga , Leishmania/genética
19.
Methods Mol Biol ; 1971: 189-210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980304

RESUMO

Postgenomic analyses of Leishmania biology benefit from rapid and precise methods for gene manipulation. Traditional methods of gene knockout or tagging by homologous recombination have limitations: they tend to be slow and require successive transfection and selection rounds to knock out multiple alleles of a gene. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems overcome these limitations. We describe here in detail a simple, rapid, and scalable method for CRISPR-Cas9-mediated gene knockout and tagging in Leishmania. This method details how to use simple PCR to generate (1) templates for single guide RNA (sgRNA) transcription in cells expressing Cas9 and T7 RNA polymerase and (2) drug-selectable editing cassettes, using a modular set of plasmids as templates. pT plasmids allow for amplification of drug resistance genes for knockouts and pPLOT plasmids provide a choice of different tags to generate N- or C-terminally tagged proteins. We describe how to use an online platform ( LeishGEdit.net ) for automated primer design and how to perform PCRs and transfections in small batches or on 96-well plates for large-scale knockout or tagging screens. This method allows generation of knockout mutants or tagged cell lines within 1 week.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Leishmania/genética , Recombinação Homóloga , Plasmídeos/genética , Transfecção
20.
Methods Mol Biol ; 1971: 211-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980305

RESUMO

Conditional gene deletion using dimerizable Cre recombinase (DiCre) is so far the best developed system for the phenotypic analysis of essential genes in Leishmania species. Here, we describe a protocol for the generation of a conditional gene deletion mutant and the subsequent inducible deletion of a target gene. Leishmania parasites are genetically modified to express two inactive Cre subunits (DiCre) and a single LoxP-flanked version of a target gene in a context where both endogenous copies of the gene have been deleted. Treatment with rapamycin dimerizes the DiCre subunits, resulting in activation of the enzyme, recombination between the LoxP sites, and excision of the LoxP-flanked target gene. Subsequent phenotyping allows for the analysis of essential gene function.


Assuntos
Deleção de Genes , Genes de Protozoários , Integrases , Leishmania/genética , Recombinação Genética
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