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1.
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
2.
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
3.
Biochim Biophys Acta Gen Subj ; 1864(6): 129558, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32061714

RESUMO

BACKGROUND: Cutaneous leishmaniasis is a parasitic disease, which is difficult to treat due to high drug resistance and adverse side effects. Photodynamic therapy by ultraviolet radiation using materials with high photocatalytic features like titanium dioxide nanoparticles (TiO2-NPs) is an emerging treatment for this disease. In this study, TiO2-NPs with ultraviolet (UV) radiation were administered as photodynamic therapy against Leishmania Major (LM) promastigotes. METHODS: Two forms of TiO2 viz. including Anatase and Rutile were administered in two UV ranges< UVA and UVB for different time periods (30 and 60 min). Finally, 24 and 48 h after incubation, the MTS test was performed and cell survival percentage was calculated. RESULTS: The mean size of Anatase and Rutile-NPs is approximately 32.5 and 50.9 nm respectively by DLS and FE-SEM, and crystal phase is emphasized by XRD. The combined treatment of LM with TiO2-NPs and UV has significant effects on LM promastigotes, which vary depending on NP and UV types. The synergistic effect was anticipated in the groups irradiated by UV-B in the presence of Rutile NPs. CONCLUSION: The combined treatment with UV- radiation and TiO2-NPs can be effective in killing the promastigotes of Leishmania major. The proper concentration of NPs and the type of UV-radiation must be taken into consideration. The results suggest improved treatment methods, after proper in vivo studies.


Assuntos
Leishmaniose Cutânea/terapia , Nanopartículas Metálicas/química , Titânio/farmacologia , Terapia Ultravioleta , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos , Leishmania major/efeitos dos fármacos , Leishmania major/patogenicidade , Leishmania major/efeitos da radiação , Leishmaniose Cutânea/parasitologia , Nanopartículas Metálicas/administração & dosagem , Microscopia Eletrônica de Transmissão , Fotoquimioterapia/métodos , Titânio/química
4.
PLoS Negl Trop Dis ; 14(1): e0006596, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31923234

RESUMO

An association between increased susceptibility to infectious diseases and obesity has been described as a result of impaired immunity in obese individuals. It is not clear whether a similar linkage can be drawn between obesity and parasitic diseases. To evaluate the effect of obesity in the immune response to cutaneous Leishmania major infection, we studied the ability of C57BL/6 mice fed a hypercaloric diet (HSB) to control leishmaniasis. Mice with diet-induced obesity presented thicker lesions with higher parasite burden and a more intense inflammatory infiltrate in the infected ear after infection with L. major. There was no difference between control and obese mice in IFN-gamma or IL-4 production by auricular draining lymph node cells, but obese mice produced higher levels of IgG1 and IL-17. Peritoneal macrophages from obese mice were less efficient to kill L. major when infected in vitro than macrophages from control mice. In vitro stimulation of macrophages with IL-17 decreased their capacity to kill the parasite. Moreover, macrophages from obese mice presented higher arginase activity. To confirm the role of IL-17 in the context of obesity and infection, we studied lesion development in obese IL-17R-/- mice infected with L. major and found no difference in skin lesions and the leukocyte accumulation in the draining lymph node is redcuced in knockout mice compared between obese and lean animals. Our results indicate that diet-induced obesity impairs resistance to L. major in C57BL/6 mice and that IL-17 is involved in lesion development.


Assuntos
Leishmania major/patogenicidade , Leishmaniose Cutânea/imunologia , Obesidade , Animais , Dieta/efeitos adversos , Orelha/parasitologia , Feminino , Interferon gama , Interleucina-17 , Leishmaniose Cutânea/parasitologia , Linfonodos/citologia , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Risco
5.
Cell Mol Life Sci ; 77(9): 1827-1845, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31372684

RESUMO

Leishmaniasis comprises a group of neglected diseases caused by the protozoan parasite Leishmania spp. As is the case for other trypanosomatid parasites, Leishmania is auxotrophic for heme and must scavenge this essential compound from its human host. In mammals, the SLC transporter FLVCR2 mediates heme import across the plasma membrane. Herein we identify and characterize Leishmania major FLVCRb (LmFLVCRb), the first member of the FLVCR family studied in a non-metazoan organism. This protein localizes to the plasma membrane of the parasite and is able to bind heme. LmFLVCRb levels in Leishmania, which are modulated by overexpression thereof or the abrogation of an LmFLVCRb allele, correlate with the ability of the parasite to take up porphyrins. Moreover, injection of LmFLVCRb cRNA to Xenopus laevis oocytes provides these cells with the ability to take up heme. This process is temperature dependent, requires monovalent ions and is inhibited at basic pH, characteristics shared by the uptake of heme by Leishmania parasites. Interestingly, LmFLVCRb is essential as CRISPR/Cas9-mediated knockout parasites were only obtained in the presence of an episomal copy of the gene. In addition, deletion of just one of the alleles of the LmFLVCRb gene markedly impairs parasite replication as intracellular amastigotes as well as its virulence in an in vivo model of cutaneous leishmaniasis. Collectively, these results show that Leishmania parasites can rescue heme through plasma membrane transporter LFLVCRb, which could constitute a novel target for therapeutic intervention against Leishmania and probably other trypanosomatid parasites in which FLVCR genes are also present.


Assuntos
Heme/metabolismo , Leishmania major/metabolismo , Leishmaniose/parasitologia , Macrófagos/parasitologia , Proteínas de Membrana Transportadoras/metabolismo , Porfirinas/metabolismo , Proteínas de Protozoários/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Leishmania major/patogenicidade , Leishmaniose/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/genética , Oócitos/metabolismo , Oócitos/parasitologia , Proteínas de Protozoários/genética , Receptores Virais/genética , Homologia de Sequência , Virulência , Xenopus laevis
6.
Biomolecules ; 9(11)2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31718000

RESUMO

The identification and clarification of the mechanisms of action of drugs used against leishmaniasis may improve their administration regimens and prevent the development of resistant strains. Herein, for the first time, we describe the structure of the putatively essential Ser/Thr kinase LmjF.22.0810 from Leishmania major. Molecular dynamics simulations were performed to assess the stability of the kinase model. The analysis of its sequence and structure revealed two druggable sites on the protein. Furthermore, in silico docking of small molecules showed that aminoglycosides preferentially bind to the phosphorylation site of the protein. Given that transgenic LmjF.22.0810-overexpressing parasites displayed less sensitivity to aminoglycosides such as paromomycin, our predicted models support the idea that the mechanism of drug resistance observed in those transgenic parasites is the tight binding of such compounds to LmjF.22.0810 associated with its overexpression. These results may be helpful to understand the complex machinery of drug response in Leishmania.


Assuntos
Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Paromomicina/efeitos adversos , Proteínas Serina-Treonina Quinases/genética , Antiprotozoários , Resistência a Medicamentos/genética , Humanos , Leishmania major/enzimologia , Leishmania major/patogenicidade , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/parasitologia , Simulação de Dinâmica Molecular , Paromomicina/química , Proteínas Serina-Treonina Quinases/química
7.
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
8.
Exp Parasitol ; 204: 107728, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31348915

RESUMO

BACKGROUND: Co-infection with Leishmania major and Schistosoma mansoni may have significant consequences for disease progression, severity and subsequent transmission dynamics. Pentavalent antimonials and Praziquantel (PZQ) are used as first line of treatment for Leishmania and Schistosoma infections respectively. However, there is limited insight on how combined therapy with the standard drugs impacts the host in comorbidity. The study aimed to determine the efficacy of combined chemotherapy using Pentostam (P) and PZQ in murine model co-infected with L. major and S. mansoni. METHODS: A 3 × 4 factorial design with three parasite infection groups (Lm, Sm, Lm + Sm to represent L. major, S. mansoni and L. major + S. mansoni respectively) and four treatment regimens [P, PZQ, P + PZQ, and PBS designating Pentostam (GlaxoSmithKline UK), Praziquantel (Biltricide®, Bayer Ag. Leverkusen, Germany), Pentostam + Praziquantel and Phosphate buffered saline] as factors was applied. RESULTS: Significant changes were observed in the serum Interferon gamma (IFN-γ), and Macrophage inflammatory protein-one alpha (MIP-1α) levels among various treatment groups between week 8 and week 10 (p < 0.05). There was increased IFN-γ in the L. major infected mice subjected to PZQ and PBS, and in L. major + S. mansoni infected BALB/c mice treated with P + PZQ. Subsequently, MIP-1α levels increased significantly in both the L. major infected mice under PZQ and PBS and in L. major + S. mansoni infected BALB/c mice undergoing concurrent chemotherapy with P + PZQ between 8 and 10 weeks (p < 0.05). In the comorbidity, simultaneous chemotherapy resulted in less severe histopathological effects in the liver. CONCLUSION: It was evident, combined first line of treatment is a more effective strategy in managing co-infection of L. major and S. mansoni. The findings denote simultaneous chemotherapy compliments immunomodulation in the helminth-protozoa comorbidity hence, less severe pathological effects following the parasites infection. Recent cases of increased incidences of polyparasitism in vertebrates call for better ways to manage co-infections. The findings presented necessitate intrinsic biological interest on examining optimal combined chemotherapeutic agents strategies in helminth-protozoa concomitance and the related infections abatement trends vis-a-vis host-parasite relationships.


Assuntos
Anti-Helmínticos/uso terapêutico , Antiprotozoários/uso terapêutico , Comorbidade , Leishmania major/patogenicidade , Leishmaniose Cutânea/complicações , Esquistossomose mansoni/complicações , Análise de Variância , Animais , Anti-Helmínticos/administração & dosagem , Gluconato de Antimônio e Sódio/administração & dosagem , Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/administração & dosagem , Quimiocina CCL3/sangue , Modelos Animais de Doenças , Quimioterapia Combinada , Interferon gama/sangue , Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Fígado/parasitologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Praziquantel/administração & dosagem , Praziquantel/uso terapêutico , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/patogenicidade , Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/patologia
9.
PLoS One ; 14(7): e0219985, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339931

RESUMO

BACKGROUND: In vitro assays are widely used in studies on pathogen infectivity, immune responses, drug and vaccine discovery. However, most in vitro assays display significant differences to the in vivo situation and limited predictive properties. We applied medium perfusion methods to mimic interstitial fluid flow to establish a novel infection model of Leishmania parasites. METHODS: Leishmania major infection of mouse peritoneal macrophages was studied within the Quasi Vivo QV900 macro-perfusion system. Under a constant flow of culture media at a rate of 360µl/min, L. major infected macrophages were cultured either at the base of a perfusion chamber or raised on 9mm high inserts. Mathematical and computational modelling was conducted to estimate medium flow speed, shear stress and oxygen concentration. The effects of medium flow on infection rate, intracellular amastigote division, macrophage phagocytosis and macropinocytosis were measured. RESULTS: Mean fluid speeds at the macrophage cell surface were estimated to be 1.45 x 10-9 m/s and 1.23 x 10-7 m/s for cells at the base of the chamber and cells on an insert, respectively. L. major macrophage infection was significantly reduced under both media perfusion conditions compared to cells maintained under static conditions; a 85±3% infection rate of macrophages at 72 hours in static cultures compared to 62±5% for cultures under slow medium flow and 55±3% under fast medium flow. Media perfusion also decreased amastigote replication and both macrophage phagocytosis (by 44±4% under slow flow and 57±5% under fast flow compared with the static condition) and macropinocytosis (by 40±4% under slow flow and 62±5% under fast flow compared with the static condition) as measured by uptake of latex beads and pHrodo Red dextran. CONCLUSIONS: Perfusion of culture medium in an in vitro L. major macrophage infection model (simulating in vivo lymphatic flow) reduced the infection rate of macrophages, the replication of the intracellular parasite, macrophage phagocytosis and macropinocytosis with greater reductions achieved under faster flow speeds.


Assuntos
Leishmania major/patogenicidade , Macrófagos/parasitologia , Cultura Primária de Células/métodos , Animais , Células Cultivadas , Macrófagos/imunologia , Camundongos , Fagocitose
10.
ACS Infect Dis ; 5(8): 1295-1305, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31094195

RESUMO

Cutaneous leishmaniasisis is the most common clinical form of leishmaniasis and one of the most relevant neglected diseases. It is known that the progress of the disease is species specific and the host's immune response plays an important role in its outcome. However, the pathways that lead to parasite clearance or survival remain unknown. In this work, skin tissue from mice experimentally infected with L. amazonensis, one of the causative agents of cutaneous leishmaniasis in the Amazon region, L. major, another causative agent of cutaneous leishmaniasis in Africa, the Middle East, China, and India, or lipopolysaccharides from Escherichia coli as an inflammation model were investigated using label-free proteomics to unveil Leishmania-specific protein alterations. Proteomics is a powerful tool to investigate host-pathogen relationships to address biological questions. In this work, proteins from mice skin biopsies were identified and quantified using nano-LC coupled with tandem mass spectrometry analyses. Integrated Proteomics Pipeline was used for peptide/protein identification and quantification. Western blot was used for validation of protein quantification by mass spectrometry, and protein pathways were predicted using Ingenuity Pathway Analysis. In this proteomics study, several proteins were pointed out as hypothetical targets to guide future studies on Leishmania-specific modulation of proteins in the host. We identified hundreds of exclusively modulated proteins after Leishmania spp. infection and 17 proteins that were differentially modulated in the host after L. amazonensis or L. major infection.


Assuntos
Interações Hospedeiro-Patógeno , Leishmania braziliensis/patogenicidade , Leishmania major/patogenicidade , Leishmaniose Cutânea/metabolismo , Proteômica , Pele/metabolismo , Animais , Biópsia , Feminino , Inflamação , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mapas de Interação de Proteínas , Proteínas/análise , Pele/parasitologia , Pele/patologia , Espectrometria de Massas em Tandem
11.
Biochem J ; 476(8): 1303-1321, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30988012

RESUMO

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania The modeled structure of N-terminal of this protein exhibits four antiparallel ß sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.


Assuntos
Autofagossomos/imunologia , Leishmania major , Macrófagos , Modelos Moleculares , Fosfoglicerato Quinase , Proteínas de Protozoários , Animais , Leishmania major/genética , Leishmania major/imunologia , Leishmania major/patogenicidade , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/deficiência , Fosfoglicerato Quinase/imunologia , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia
12.
PLoS Negl Trop Dis ; 12(10): e0006921, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30372439

RESUMO

Leishmania species are sand fly-transmitted protozoan parasites that cause leishmaniasis, neglected tropical diseases that affect millions of people. Leishmania amastigotes must overcome a variety of host defenses, including reactive oxygen species (ROS) produced by the NADPH oxidase. Leishmania species encode three superoxide dismutases (SODs): the mitochondrial SODA and two glycosomal SODs (SODB1 and SODB2). SODs are metalloenzymes that function in antioxidant defense by converting superoxide to oxygen and hydrogen peroxide. Here, we investigated a role for SODB1 in Leishmania infection of macrophages and virulence in mice. We found that a single allele deletion of SODB1 (SODB1/Δsodb1) had minimal effects on the replication of axenically-grown L. major promastigotes or differentiation to infective metacyclic promastigotes. Disruption of a single SODB1 allele also did not affect L. donovani differentiation to amastigotes induced axenically, or the replication of axenically-grown L. donovani promastigotes and amastigotes. In contrast, the persistence of SODB1/Δsodb1 L. major in WT macrophages was impaired, and the development of cutaneous lesions in SODB1/Δsodb1 L. major-infected C57BL/6 and BALB/c mice was strongly reduced. The reduced disease severity in mice was associated with reduced burdens of SODB1/Δsodb1 L. major parasites in the foot at late, but not early times post-inoculation, as well as an impaired capacity to disseminate from the site of inoculation. Collectively, these data suggest that SODB1 is critical for L. major persistence in macrophages and virulence in mice.


Assuntos
Leishmania major/enzimologia , Leishmania major/patogenicidade , Leishmaniose Cutânea/patologia , Macrófagos/imunologia , Macrófagos/parasitologia , Superóxido Dismutase/metabolismo , Fatores de Virulência/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Deleção de Genes , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leishmania donovani/patogenicidade , Leishmania major/genética , Leishmaniose Cutânea/parasitologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Carga Parasitária , Superóxido Dismutase/genética , Virulência , Fatores de Virulência/genética
13.
BMC Res Notes ; 11(1): 642, 2018 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-30180875

RESUMO

OBJECTIVE: The numbers of Leishmania major parasites in foot lesions of C57Bl/6, BALB/c or SCID mice can be significantly reduced by pre-exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). One potential mechanism to explain this enhanced resistance to infection is that TCDD is directly toxic to L. major. This potential mechanism was addressed by exposing L. major promastigotes and amastigotes to TCDD in vitro and examining their subsequent proliferation and infectivity. RESULTS: We found no significant change in the rate of in vitro L. major proliferation (promastigotes or amastigotes) after TCDD exposure at concentrations up to 100 nM. Moreover, in vitro TCDD exposure did not significantly alter the ability of L. major to infect mice, trigger lesion formation, or survive in those lesions.


Assuntos
Leishmania major/efeitos dos fármacos , Dibenzodioxinas Policloradas/farmacologia , Animais , Feminino , Leishmania major/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Dibenzodioxinas Policloradas/toxicidade
14.
Artigo em Inglês | MEDLINE | ID: mdl-30104270

RESUMO

The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side effects, and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism of action to avoid cross-resistance with the current drugs. As cyclic nucleotide-specific phosphodiesterases (PDEs) of Leishmania major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs were explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and to increase the cellular cyclic AMP (cAMP) level at in a dose-dependent manner at just 2× and 5× the 50% inhibitory concentration (IC50), indicating a correlation between antileishmanial activity and increased cellular cAMP levels. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy.


Assuntos
Leishmaniose/tratamento farmacológico , Leishmaniose/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Antiprotozoários/uso terapêutico , AMP Cíclico/metabolismo , Leishmania major/efeitos dos fármacos , Leishmania major/enzimologia , Leishmania major/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Fosfodiesterase/uso terapêutico
15.
Mediators Inflamm ; 2018: 9787128, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30150896

RESUMO

Certain cytokines modulate the expression of insulin-like growth factor- (IGF-) I. Since IL-4 and IGF-I promote growth of the protozoan Leishmania major, we here addressed their interaction in downregulating the expression of Igf-I mRNA using small interfering RNA (siRNA) in Leishmania major-infected macrophages. Parasitism was decreased in the siRNA-treated cells compared with the nontreated cells, reversed by the addition of recombinant IGF-I (rIGF-I). In IL-4-stimulated macrophages, parasitism and the Igf-I mRNA amount were increased, and the effects were nullified upon siRNA transfection. IGF-I downregulation inhibited both parasite and macrophage arginase activation even in IL-4-stimulated cells. Searching for intracellular signaling components shared by IL-4 and IGF-I, upon siRNA transfection, phosphorylated p44, p38, and Akt proteins were decreased, affecting the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. In L. major-infected C57BL6-resistant mice, the preincubation of the parasite with rIGF-I changed the infection profile to be similar to that of susceptible mice. We conclude that IGF-I constitutes an effector element of IL-4 involving the PI3K/Akt pathway during L. major infection.


Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-4/farmacologia , Leishmania major/metabolismo , Leishmania major/patogenicidade , Leishmaniose Cutânea/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Insulin-Like I/genética , Leishmaniose Cutânea/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7
16.
Parasitol Res ; 117(9): 2949-2956, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29978420

RESUMO

Leishmania (L.) tropica is a causative agent of cutaneous and occasionally visceral or viscerotropic leishmaniasis in humans. The dose of parasites influences the course and outcome of disease in some Leishmania species. The effect of parasite dose on L. tropica infection in an experimental model was studied in the current paper. High and low doses of L. tropica were used for ear infection of BALB/c mice and lesion development, parasite load, and cytokine responses were assessed. L. major infection was used for comparison. Pre-infected mice were challenged in the footpad by a fixed high dose of L. tropica, and immune response and protection level were evaluated. High dose L. tropica infection in comparison to low dose results in higher lesion diameters, higher load of parasite in draining lymph node, higher levels of interferon-γ and interleukin-10, dissemination of parasite to spleen, and induction of protection against further L. tropica challenge. Comparison of L. tropica with L. major showed that L. tropica results in lower lesion diameters, more potential for growth in lymph nodes at early phases of infection, parasite dissemination to spleen, lower levels of IL-10, and a permanent lower cytokine response against low parasite dose in comparison to high dose. Our findings suggest that for L. tropica infection, only the high dose results in visceralization of the parasite and protection against further challenge of L. tropica. Therefore, the parasite dose may be an important factor in pathogenesis and immunity in L. tropica infection.


Assuntos
Leishmania tropica/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Visceral/imunologia , Linfonodos/parasitologia , Carga Parasitária , Baço/parasitologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Interferon gama/sangue , Interleucina-10/sangue , Leishmania major/imunologia , Leishmania major/patogenicidade , Leishmania tropica/crescimento & desenvolvimento , Leishmania tropica/patogenicidade , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Pele/parasitologia , Virulência
17.
Cell Mol Biol (Noisy-le-grand) ; 64(8): 44-49, 2018 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-29981682

RESUMO

Leishmania major is an intracellular parasite generally responsible for cutaneous leishmaniasis (CL), one of the most encountered skin diseases especially in Pakistan, Iran, Iraq, and Saudi Arabia. Current treatment options are not ideal, due to unwanted side effects and increasing resistance and availability is often limited in developing countries. Medicinal plants continue to attract attention because of their beneficial effects in the prevention or/and accelerating the healing process of various diseases. In this study, in vitro and in vivo susceptibility of L. major to Veronica persica Poir. extract, a medicinal plant with many applications, has been evaluated. Antileishmanial activity of plant extract was investigated both on cultured L. major promastigotes and in mice challenged with L. major. Animals were divided into three groups including control (without any treatment), test (treated with plant extract) and glucantime (the reference drug) treated groups. After treatments, skin lesion sizes and body weights of animals were checked during 4 weeks. The potential of the plant extract in decreasing the number of parasites in spleen cells of animals as well as inducing the nitric oxide (NO) production by macrophage cells was also investigated. In vitro tests showed that the plant extract was able to reduce the survival time of promastigotes in a concentration-dependent manner. In vivo experiments also revealed a significant influence of V. persica extracts on accelerating the healing process as well as reducing the overall disease burden in animal model by inducing NO production in macrophage cells. Our findings indicated the promising potential of V. persica extract as an ideal candidate in the treatment of CL caused by L. major.


Assuntos
Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Extratos Vegetais/farmacologia , Veronica/química , Animais , Modelos Animais de Doenças , Humanos , Irã (Geográfico)/epidemiologia , Leishmania major/patogenicidade , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Paquistão , Extratos Vegetais/química , Plantas Medicinais/química
18.
J Dermatol Sci ; 92(1): 78-88, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30037731

RESUMO

BACKGROUND: Cutaneous leishmaniasis (CL) skin lesions are the result of a deregulated immune response, which is unable to eliminate Leishmania parasites. The control of both, parasites and host immune response, is critical to prevent tissue destruction. The skin ulceration has been correlated with high TNF-α level. OBJECTIVE: Because human anti-TNF-α antibodies (Ab) have been successfully assayed in several mice inflammatory diseases, we hypothesized that their anti-inflammatory effect could optimize the healing of CL lesions achieved after topical application of paromomycin (PM), the current chemotherapy against CL. METHODS AND RESULTS: We first compared the in vitro efficacy of PM and Ab alone and the drug given in combination with Ab to assess if the Ab could interfere with PM leishmanicidal activity in L. major-infected bone marrow-derived macrophages. The combination therapy had similar antileishmanial activity to the drug alone and showed no influence on NO production, which allows macrophage-mediated parasite killing. Next, we demonstrated in an in vivo model of Imiquimod®-induced inflammation that topical Ab and PM inhibit the infiltration of inflammatory cells in the skin. In the efficacy studies in L. major-infected BALB/c mice, PM combined with Ab led to a sharp infection reduction and showed a stronger anti-inflammatory activity than PM alone. This was confirmed by the down-regulation of TNF-α, IL-1ß, iNOS, IL-17, and CCL3 as well as by a decrease of the neutrophilic infiltrate during infection upon treatment with the Ab. CONCLUSIONS: In terms of parasite elimination and inflammation reduction, topical application of Ab in combination with PM was more effective than the drug alone.


Assuntos
Anticorpos/farmacologia , Antiprotozoários/farmacologia , Dermatite/tratamento farmacológico , Mediadores da Inflamação/antagonistas & inibidores , Leishmania major/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Paromomicina/farmacologia , Pele/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Células Cultivadas , Dermatite/etiologia , Dermatite/imunologia , Dermatite/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Interações Hospedeiro-Patógeno , Imiquimode , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Leishmania major/imunologia , Leishmania major/patogenicidade , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos Endogâmicos BALB C , Infiltração de Neutrófilos/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Pele/parasitologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
19.
Exp Parasitol ; 192: 73-84, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30040961

RESUMO

The current study was designed to assess the anti-leishmanial effect of a semi-purified fraction of wild mushroom Grifola frondosa against Leishmania donovani, in vitro. A total of five extracts from three wild mushrooms [Grifola frondosa (family, Meripilaceae) Laetiporus sulphurous (family, Polyporaceae) and Meripilus giganteus (family, Meripilaceae) were explored for novel anti-leishmanial leads against promastigotes. The ethanol extract of G. frondosa was selected as the most efficient against L. donovani promastigotes (IC50: 93.9 µg/mL). A semi-purified fraction was obtained from an active ethanol extract of G. frondosa and found to inhibit the survival of promastigotes of L. donovani (MHOM/IN/83/AG83) significantly (IC50: 20.37 µg/mL) and it also had some effect against L. major LV39 (MRHO/Sv/59/P strain) and L. tropica WR683 (MHOM/SU/58/OD) strains at higher concentrations (IC50: 46.08 µg/mL and 53.79 µg/mL respectively). The semi-purified fraction also interfered in lipid biosynthesis, altered parasite morphology and induced apoptosis in L. donovani promastigotes. The semi-purified fraction was also effective against intracellular amastigotes in infected macrophages and enhanced the release of nitric oxide and pro-inflammatory cytokines, in vitro. Interestingly, the 50% inhibitory concentration of the semi-purified fraction against the intracellular amastigotes (IC50: 2.48 µg/mL) was much lower in comparison to promastigotes (IC50: 20.37 µg/mL). The semi-purified fraction was found to inhibit the intracellular amastigotes slightly more efficiently in comparison to conventional anti-leishmanial drugs; sodium antimony gluconate, amphotericin B, miltefosine and paromomycin and noticeably non-toxic towards host splenocytes. The findings of the present study established that G. frondosa might be a natural resource for development of a new anti-leishmanial lead.


Assuntos
Grifola/química , Leishmania donovani/efeitos dos fármacos , Animais , Cromatografia em Gel , Citocinas/genética , Citocinas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Concentração Inibidora 50 , Leishmania donovani/patogenicidade , Leishmania donovani/ultraestrutura , Leishmania major/patogenicidade , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Polyporaceae/química , Polyporales/química , Baço/citologia , Baço/efeitos dos fármacos , Virulência
20.
Biochem Biophys Res Commun ; 503(1): 371-377, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-29906460

RESUMO

Leishmania promastigotes have the ability to synthesize essential polyunsaturated fatty acids de novo and can grow in lipid free media. Recently, we have shown that NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) enzyme in Leishmania acts as the redox partner for Δ12 fatty acid desaturase, which catalyses the conversion of oleate to linoleate. So far, the exact role of Leishmania derived linoleate synthesis is still incomplete in the literature. The viability assay by flow cytometry as well as microscopic studies suggests that linoleate is an absolute requirement for Leishmania promastigote survival in delipidated media. Western blot analysis suggested that infection with log phase linoleate deficient mutant (KO) results in increased level of NF-κBp65, IκB and IKKß phosphorylation in RAW264.7 cells. Similarly, the log phase KO infected RAW264.7 cells show dramatic increment of COX-2 expression and TNF-α secretion, compared to control or Ncb5or complement (CM) cell lines. The activation of inflammatory signaling pathways by KO mutant is significantly reduced when the RAW264.7 cells are pre-treated with BSA bound linoleate. Together, these findings confirmed that the leishmanial linoleate inhibits both COX-2 and TNF-α expression in macrophage via the inactivation of NF-κB signaling pathway. The stationary phase of KO promastigotes shows avirulence after infection in macrophages as well as inoculation into BALB/c mice; whereas CM cell lines show virulence. Collectively, these data provide strong evidence that de novo linoleate synthesis in Leishmania is an essential for parasite survival at extracellular promastigote stage as well as intracellular amastigote stage.


Assuntos
Citocromo-B(5) Redutase/genética , Deleção de Genes , Leishmania major/genética , Leishmania major/patogenicidade , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/genética , Animais , Ciclo-Oxigenase 2/genética , Feminino , Regulação da Expressão Gênica , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/patologia , Ácido Linoleico/genética , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Fator de Necrose Tumoral alfa/genética , Virulência
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