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1.
Nat Commun ; 12(1): 215, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431825

RESUMO

Leishmaniasis is widely regarded as a vaccine-preventable disease, but the costs required to reach pivotal Phase 3 studies and uncertainty about which candidate vaccines should be progressed into human studies significantly limits progress in vaccine development for this neglected tropical disease. Controlled human infection models (CHIMs) provide a pathway for accelerating vaccine development and to more fully understand disease pathogenesis and correlates of protection. Here, we describe the isolation, characterization and GMP manufacture of a new clinical strain of Leishmania major. Two fresh strains of L. major from Israel were initially compared by genome sequencing, in vivo infectivity and drug sensitivity in mice, and development and transmission competence in sand flies, allowing one to be selected for GMP production. This study addresses a major roadblock in the development of vaccines for leishmaniasis, providing a key resource for CHIM studies of sand fly transmitted cutaneous leishmaniasis.


Assuntos
Leishmania major/fisiologia , Leishmaniose Cutânea/parasitologia , Animais , Modelos Animais de Doenças , Humanos , Insetos Vetores/parasitologia , Israel , Leishmania major/genética , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/transmissão , Camundongos Endogâmicos BALB C , Parasitos/genética , Filogenia , Psychodidae/parasitologia , Sequenciamento Completo do Genoma
2.
Nat Commun ; 11(1): 3461, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651371

RESUMO

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Leishmania major/genética , Leishmania major/patogenicidade , Vacinas Atenuadas/uso terapêutico , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Dexametasona/farmacologia , Feminino , Citometria de Fluxo , Edição de Genes , Engenharia Genética , Humanos , Imunossupressão , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Psychodidae/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
PLoS Genet ; 16(7): e1008828, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609721

RESUMO

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.


Assuntos
Recombinação Homóloga/genética , Leishmania major/genética , Leishmaniose Cutânea/genética , Rad51 Recombinase/genética , Sistemas CRISPR-Cas/genética , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Técnicas de Inativação de Genes , Genoma/genética , Humanos , Leishmania major/patogenicidade , Leishmaniose Cutânea/parasitologia
4.
Ann Parasitol ; 66(2): 251-254, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32592548

RESUMO

Ouagadougou, the capital city of Burkina Faso, was recognized as a focus of zoonotic cutaneous leishmaniosis in April 2000. Leishmania major was the only strain isolated in this focus. We conducted a prospective study to detect L. major in rodents, animals which are described as reservoir of the parasite. Rodents were caught in five city areas from November 2005 to October 2006. Giemsa stained smears were realized from the cutaneous lesions when present after macroscopic examination of external lesions. The spleen of each rodent was sterilely removed and split into 3 parts for microscopic examination of smears, culture on NNN media and PCR, respectively. A total of 101 rodents belonging to 9 genera were trapped. All the direct examinations and cultures were negative. By using PCR of lesions and spleen samples, three animals were found infected by L. major: one out of 24 (4.2%) Mastomys natalensis; one out of 8 (12.5%) Taterillus sp. and one out of three Cricetomys gambianus. This is the first detection of L. major in rodent species in Burkina Faso. Further studies are needed to confirm their role as reservoirs of L. major.


Assuntos
Reservatórios de Doenças , Leishmania major , Leishmaniose Cutânea , Reação em Cadeia da Polimerase , Doenças dos Roedores , Roedores , Animais , Burkina Faso , Reservatórios de Doenças/parasitologia , Leishmania major/genética , Leishmaniose Cutânea/diagnóstico , Estudos Prospectivos , Doenças dos Roedores/parasitologia , Roedores/parasitologia , Baço/parasitologia
5.
Nucleic Acids Res ; 48(10): 5511-5526, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32365184

RESUMO

RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.


Assuntos
Leishmania major/enzimologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica , Leishmania major/genética , Metilação , Estabilidade Proteica
6.
Int J Nanomedicine ; 14: 7593-7607, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31802863

RESUMO

Background: Amphotericin B (Amp) and Betulinic acid (BA) as antileishmanial agents have negligible water solubility and high toxicity. To solve these problems, for the first time, chitosan nanoparticles and Anionic Linear Globular Dendrimer (D) were synthesized for the treatment of Leishmania major (L. major). Method: Chitosan and dendrimer nanoparticles were synthesized, and Amp and BA were loaded into the nanoparticles. The particles were then characterized using various methods and their efficacy was evaluated in vitro and in vivo environments (parasite burden was confirmed using pathological studies and real-time PCR methods). Result: The results of docking showed that Amp and BA can be loaded into chitosan and dendrimer nanoparticles. The results of physically drug loading efficiency for AK (Amphotericin B-chitosan), BK (Betulinic acid-chitosan), AD (Amphotericin B-Dendrimer) and BD (Betulinic acid- Dendrimer) were 90, 93, 84 and 96 percent, respectively. The characterization results indicated that the drugs were loaded into nanoparticles physically. Moreover, the increased solubility rate for AD=478, BD=790, AK=80 and BK=300 folds. Furthermore, the results of the drug delivery system showed the slow controlled drug release pattern with cellular uptake of more than 90%. The treatment results showed a 100 percent decrease of toxicity for the all nanodrugs was observed in vivo and in vitro environments. Moreover, AK10 and BK20 mg/kg reduced parasite burden by 83 percent (P<0.001), while AD50 and BD40 mg/kg reduced it to a lesser extent compared to glucantime. Conclusion: All the synthesized nanodrugs were completely succeeded by 100% to recovery the L. major induced pathological effects in the infected footpad. Also, the results of present study were confirmed with real-time PCR and the results showed that AK and BK were succeeded in a large extent to the treatment of L. major infection (P<0.001), therefore AK and BK could be considered as proper alternatives of choices drugs.


Assuntos
Anfotericina B/farmacologia , Quitosana/química , Dendrímeros/química , Leishmania major/efeitos dos fármacos , Leishmania major/genética , Nanopartículas/química , Reação em Cadeia da Polimerase em Tempo Real/métodos , Triterpenos/química , Anfotericina B/química , Animais , Antiprotozoários/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Simulação de Acoplamento Molecular , Nanopartículas/uso terapêutico , Nanopartículas/toxicidade , Parasitos/efeitos dos fármacos , Parasitos/genética , Triterpenos Pentacíclicos , Solubilidade , Termodinâmica
7.
Bull Soc Pathol Exot ; 112(3): 147-152, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31825568

RESUMO

Three distinct noso-epidemiological cutaneous leishmaniasis (LC) entities coexist in Algeria: the so-called sporadic form of the North (LCN), the zoonotic form (LCZ) and the chronic form (LCC). The precise identification of the parasitic species involved in each of the forms makes it possible to specify the geographical distribution of each of the forms raised, to distinguish their clinical aspects, to guide the therapeutic behaviors and to adapt the control programs. Ninety-seven (97) human strains from 97 cases of LC were subjected to molecular characterization by PCR-ITS1 followed by sequencing of this inter-gene space. Our results confirm the endemicity of the three forms. The LCN, caused by L. infantum (17 isolates/97 i.e. 17.52%) is limited to the North of the country mainly (16 isolates/17). Its geographical distribution is superimposable to that of visceral leishmaniasis with an extension more and more reported in previously unaffected areas, such as the regions of Tlemcen and Oran in the West, Setif, Annaba and Collo in the East. The LCZ, due to L. major (70 strains/97 i.e. 72.16%), remains the dominant form in the arid and semi-arid zones (47 strains/70) with a progression towards the North (20/70 strains). Indeed, long confined to the Sahara, it shows a geographical extension outside its historic homes of Biskra and Abadla. This form is progressing dangerously towards the highlands and the steppe regions of the country. The most interesting fact was the identification of L. tropica for the first time in North-Central and North-West Algeria in Algerian patients who had never left the national territory. Out of the 10 strains of L. tropica identified, 8 belonged to patients of Syrian origin and 2 to Algerian patients. L. tropica was reported for the first time in 2008 in 6 patients living in Constantine (North-East Algeria) and in 2017, still in the North-East of the country, in Annaba. The observation of L. tropica in the North and Northeast center of the country, where L. infantum and L. major coexist, suggests changes in the epidemiology of cutaneous leishmaniasis in Algeria, which highlights the interest of more investigations to better understand the transmission cycle of the different entities.


Assuntos
Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Argélia/epidemiologia , Animais , Humanos , Leishmania infantum/genética , Leishmania infantum/isolamento & purificação , Leishmania major/genética , Leishmania major/isolamento & purificação , Leishmania tropica/genética , Leishmania tropica/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Polimorfismo de Fragmento de Restrição , Zoonoses/epidemiologia , Zoonoses/parasitologia
8.
Nucleic Acids Res ; 47(22): 11637-11648, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31722422

RESUMO

Immunopathologies caused by Leishmania cause severe human morbidity and mortality. This protozoan parasite invades and persists inside host cells, resulting in disease development. Leishmania modifies the epigenomic status of the host cells, thus probably averting the host cell defense mechanism. To accomplish this, Leishmania may change the host cell chromatin structure. However, the mechanism by which the parasite changes the host cell chromatin has not been characterized. In the present study, we found that ectopically produced Leishmania histone H3, LmaH3, which mimics the secreted LmaH3 in infected cells, is incorporated into chromatin in human cells. A crystallographic analysis revealed that LmaH3 forms nucleosomes with human histones H2A, H2B and H4. We found that LmaH3 was less stably incorporated into the nucleosome, as compared to human H3.1. Consistently, we observed that LmaH3-H4 association was remarkably weakened. Mutational analyses revealed that the specific LmaH3 Trp35, Gln57 and Met98 residues, which correspond to the H3.1 Tyr41, Arg63 and Phe104 residues, might be responsible for the instability of the LmaH3 nucleosome. Nucleosomes containing LmaH3 resisted the Mg2+-mediated compaction of the chromatin fiber. These distinct physical characteristics of LmaH3 support the possibility that histones secreted by parasites during infection may modulate the host chromatin structure.


Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Leishmania major/imunologia , Nucleossomos/metabolismo , Linhagem Celular Tumoral , Células HeLa , Histonas/genética , Humanos , Leishmania major/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Processamento de Proteína Pós-Traducional/fisiologia
9.
Biochemistry ; 58(49): 5011-5021, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31743022

RESUMO

Class I fumarate hydratases (FHs) are central metabolic enzymes that use a [4Fe-4S] cluster to catalyze the reversible conversion of fumarate to S-malate. The parasite Leishmania major, which is responsible for leishmaniasis, expresses two class I FH isoforms: mitochondrial LmFH-1 and cytosolic LmFH-2. In this study, we present kinetic characterizations of both LmFH isoforms, present 13 crystal structures of LmFH-2 variants, and employ site-directed mutagenesis to investigate the enzyme's mechanism. Our kinetic data confirm that both LmFH-1 and LmFH-2 are susceptible to oxygen-dependent inhibition, with data from crystallography and electron paramagnetic resonance spectroscopy showing that oxygen exposure converts an active [4Fe-4S] cluster to an inactive [3Fe-4S] cluster. Our anaerobically conducted kinetic studies reveal a preference for fumarate over S-malate. Our data further reveal that single alanine substitutions of T467, R421, R471, D135, and H334 decrease kcat values 9-16000-fold without substantially affecting Km values, suggesting that these residues function in catalytic roles. Crystal structures of LmFH-2 variants are consistent with this idea, showing similar bidentate binding to the unique iron of the [4Fe-4S] cluster for substrate S-malate as observed in wild type FH. We further present LmFH-2 structures with substrate fumarate and weak inhibitors succinate and malonate bound in the active site and the first structure of an LmFH that is substrate-free and inhibitor-free, the latter showing increased mobility in the C-terminal domain. Collectively, these data provide insight into the molecular basis for the reaction catalyzed by LmFHs, enzymes that are potential drug targets against leishmaniasis.


Assuntos
Fumarato Hidratase/química , Fumarato Hidratase/metabolismo , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/metabolismo , Leishmania major/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Fumarato Hidratase/genética , Proteínas com Ferro-Enxofre/genética , Cinética , Leishmania major/química , Leishmania major/genética , Família Multigênica , Oxigênio/química , Oxigênio/metabolismo , Proteínas de Protozoários/genética
10.
PLoS Negl Trop Dis ; 13(11): e0007865, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738761

RESUMO

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis and is caused by several species of Leishmania parasite. Clinical presentation of CL varies from a self-healing infection to a chronic form of the disease determined by the virulence of infecting Leishmania species and host immune responses to the parasite. Mouse models of CL show contradictory roles of lymphocytes in pathogenesis, while acquired immune responses are responsible for host protection from diseases. To reconcile the inconclusive roles of acquired immune responses in pathogenesis, we infected mice from various genetic backgrounds with two pathogenic strains of Leishmania major, Friedlin or 5ASKH, and assessed the outcome of the infections. Our findings showed that the genetic backgrounds of L. major determine the impact of lymphocytes for pathogenesis. In the absence of lymphocytes, L. major Friedlin induced the lowest inflammatory reaction and pathology at the site of infection, while 5ASKH infection induced a strong inflammatory reaction and severe pathology. Lymphocytes ameliorated 5ASKH mediated pathology, while it exacerbated pathology during Friedlin infection. Excess inflammatory reactions, like the recruitment of macrophages, neutrophils, eosinophils and production of pro-inflammatory cytokines, together with uncontrolled parasite growth in the absence of lymphocytes during 5ASKH infection may induce severe pathology development. Taken together our study provides insight into the impact of differences in the genetic background of Leishmania on CL pathogenesis.


Assuntos
Leishmania major/crescimento & desenvolvimento , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Linfócitos/imunologia , Imunidade Adaptativa , Animais , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Leishmania major/genética , Camundongos Endogâmicos BALB C
11.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527128

RESUMO

The molecular mechanisms underlying biological differences between two Leishmania species that cause cutaneous disease, L. major and L. amazonensis, are poorly understood. In L. amazonensis, reactive oxygen species (ROS) signaling drives differentiation of nonvirulent promastigotes into forms capable of infecting host macrophages. Tight spatial and temporal regulation of H2O2 is key to this signaling mechanism, suggesting a role for ascorbate-dependent peroxidase (APX), which degrades mitochondrial H2O2 Earlier studies showed that APX-null L. major parasites are viable, accumulate higher levels of H2O2, generate a greater yield of infective metacyclic promastigotes, and have increased virulence. In contrast, we found that in L. amazonensis, the ROS-inducible APX is essential for survival of all life cycle stages. APX-null promastigotes could not be generated, and parasites carrying a single APX allele were impaired in their ability to infect macrophages and induce cutaneous lesions in mice. Similar to what was reported for L. major, APX depletion in L. amazonensis enhanced differentiation of metacyclic promastigotes and amastigotes, but the parasites failed to replicate after infecting macrophages. APX expression restored APX single-knockout infectivity, while expression of catalytically inactive APX drastically reduced virulence. APX overexpression in wild-type promastigotes reduced metacyclogenesis, but enhanced intracellular survival following macrophage infection or inoculation into mice. Collectively, our data support a role for APX-regulated mitochondrial H2O2 in promoting differentiation of virulent forms in both L. major and L. amazonensis Our results also uncover a unique requirement for APX-mediated control of ROS levels for survival and successful intracellular replication of L. amazonensis.


Assuntos
Ascorbato Peroxidases/metabolismo , Leishmania major/patogenicidade , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/patologia , Macrófagos/parasitologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Ascorbato Peroxidases/genética , Células Cultivadas , Leishmania major/genética , Leishmania major/metabolismo , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , Virulência
12.
J Vector Borne Dis ; 56(2): 98-104, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31397384

RESUMO

Background & objectives: Leishmania parasites cause various clinical symptoms in humans such as cutaneous ulcers and fatal visceral diseases. These parasites cannot synthesize purine rings de novo and must uptake purines from their hosts via salvage. Salvage is regulated by permeases in the cell membrane. There are hundreds of membrane transporter proteins to receive nutrients in Leishmania. Nucleoside transporter 4 (NT4) is one of the purine transporters that is involved in enhancing the uptake of adenine in Leishmania major. They are important new drug targets for the treatment of leishmaniasis because they can be used to transport toxic purine analogs to kill parasitic cells, thus preventing the progression of the infection. The present study was conducted to silence the NT4 nucleobase involved in the salvage pathway to interrupt purine nucleotide membrane transport in the cells of L. major. Methods: In this study, a 502 bp segment of NT4 gene sequence was selected and designed as antisense transcripts after insertion in the parasite. The NT4 construct was transfected into L. major promastigotes for in vitro study of gene expression. Then, BALB/c mice infected with transgenic Leishmania and wild-type strain along with the number and size of lesions were studied in vivo. Results: The study showed that relative expression of NT4 gene in mutant Leishmania was lower than in the control on Day 3 to 20. The percentages and the number of amastigotes in infected macrophages with wild-type strain L. major were more than infected macrophages with mutant parasites. Infected BALB/c mice with transgenic Leish- mania showed a lower number and size of lesions than the BALB/c mice infected with wild-type strain. Interpretation & conclusion: The results of the study indicated that the use of antisense RNA reduced NT4 gene expression in L. major. Further, studies are needed to ascertain that the use of antisense can be considered as a new treatment for leishmaniasis.


Assuntos
Leishmania major/genética , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Protozoários/genética , RNA Antissenso , Animais , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Leishmaniose Cutânea/terapia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C
13.
Int J Infect Dis ; 88: 14-20, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31442631

RESUMO

OBJECTIVES: Local health personnel have drawn attention to an apparent increase in incidence and severity of cutaneous leishmaniasis (CL) in Sudan. The objective of this study was to investigate CL burden and surveillance. METHODS: Surveillance data were compiled from the KalaCORE programme, Leishmania coordinators in Northern Kordofan and Southern Darfur, and Khartoum Dermatology Hospital. CL lesions were sampled from 14 suspected cases from Northern Kordofan and the Hospital for Tropical Diseases in Omdurman. PCR-restriction fragment length polymorphism analysis and multilocus sequencing were used to characterize the disease agent. RESULTS: All sites reported substantial increases from 2014 to 2016/7, far exceeding World Health Organization case reports for 2014, consistent with a widespread outbreak. Single seasonal peak incidence was observed, except for two peaks in Southern Darfur. In Northern Kordofan, the odds ratio for CL in the 35-44 years age group was 2.6 times higher than in the >45 years age group (p<0.0001); in Southern Darfur, the OR was 2.38 greater in males than females (p<0.0001). Lesions included severe presentations, despite chemotherapy. Leishmania major was identified as the agent. CONCLUSIONS: Active surveillance is required to understand the extent of CL in Sudan, as well as training to standardize surveillance, diagnosis, reporting, and quality control. Point-of-care rapid diagnosis would be valuable. Genotyping and phenotyping are required to monitor the emergence of pathogenic strains, drug resistance, outbreaks, and changes in severity.


Assuntos
Surtos de Doenças , Leishmania major/genética , Leishmaniose Cutânea/epidemiologia , Adolescente , Adulto , Criança , Pré-Escolar , Monitoramento Epidemiológico , Feminino , Humanos , Incidência , Lactente , Leishmania major/isolamento & purificação , Leishmaniose Cutânea/parasitologia , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Fragmento de Restrição , Sudão/epidemiologia , Organização Mundial da Saúde , Adulto Jovem
14.
Mol Biol Rep ; 46(5): 5371-5388, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385238

RESUMO

Drug resistance is a complex phenomenon during leishmaniasis chemotherapy. In this study, the genes and pathways involved in miltefosine (MIL)-resistant Leishmania were identified using microarray data and in silico approaches. GSE30685 and GSE45496 were obtained from GEO database and analyzed with GEO2R tool to identify genes involved in MIL-resistant Leishmania. 177 differentially expressed genes (DEGs) were selected from these GSEs, which about half of them were uncharacterized/hypothetical proteins. The interactions between DEGs were investigated using STRING database and protein-protein interaction (PPI) networks. Five hub nodes were found in the PPI network. The gene ontology (GO) analysis of the resulting network revealed that DNA replication (GO:0006260) and ATP hydrolysis coupled proton transport (GO:0015991) were the most enriched GO term. Iranian MIL-resistant Leishmania major (L. major) parasites were generated by exposure of wild-type isolates to the increasing concentrations of MIL over a period of 5 months. Proof of mRNA expression levels of the obtained hub genes was assessed in Iranian wild-type and acquired resistant L. major parasites by real-time PCR. A significant higher expression level of LDBPK_150170 (encoding protein phosphatase 2C, PP2C), was only observed in Iranian L. major parasites resistance to MIL. Moreover, the RT-PCR results showed that the expression of metacyclic marker (small hydrophilic endoplasmic reticulum-associated protein, SHERP) and MIL-resistant marker (Leishmania MIL-transporter, LMT) was significantly increased and decreased, respectively, in Iranian MIL-resistant L. major parasites. Taken together, these data suggested that PP2C as well as SHERP and LMT genes may be prospective targets for the treatment of MIL-resistant Leishmania.


Assuntos
Farmacorresistência Fúngica/genética , Leishmania major/genética , Mapas de Interação de Proteínas/genética , Biologia Computacional/métodos , Simulação por Computador , Resistência a Medicamentos/genética , Ontologia Genética , Irã (Geográfico) , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia
15.
Nat Chem ; 11(7): 629-637, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209299

RESUMO

In DNA, the loss of a nucleobase by hydrolysis generates an abasic site. Formed as a result of DNA damage, as well as a key intermediate during the base excision repair pathway, abasic sites are frequent DNA lesions that can lead to mutations and strand breaks. Here we present snAP-seq, a chemical approach that selectively exploits the reactive aldehyde moiety at abasic sites to reveal their location within DNA at single-nucleotide resolution. Importantly, the approach resolves abasic sites from other aldehyde functionalities known to exist in genomic DNA. snAP-seq was validated on synthetic DNA and then applied to two separate genomes. We studied the distribution of thymine modifications in the Leishmania major genome by enzymatically converting these modifications into abasic sites followed by abasic site mapping. We also applied snAP-seq directly to HeLa DNA to provide a map of endogenous abasic sites in the human genome.


Assuntos
DNA/genética , Genoma/genética , Análise de Sequência de DNA/métodos , Aldeídos/química , Sequência de Bases , DNA/química , Dano ao DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Leishmania major/genética , Sondas Moleculares/síntese química , Sondas Moleculares/química , Timina/química , Uracila-DNA Glicosidase/química
16.
Acta Parasitol ; 64(3): 645-651, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31111360

RESUMO

PURPOSE: Leishmaniasis, as one of the most important vector-borne and zoonotic diseases, can be seen in different forms and is more prevalent in developing countries worldwide. Due to the absence of effective strategies in its prevention, treatment, and control, investigation of effective control strategies against the disease is necessary. In this research, we evaluated the immunogenicity of a cold-adapted laboratory strain of Leishmania major (LMC) in the mouse model. METHODS: Twenty BALB/c mice were divided into two groups. LMC group received 4 × 106 of LMC strain in 0.5 ml DMEM, and VLM group, as the control group, received 0.5 ml Dulbecco's modified Eagle's medium. Both groups were challenged with virulent L. major 3 weeks after inoculation. RESULTS: The data obtained from the analysis of immune responses and histopathological changes interestingly revealed protection against L. major in immunized mice. Compared with the VLM group, the mice immunized with LMC strain of L. major in the LMC group showed a significant increase in IFN-γ and IgG2a levels (P < 0.05) which are important indexes for Th1-related immune responses. Additionally, significant differences in concentration of IgG1 and IgG total before and after the challenge was observed in LMC group (P < 0.05). Furthermore, the immunized mice showed a significant reduction in mean sizes of skin lesion and liver damage compared to the VLM group. CONCLUSION: Based on the present findings on immunogenicity of LMC strain, it seems this strain is able to induce both humoral and cellular immunity and a significant protection against L. major in the mouse model.


Assuntos
Leishmania major/imunologia , Leishmaniose/imunologia , Leishmaniose/prevenção & controle , Animais , Anticorpos Antiprotozoários/imunologia , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Imunização , Leishmania major/genética , Leishmaniose/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Células Th1/imunologia
17.
Am J Trop Med Hyg ; 101(1): 101-107, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31094311

RESUMO

Cutaneous leishmaniasis (CL) remains one of the world's most prevalent neglected diseases, particularly in developing countries. Identification of the involved Leishmania species is an important step in the diagnosis and case management process. In this study, we tested simple, rapid, and highly sensitive loop-mediated isothermal amplification (LAMP) assays for Leishmania DNA species-specific detection from cutaneous lesions. Two LAMP assays, targeting cysteine protease B (cpb) gene, were developed to detect and identify Leishmania major and Leishmania tropica species. Loop-mediated isothermal amplification specificity was examined using DNA samples from other Leishmania species and Trypanosoma species. No cross-reactions were detected. The developed LAMP assays exhibited sensitivity with a detection limit of 20 fg and 200 fg for L. major and L. tropica, respectively. Both tests were applied on clinical samples of CL suspected patients living in endemic Tunisian regions and compared with kinetoplast DNA quantitative PCR (qPCR), microscopic, and conventional cpb-based polymerase chain reaction (PCR) assays. Our LAMP tests were able to discriminate between L. major and L. tropica species and showed a sensitivity of 84% and a specificity of 100%. However, when compared with the performance of the diagnostic tests with latent class analysis (LCA), our LAMP assays show a sensitivity of 100%. These assays can be used as a first-line molecular test for early diagnosis and prompt management of CL cases in public health programs.


Assuntos
Leishmania major/genética , Leishmania tropica/genética , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , Humanos , Leishmaniose Cutânea/parasitologia , Doenças Negligenciadas/diagnóstico , Doenças Negligenciadas/epidemiologia , Doenças Negligenciadas/parasitologia , Técnicas de Amplificação de Ácido Nucleico , Especificidade da Espécie , Tunísia/epidemiologia
18.
PLoS Genet ; 15(5): e1008042, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31091230

RESUMO

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.


Assuntos
Genoma de Protozoário , Leishmania infantum/genética , Leishmania major/genética , Leishmania tropica/genética , Animais , Sequência de Bases , Quimera , Mapeamento Cromossômico , Cruzamentos Genéticos , Genótipo , Padrões de Herança , Insetos Vetores/parasitologia , Leishmania infantum/metabolismo , Leishmania major/metabolismo , Leishmania tropica/metabolismo , Meiose , Psychodidae/parasitologia , Recombinação Genética , Sequenciamento Completo do Genoma
19.
Sci Rep ; 9(1): 6919, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061406

RESUMO

Besides their medical relevance, Leishmania is an adequate model for studying post-transcriptional mechanisms of gene expression. In this microorganism, mRNA degradation/stabilization mechanisms together with translational control and post-translational modifications of proteins are the major drivers of gene expression. Leishmania parasites develop as promastigotes in sandflies and as amastigotes in mammalians, and during host transmission, the parasite experiences a sudden temperature increase. Here, changes in the transcriptome of Leishmania major promastigotes after a moderate heat shock were analysed by RNA-seq. Several of the up-regulated transcripts code for heat shock proteins, other for proteins previously reported to be amastigote-specific and many for hypothetical proteins. Many of the transcripts experiencing a decrease in their steady-state levels code for transporters, proteins involved in RNA metabolism or translational factors. In addition, putative long noncoding RNAs were identified among the differentially expressed transcripts. Finally, temperature-dependent changes in the selection of the spliced leader addition sites were inferred from the RNA-seq data, and particular cases were further validated by RT-PCR and Northern blotting. This study provides new insights into the post-transcriptional mechanisms by which Leishmania modulate gene expression.


Assuntos
Perfilação da Expressão Gênica , Resposta ao Choque Térmico/genética , Leishmania major/genética , Leishmania major/fisiologia , RNA-Seq , Processamento Alternativo , Regulação para Baixo , Fases de Leitura Aberta/genética , RNA Mensageiro/genética
20.
Biomed Res Int ; 2019: 1425281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058184

RESUMO

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.


Assuntos
Leishmania major/genética , RNA Polimerase III/genética , Fator de Transcrição TFIIIB/genética , Transcrição Genética , Simulação por Computador , Sequência Conservada/genética , Regulação da Expressão Gênica/genética , Recombinação Homóloga/genética , Proteínas Mutantes/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Subunidades Proteicas/genética , RNA Ribossômico 5S/biossíntese , RNA Nuclear Pequeno/biossíntese , RNA de Transferência/biossíntese
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