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1.
Mem Inst Oswaldo Cruz ; 117: e220407, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35384972

RESUMO

A significant percentage of exogenous cholesterol was found in promastigotes and amastigotes of all studied species of Leishmania, suggesting a biological role for this molecule. Previous studies have shown that promastigotes of Leishmania uptake more low-density lipoprotein (LDL) particles under pharmacological pressure and are more susceptible to ergosterol inhibition in the absence of exogenous sources of cholesterol. This work shows that the host's LDL is available to intracellular amastigotes and that the absence of exogenous cholesterol enhances the potency of sterol biosynthesis inhibitors in infected macrophages. A complete understanding of cholesterol transport to the parasitophorous vacuole can guide the development of a new drug class to be used in combination with sterol biosynthesis inhibitors for the treatment of leishmaniases.


Assuntos
Leishmania mexicana , Leishmania , Leishmaniose , Animais , Colesterol , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C
2.
Sci Rep ; 12(1): 3266, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-35228627

RESUMO

Kinesins are motor proteins present in organisms from protists to mammals playing important roles in cell division, intracellular organisation and flagellum formation and maintenance. Leishmania mexicana is a protozoan parasite of the order Kinetoplastida causing human cutaneous leishmaniasis. Kinetoplastida genome sequence analyses revealed a large number of kinesins showing sequence and structure homology to eukaryotic kinesins. Here, we investigate the L. mexicana kinesin LmxKIN29 (LmxM.29.0350), also called DEATH kinesin. The activated MAP kinase LmxMPK3, a kinase affecting flagellum length in Leishmania, is able to phosphorylate recombinant full length LmxKIN29 at serine 554. Insect promastigote LmxKIN29 Leishmania null mutants showed no obvious phenotype. However, in mouse infection experiments, the null mutants were unable to cause the disease, whereas LmxKIN29 add-backs and single allele knockouts caused footpad lesions. Localisation using promastigotes expressing GFP-tagged LmxKIN29 revealed that the kinesin is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells the GFP-fusion protein was found at the anterior and posterior ends of the cells indicating a role in cytokinesis. The inability to cause lesions in infected animals and the amino acid sequence divergence from mammalian kinesins suggests that LmxKIN29 is a potential drug target against leishmaniasis.


Assuntos
Leishmania mexicana , Leishmaniose Cutânea , Animais , Flagelos/metabolismo , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Mamíferos/metabolismo , Camundongos , Proteínas de Protozoários/metabolismo
3.
World J Microbiol Biotechnol ; 38(4): 66, 2022 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-35246768

RESUMO

We evaluated the leishmanicidal activity of commercially available 5α-cholest-7-en-3ß-ol [5α-chol], (+)-4-cholesten-3-one [(+)-4-chol] and the equimolar mixture of the two of them in promastigotes and amastigotes of two different strains of Leishmania mexicana (LCL) and (DCL). The leishmanicidal effectiveness of these sterols was determined by promastigote growth-kinetic experiments and promastigote viability using the propidium iodide staining procedure. The proliferation test was performed using the CFSE (5-Carboxyfluorescein N-succinimidyl ester) staining of parasites at different time points. To determine the leishmanicidal effectiveness of these sterols in amastigotes, we evaluated parasite killing inside of macrophages at different time points. The trypan blue exclusion test was used to determine cytotoxicity of sterols in uninfected macrophages. We included in all experiments a control group of parasites treated with 2% DMSO (Dimethyl Sulfoxide) and another one treated with the reference drug sodium stibogluconate (Sb). Our results showed that the equimolar mixture at 2000 times lower concentration presented similar leishmanicidal activity as Sb. This mixture was similarly effective at 100 times lower concentration than individual sterols tested separately indicating the existence of a synergistic effect against LCL and DCL parasites. The therapeutic index of the equimolar mixture was 10,000-16,000 times higher than the one recorded by Sb and was not cytotoxic to macrophages. Therefore, the equimolar mixture of 5α-Chol and (+)-4-chol may represent a potential alternative for the treatment of cutaneous leishmaniasis.


Assuntos
Leishmania mexicana , Leishmaniose Cutânea , Gluconato de Antimônio e Sódio/farmacologia , Colesterol , Humanos , Leishmaniose Cutânea/tratamento farmacológico , Esteróis/farmacologia
4.
Infect Immun ; 90(3): e0018321, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35130453

RESUMO

To colonize mammalian phagocytic cells, the parasite Leishmania remodels phagosomes into parasitophorous vacuoles that can be either tight-fitting individual or communal. The molecular and cellular bases underlying the biogenesis and functionality of these two types of vacuoles are poorly understood. In this study, we investigated the contribution of host cell soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins to the expansion and functionality of communal vacuoles as well as the replication of the parasite. The differential patterns of recruitment of soluble N-ethylmaleimide-sensitive-factor attachment protein receptor to communal vacuoles harboring Leishmania amazonensis and to individual vacuoles housing L. major led us to further investigate the roles of VAMP3 and VAMP8 in the interaction of Leishmania with its host cell. We show that whereas VAMP8 contributes to the optimal expansion of communal vacuoles, VAMP3 negatively regulates L. amazonensis replication, vacuole size, as well as antigen cross-presentation. In contrast, neither protein has an impact on the fate of L. major. Collectively, our data support a role for both VAMP3 and VAMP8 in the development and functionality of L. amazonensis-harboring communal parasitophorous vacuoles.


Assuntos
Leishmania mexicana , Leishmania , Animais , Habitação , Leishmania/fisiologia , Macrófagos/metabolismo , Mamíferos , Vacúolos/parasitologia , Proteína 3 Associada à Membrana da Vesícula/metabolismo
5.
Parasitology ; 149(4): 490-495, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35109958

RESUMO

Cutaneous leishmaniasis (CL) is a spectrum of clinical manifestations characterized by severe skin ulcerations that leads to social stigma. There are limited treatment options for CL, and the available drugs are becoming less efficacious due to drug resistance. More efficacious and safer antileishmanial drugs are needed. In this study, the biological effect of seven synthetically accessible nitroaromatic compounds was evaluated in vitro against amastigotes of Leishmania amazonensis, followed by in vivo evaluation using mouse models of CL. Two compounds (6 and 7) were active against amastigotes in vitro [half-maximal effective concentration (EC50): 4.57 ± 0.08 and 9.19 ± 0.68 µm, respectively], with selectivity indexes >50, and the other compounds were not selective. In vivo, compounds 6 and 7 (10 mg kg−1, twice a day for 14 days) failed to reduce skin lesion sizes and parasite loads determined by light microscopy of lesion imprints and quantitative polymerase chain reaction. Nevertheless, the in vitro leishmanicidal efficacy sustained their use as templates for nitroimidazole-based antileishmanial drug discovery programmes focusing on analogues with more suitable properties.


Assuntos
Antiprotozoários , Leishmania mexicana , Leishmaniose Cutânea , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Leishmaniose Cutânea/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Nitrocompostos/uso terapêutico
6.
Microbiol Spectr ; 10(1): e0242221, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35138191

RESUMO

Proteomic profiling of RNA-binding proteins in Leishmania is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labeling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main Leishmania life cycle stages. Eight out of 20 Leishmania deubiquitinases, including the recently characterized L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the Leishmania non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in Leishmania. We also characterize the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 142 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the Leishmania protein kinome. Additionally, we characterize the quantitative changes in RNA-protein interactions in hundreds of Leishmania proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in Leishmania causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in Leishmania. This study defines the comprehensive RNA interactome of Leishmania and provides in-depth insight into the widespread involvement of RNA-protein interactions in Leishmania biology. IMPORTANCE Advances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites Leishmania spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and subtropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In Leishmania, RNA-binding proteins serve as primary gene regulators. Therefore, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in Leishmania. Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labeling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main Leishmania life cycle stages. In addition, we characterize for the first time the quantitative changes in RNA-protein interactions in Leishmania following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in Leishmania, thus significantly expanding our knowledge of the emergence of RNA-protein interactions in Leishmania biology.


Assuntos
Leishmania mexicana/genética , Proteínas de Protozoários/genética , RNA de Protozoário/genética , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Transcriptoma , Leishmania mexicana/metabolismo , Espectrometria de Massas , Fases de Leitura Aberta , Ligação Proteica , Proteômica , Proteínas de Protozoários/metabolismo , RNA de Protozoário/metabolismo , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
7.
Biomed Res Int ; 2022: 9928362, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35187176

RESUMO

Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals in vivo. Extracellularly released nucleotides such as ATP are rapidly hydrolyzed to adenosine by the coordinated ectonucleotidase activities of CD39 and CD73. Leishmania is an obligate intracellular parasite of macrophages and capable of modulating host immune response in order to survive and multiply within host cells. In this study, the activity of CD73 induced by Leishmania amazonensis in infected macrophages has been investigated and correlated with parasite survival and infection in vitro. For this, the expression of CD39 and CD73, by flow cytometry, in murine peritoneal macrophages infected with metacyclic promastigotes of L. amazonensis has been analyzed. Our results showed that L. amazonensis-infected macrophages, unlike LPS-treated macrophages, increased CD73 expression. It was also noted that when CD73 enzymatic activity was blocked by α, ß-methyleneadenosine 5'-diphosphate sodium salt (APCP), macrophage parasitism was significantly decreased. Interestingly, these effects were not associated with the production of TNF-α, IL-10, or nitric oxide (NO). Together, these data demonstrate that L. amazonensis induces a regulatory phenotype in macrophages, which by activating the CD39/CD73 pathway allows parasite survival through the action of immunomodulatory adenosine receptors.


Assuntos
5'-Nucleotidase/metabolismo , Adenosina Trifosfatases/metabolismo , Leishmania mexicana/enzimologia , Macrófagos Peritoneais/parasitologia , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
8.
Front Immunol ; 13: 762080, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35145518

RESUMO

Leishmania spp. infection outcomes are dependent on both host and parasite factors. Manipulation of host signaling pathways involved in the generation of immune responses is thought to be one of the most common mechanisms used by parasites for persistence within the host. Considering the diversity of pathologies caused by different Leishmania spp., it is plausible that significant differences may exist in the mechanisms of host cell manipulation by each parasite species, which may have implications when developing new vaccine or treatment strategies. Here we show that in L. braziliensis-infection in BALB/c mice, a model of resistance, activation of ERK1/2 coincides with the peak of inflammatory responses and resolution of tissue parasitism. In contrast, in the susceptibility model of L. amazonensis-infection, an early silent phase of infection is observed, detected solely by quantification of parasite loads. At this early stage, only basal levels of P-ERK1/2 are observed. Later, after a brief shutdown of ERK1/2 phosphorylation, disease progression is observed and is associated with increased inflammation, lesion size and tissue parasitism. Moreover, the short-term down-regulation of ERK1/2 activation affected significantly downstream inflammatory pathways and adaptive T cell responses. Administration of U0126, a MEK/ERK inhibitor, confirmed this phenomenon, since bigger lesions and higher parasite loads were seen in infected mice that received U0126. To investigate how kinetics of ERK1/2 activation could affect the disease progression, U0126 was administered to L. amazonensis-infected animals earlier than the P-ERK1/2 switch off time-point. This intervention resulted in anticipation of the same effects on inflammatory responses and susceptibility phenotype seen in the natural course of infection. Additionally, in vitro inhibition of ERK1/2 affected the phagocytosis of L. amazonensis by BMDMs. Collectively, our findings reveal distinct temporal patterns of activation of inflammatory responses in L. braziliensis and L. amazonensis in the same animal background and a pivotal role for a brief and specific shutdown of ERK1/2 activation at late stages of L. amazonensis infection. Since activation of inflammatory responses is a crucial aspect for the control of infectious processes, these findings may be important for the search of new and specific strategies of vaccines and treatment for tegumentary leishmaniasis.


Assuntos
Imunidade Celular , Leishmania mexicana/imunologia , Leishmaniose/imunologia , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Biomarcadores , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Interações Hospedeiro-Patógeno/imunologia , Mediadores da Inflamação/metabolismo , Leishmaniose/patologia , Camundongos , Carga Parasitária , Fagocitose/imunologia , Fosforilação , Transdução de Sinais
9.
Nat Prod Res ; 36(6): 1599-1603, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33586545

RESUMO

Natural products have been largely explored as treatments for leishmaniasis, neglected diseases with few toxic therapeutic options, as scaffolds for the development of new drugs. Herein, derivatives from the aerial parts of Baccharis trimera (Less.) DC (extract and its fractions) were evaluated against Leishmania amazonensis and macrophage cells. The ethyl acetate extract was fractionated by solid-phase extraction, resulting in eight fractions (F1-F8). Fractions F3-4 were further separated into 149 subfractions; subfraction 148 (IC50-PRO = 1.56 ± 0.1 µg mL-1) was selected for purification and constituent(s) characterization by high-performance liquid chromatography, as well as 1H and 13C nuclear magnetic resonance spectroscopy. The flavonoid eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) was identified. This compound was 3.7 times more effective against intracellular amastigotes (IC50-AMA = 1.6 ± 0.1 µM) than amphotericin B and presented low cytotoxicity (CC50 > 100 µM), being almost 62 times more selective for the parasite, showing great potential in drug development for cutaneous leishmaniasis treatment.


Assuntos
Antiprotozoários , Baccharis , Leishmania mexicana , Leishmaniose Cutânea , Antiprotozoários/farmacologia , Baccharis/química , Flavonoides/análise , Leishmaniose Cutânea/tratamento farmacológico , Extratos Vegetais/química , Folhas de Planta/química
10.
Acta Trop ; 226: 106272, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34896324

RESUMO

Cutaneous leishmaniasis is the most common form of leishmaniasis in humans, factors such as poverty, poor housing, inadequate domestic hygiene, malnutrition, mobility, and occupational exposure are risk factors associated with the condition, however, there are few studies focused on determining the immune mechanism involved in the resolution of cutaneous leishmaniasis caused by the species Leishmania mexicana, as well as possible environmental factors such as solar radiation, which could contribute to its establishment. through mechanisms immunosuppressants, of which to date is unknown. In this study, the effect of UV-B light was evaluated as a risk factor affecting components of the innate immune response 3 days after infection with L. mexicana. A delayed-type hypersensitivity reaction (DTH) was used to evaluate immunosuppression induced by UV-B light. Through a histological analysis, the skin lesions of the mice (Hematoxylin & Eosin) were evaluated, the presence of mast cells and their level of degranulation (toluidine blue staining), the presence of IL-10+ and MOMA2+ cells were analyzed by immunohistochemistry and finally, the cytokine profile was evaluated by qPCR in the skin lesions tissue. An alteration in the architecture of the tissue was observed, as well as a greater number of mast cells, both complete and degranulated, as well as an increase in IL-10+ and MOMA2+ cells in the skin lesions of the mice that were irradiated and subsequently infected, when compared with the lesions of infected mice (P> 0.0001), immunomodulation was also observed in the profile of cytokines expressed between both groups analyzed. This is the first study to demonstrate the effects of UV-B radiation on components of the innate immune response at short times of infection by L. mexicana.


Assuntos
Leishmania mexicana , Leishmaniose Cutânea , Animais , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C
11.
Parasitol Int ; 86: 102458, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34509671

RESUMO

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC50 = 28.5 ± 2.0 µM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.


Assuntos
Biflavonoides/farmacologia , Leishmania mexicana/fisiologia , Mitocôndrias/fisiologia , Selaginellaceae/química , Biflavonoides/química , Leishmania mexicana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Tripanossomicidas
12.
PLoS Negl Trop Dis ; 15(12): e0010041, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34965251

RESUMO

BACKGROUND: Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. CONCLUSION/SIGNIFICANCE: Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.


Assuntos
Instabilidade Genômica , Autoantígeno Ku/metabolismo , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Proteínas de Protozoários/metabolismo , Telômero/metabolismo , Genoma de Protozoário , Humanos , Autoantígeno Ku/genética , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/genética , Telômero/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo
13.
Int J Mol Sci ; 22(24)2021 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-34948408

RESUMO

Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 µM and 82 µM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.


Assuntos
Antiprotozoários/farmacologia , Arginase/antagonistas & inibidores , Benzimidazóis/farmacologia , Leishmania mexicana/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/química , Arginase/metabolismo , Benzimidazóis/química , Linhagem Celular , Descoberta de Drogas , Humanos , Leishmania mexicana/enzimologia , Leishmania mexicana/fisiologia , Leishmaniose Cutânea/tratamento farmacológico , Camundongos , Modelos Moleculares , Proteínas de Protozoários/metabolismo
14.
An. bras. dermatol ; 96(5): 602-604, Sept.-Oct. 2021. graf
Artigo em Inglês | LILACS | ID: biblio-1345132

RESUMO

Abstract Diffuse cutaneous leishmaniasis is a rare universal disease associated with an inadequate host cell immune response, caused by different species: infantum, aethiopica, major, mexicana, and others, which presents the challenge of a poor therapeutic response. In Brazil, it is caused by L. amazonensis. A case confirmed by histopathology with an abundance of vacuolated macrophages full of amastigotes and lymphocyte scarcity, identified by RFLP-ITS1PCR and in vitro decrease and exhaustion of the host cell immune response to L. amazonensis antigen, was treated early (3 months after the onset) with Glucantime (2 months) and allopurinol (29 months) with clinical cure, after a follow-up for 30 months after treatment.


Assuntos
Humanos , Leishmania mexicana , Leishmaniose Tegumentar Difusa/tratamento farmacológico , Leishmaniose Cutânea/tratamento farmacológico , Antiprotozoários/uso terapêutico , Brasil , Antimoniato de Meglumina
15.
Trop Biomed ; 38(3): 338-342, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34508341

RESUMO

Leishmaniasis is a neglected and endemic disease that affects poorest population mainly in developing countries. Thymus provides an essential complex environment for T cell maturation and differentiation during leishmania infection. The aim of this study was to investigate the pathological alterations of the Thymus during early Leishmania amazonensis murine infection. BALB/c mice were infected with 105 amastigotes for 24 h, 3 days, 7 days, 15 days or 30 days. At different times of infection, the relative weight of the Thymus was obtained, and the Thymus cellularity was determined by counting total cells of one thymic lobe. The thymic lobe was, alternatively, processed for standard Haematoxylin and Eosin protocol. Our results suggest thymic alteration during the early days of BALB/c mice infection with L. amazonensis. The thymic hypertrophy was accompanied by histological alterations in Thymus architecture with thickening cortex at 3 days p.i. and loss of an evident delimitation between the cortex and medulla at 7 days p.i. when compared to the control mice. That is the first time that Thymus hypertrophy was observed during the early leishmaniasis. However, how it may contribute to infection susceptibility requires further investigation.


Assuntos
Leishmaniose , Timo , Animais , Hipertrofia , Leishmania mexicana , Leishmaniose/patologia , Camundongos , Camundongos Endogâmicos BALB C , Timo/parasitologia , Timo/patologia
16.
ChemistryOpen ; 10(9): 896-903, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34499412

RESUMO

Research for innovative drugs is crucial to contribute to parasitic infections control and eradication. Inspired by natural antiprotozoal triterpenes, a library of 12 hemisynthetic 3-O-arylalkyl esters was derived from ursolic and oleanolic acids through one-step synthesis. Compounds were tested on Trypanosoma, Leishmania and the WI38 cell line alongside with a set of triterpenic acids. Results showed that the triterpenic C3 esterification keeps the antitrypanosomal activity (IC50 ≈1.6-5.5 µm) while reducing the cytotoxicity compared to parent acids. Unsaturation of the ester alkyl chain leads to an activity loss interestingly kept when a sterically hindered group replaces the double bond or shields the ester group. An ursane/oleanane C3 hydroxylation was the only important feature for antileishmanial activity. Two candidates, dihydrocinnamoyl and 2-fluorophenylpropionyl ursolic acids, were tested on an acute mouse model of African trypanosomiasis with significant parasitemia reduction at day 5 post-infection for the dihydrocinnamoyl derivative. Further evaluation on other alkyl/protective groups should be investigated both in vitro and in vivo.


Assuntos
Ésteres/farmacologia , Triterpenos/farmacologia , Tripanossomicidas/farmacologia , Animais , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ésteres/síntese química , Ésteres/toxicidade , Feminino , Leishmania mexicana/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Parasitária , Triterpenos/síntese química , Triterpenos/toxicidade , Tripanossomicidas/síntese química , Tripanossomicidas/toxicidade , Trypanosoma brucei brucei/efeitos dos fármacos
17.
Exp Parasitol ; 229: 108154, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34481863

RESUMO

The compound 3-bromopyruvate (3-BrPA) is well-known and studies from several researchers have demonstrated its involvement in tumorigenesis. It is an analogue of pyruvic acid that inhibits ATP synthesis by inhibiting enzymes from the glycolytic pathway and oxidative phosphorylation. In this work, we investigated the effect of 3-BrPA on energy metabolism of L. amazonensis. In order to verify the effect of 3-BrPA on L. amazonensis glycolysis, we measured the activity level of three glycolytic enzymes located at different points of the pathway: (i) glucose kinases, step 1, (ii) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), step 6, and (iii) enolase, step 9. 3-BrPA, in a dose-dependent manner, significantly reduced the activity levels of all the enzymes. In addition, 3-BrPA treatment led to a reduction in the levels of phosphofruto-1-kinase (PFK) protein, suggesting that the mode of action of 3-BrPA involves the downregulation of some glycolytic enzymes. Measurement of ATP levels in promastigotes of L. amazonensis showed a significant reduction in ATP generation. The O2 consumption was also significantly inhibited in promastigotes, confirming the energy depletion effect of 3-BrPA. When 3-BrPA was added to the cells at the beginning of growth cycle, it significantly inhibited L. amazonensis proliferation in a dose-dependent manner. Furthermore, the ability to infect macrophages was reduced by approximately 50% when promastigotes were treated with 3-BrPA. Taken together, these studies corroborate with previous reports which suggest 3-BrPA as a potential drug against pathogenic microorganisms that are reliant on glucose catabolism for ATP supply.


Assuntos
Leishmania mexicana/efeitos dos fármacos , Leishmaniose Tegumentar Difusa/parasitologia , Piruvatos/farmacologia , Animais , Western Blotting , Brasil , Cricetinae , Humanos , Leishmania mexicana/enzimologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/metabolismo , Macrófagos/parasitologia , Camundongos , Consumo de Oxigênio/efeitos dos fármacos , Fosfopiruvato Hidratase/metabolismo , Células RAW 264.7
18.
Int Immunopharmacol ; 100: 108130, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34500286

RESUMO

Leishmaniasis is a set of infectious diseases with high rates of morbidity and mortality, it affects millions of people around the world. Treatment, mainly with pentavalent antimonials, presents significant toxicity and many cases of resistance. In previous works we have demonstrated the effective and selective antileishmanial activity of Eugenia uniflora L. essential oil, being constituted (47.3%) by the sesquiterpene curzerene. Considering the high rate of parasite inhibition demonstrated for E. uniflora essential oil, and the significant presence of curzerene in the oil, this study aimed to evaluate its antileishmania activity and possible mechanisms of action. Curzerene was effective in inhibiting the growth of promastigotes (IC50 3.09 ± 0.14 µM) and axenic amastigotes (EC50 2.56 ± 0.12 µM), with low cytotoxicity to RAW 264.7 macrophages (CC50 83.87 ± 4.63 µM). It was observed that curzerene has direct effects on the parasite, inducing cell death by apoptosis with secondary necrotic effects (producing pores in the plasma membrane). Curzerene proved to be even more effective against intra-macrophage amastigote forms, with an EC50 of 0.46 ± 0.02 µM. The selectivity index demonstrated by curzerene on these parasite forms was 182.32, being respectively 44.15 and 8.47 times more selective than meglumine antimoniate and amphotericin B. The antiamastigote activity of curzerene was associated with immunomodulatory activity, as it increased TNF-α, IL-12, and NO levels, and lysosomal activity, and decreased IL-10 and IL-6 cytokine levels detected in macrophages infected and treated. In conclusion, our results demonstrate that curzerene is an effective and selective antileishmanial agent, a candidate for in vivo investigation in models of antileishmanial activity.


Assuntos
Antiprotozoários/farmacologia , Leishmania mexicana/efeitos dos fármacos , Sesquiterpenos/farmacologia , Animais , Antiprotozoários/uso terapêutico , Apoptose/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo
19.
PLoS Negl Trop Dis ; 15(8): e0009682, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34449767

RESUMO

Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.


Assuntos
Insetos Vetores/anatomia & histologia , Insetos Vetores/parasitologia , Leishmania mexicana/fisiologia , Proteínas de Protozoários/metabolismo , Psychodidae/anatomia & histologia , Psychodidae/parasitologia , Animais , Feminino , Trato Gastrointestinal/anatomia & histologia , Trato Gastrointestinal/parasitologia , Leishmania mexicana/genética , Projetos Piloto , Proteínas de Protozoários/genética , Microtomografia por Raio-X
20.
Parasitol Res ; 120(9): 3307-3317, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34370070

RESUMO

The aim of this study was to synthesize several small molecules of the type 5-nitroimidazole-sulfanyl and evaluate biological properties against the main Leishmania species that cause cutaneous leishmaniasis in Venezuela. Final compounds 4-7 were generated through simple nucleophilic substitution of 1-(2-chloroethyl)-2-methyl-5-nitroimidazole 3 with 2-mercaptoethanol, 1-methyl-2-mercaptoethanol, and 2-thyolacetic acid derivative. Compound 8 was synthesized via a coupling reaction between 7 and (S)-Methyl 2-amino-4-methylpentanoate hydrochloride. The inhibitory concentrations of (3, 4, 7, 8) against Leishmania (L.) mexicana and (V.) braziliensis in promastigotes and experimentally infected macrophages were determined by in vitro activity assays. Compounds 7 and 8 shown high activity against both species of Leishmania and were selected for the in vivo evaluation. Animals were infected with promastigotes of the two species and divided into four groups of ten (10) animals and a control group. Intralesional injection way was used for the treatment. The parasitological diagnostic after treatment was obtained by PCR using species specific oligonucleotides. The two Leishmania species were susceptible to compounds 7 and 8 in vivo assays. The results indicated that both compounds reduce significantly (96%) the size of the lesion and cure 63% of the mice infected with L (L) mexicana or L (V) braziliensis as was determined by PCR. The results are indicating that both compounds may represent an alternative treatment for these two Leishmania species.


Assuntos
Antiprotozoários , Leishmania braziliensis , Leishmania mexicana , Leishmaniose Cutânea , Nitroimidazóis , Animais , Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Camundongos , Nitroimidazóis/farmacologia
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