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1.
PLoS Negl Trop Dis ; 14(7): e0008439, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628683

RESUMO

Leishmaniasis constitutes the 9th largest disease burden among all infectious diseases. Control of this disease is based on a short list of chemotherapeutic agents headed by pentavalent antimonials, followed by miltefosine and amphotericin B; drugs that are far from ideal due to host toxicity, elevated cost, limited access, and high rates of drug resistance. Knowing that the composition of extracellular vesicles (EVs) can vary according to the state of their parental cell, we hypothesized that EVs released by drug-resistant Leishmania infantum parasites could contain unique and differently enriched proteins depending on the drug-resistance mechanisms involved in the survival of their parental cell line. To assess this possibility, we studied EV production, size, morphology, and protein content of three well-characterized drug-resistant L. infantum cell lines and a wild-type strain. Our results are the first to demonstrate that drug-resistance mechanisms can induce changes in the morphology, size, and distribution of L. infantum EVs. In addition, we identified L. infantum's core EV proteome. This proteome is highly conserved among strains, with the exception of a handful of proteins that are enriched differently depending on the drug responsible for induction of antimicrobial resistance. Furthermore, we obtained the first snapshot of proteins enriched in EVs released by antimony-, miltefosine- and amphotericin-resistant parasites. These include several virulence factors, transcription factors, as well as proteins encoded by drug-resistance genes. This detailed study of L. infantum EVs sheds new light on the potential roles of EVs in Leishmania biology, particularly with respect to the parasite's survival in stressful conditions. This work outlines a crucial first step towards the discovery of EV-based profiles capable of predicting response to antileishmanial agents.


Assuntos
Antiprotozoários/farmacologia , Resistência a Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/metabolismo , Biologia Computacional , Vesículas Extracelulares , Regulação da Expressão Gênica/fisiologia , Proteoma , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
2.
Nucleic Acids Res ; 48(11): 6081-6091, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32402089

RESUMO

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.


Assuntos
DNA/química , DNA/metabolismo , Leishmania infantum/metabolismo , Macrófagos/microbiologia , Macrófagos/parasitologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , DNA/análise , Replicação do DNA , Fluorescência , Meia-Vida , Humanos , Leishmaniose/terapia , Macrófagos/citologia , Macrófagos/imunologia , Nanoestruturas/análise , Nanoestruturas/química , Técnicas de Amplificação de Ácido Nucleico , Fagocitose , Fagossomos/química , Fagossomos/microbiologia , Fagossomos/parasitologia , Tuberculose/terapia
3.
Acta Trop ; 201: 105217, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605692

RESUMO

Glycosomes of trypanosomatids are peroxisome-like organelles comprising unique metabolic features, among which the lack of the hallmark peroxisomal enzyme catalase. The absence of this highly efficient peroxidase from glycosomes is presumably compensated by other antioxidants, peroxidases of the peroxiredoxin (PRX) family being the most promising candidates for this function. Here, we follow on this premise and investigate the product of a Leishmania infantum gene coding for a putative glycosomal PRX (LigPRX). First, we demonstrate that LigPRX localizes to glycosomes, resorting to indirect immunofluorescence analysis. Second, we prove that purified recombinant LigPRX is an active peroxidase in vitro. Third, we generate viable LigPRX-depleted L. infantum promastigotes by classical homologous recombination. Surprisingly, phenotypic analysis of these knockout parasites revealed that promastigote survival, replication, and protection from oxidative and nitrosative insults can proceed normally in the absence of LigPRX. Noticeably, we also witness that LigPRX-depleted parasites can infect and thrive in mice to the same extent as wild type parasites. Overall, by disclosing the dispensable character of the glycosomal peroxiredoxin in L. infantum, this work excludes this enzyme from being a key component of the glycosomal hydroperoxide metabolism and contemplates alternative players for this function.


Assuntos
Leishmania infantum/genética , Leishmania infantum/metabolismo , Microcorpos/metabolismo , Oxirredutases/metabolismo , Peroxirredoxinas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Animais , Camundongos , Microcorpos/genética , Oxirredutases/genética , Peroxirredoxinas/genética
4.
Artigo em Inglês | MEDLINE | ID: mdl-31563118

RESUMO

DNA topoisomerases are considered consolidated druggable targets against diseases produced by trypanosomatids. Several reports indicated that indenoisoquinolines, a family of non-camptothecinic based topoisomerase poisons, have a strong leishmanicidal effect both in vitro and in vivo in murine models of visceral leishmaniasis. The antileishmanial effect of the indenoisoquinolines implies several mechanisms that include the stabilization of the cleavage complex, histone H2A phosphorylation and DNA fragmentation. A series of 20 compounds with the indenoisoquinoline scaffold and several substituents at positions N6, C3, C8 and C9, were tested both in promastigotes and in intramacrophage splenic amastigotes obtained from an experimental murine infection. The antileishmanial effect of most of these compounds was within the micromolar or submicromolar range. In addition, the introduction of an N atom in the indenoisoquinoline ring (7-azaindenoisoquinolines) produced the highest selectivity index along with strong DNA topoisomerase IB inhibition, histone H2A phosphorylation and DNA-topoisomerase IB complex stabilization. This report shows for the first time the effect of a series of synthetic indenoisoquinolines on histone H2A phosphorylation, which represents a primary signal of double stranded DNA break in genus Leishmania.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Dano ao DNA , DNA Topoisomerases/farmacologia , Histonas/metabolismo , Isoquinolinas/farmacologia , Leishmania infantum/efeitos dos fármacos , Animais , Western Blotting , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Feminino , Histonas/genética , Isoquinolinas/química , Leishmania infantum/citologia , Leishmania infantum/genética , Leishmania infantum/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Fosforilação/efeitos dos fármacos , Coelhos , Fase S/efeitos dos fármacos , Baço/citologia
5.
Eur J Med Chem ; 180: 28-40, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31299585

RESUMO

Among neglected tropical diseases, leishmaniasis is one of the most relevant with an estimated 30,000 deaths annually. Existing therapies have serious drawbacks in safety, drug resistance, field-adapted application and cost; therefore, new safer and shorter treatments are needed for this disease. Here we report on the synthesis of novel 4-amino-7-chloroquinoline-based compounds with leishmanicidal activity, together with deeper insight into the mechanism of action of our previously published hit compound 1. New derivatives showed comparable activity to 1 against both promastigote and intracellular amastigote forms of Leishmania infantum, with IC50 < 1 µM. Furthermore, we have determined that compound 1 induced a decrease of intracellular ATP levels, as well as a mitochondrial depolarization, together with an alteration of plasma membrane permeability and a significant ROS production. The inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal mechanism of this compound. In all, these results support the consideration of compound 1 for the future development of new leishmanicidal drugs.


Assuntos
Aminoquinolinas/farmacologia , Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Aminoquinolinas/síntese química , Aminoquinolinas/química , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Metabolismo Energético , Leishmania infantum/metabolismo , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
6.
Exp Parasitol ; 203: 1-7, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31128104

RESUMO

Fucose-mannose ligand (FML) is a soluble antigen purified from Leishmania donovani complex and used for diagnosis, prognosis, and vaccine development against visceral leishmaniasis (VL). We aimed to explore the effects of FML on the production of cytokines, chemokines and nitric oxide (NO) by macrophages in vitro. Peritoneal macrophages from BALB/c mice were treated with various concentrations of FML purified from Leishmania infantum in the absence or presence of LPS Peritoneal macrophages. After 48hr, cell culture supernatants were recovered and the levels of TNF-α, IL-10, IL-12p70 and IP-10 measured by Sandwich ELISA and NO concentration by Griess reaction. We found that FML significantly increase NO, IL-12p70 and IP-10 production in both LPS-treated and untreated macrophages and increase IL-10 levels only in LPS-treated macrophages. However, FML could not alert TNF-α levels in both LPS-treated and untreated macrophages. Further analysis revealed that FML can also increase IL-12p70/IL-10 ratio in LPS-treated macrophages. We concluded that FML can polarize macrophages to an appropriate phenotype similar to M1 phenotype against Leishmania donovani complex, although IL10 and TNF results are controversial.


Assuntos
Citocinas/metabolismo , Lectinas/farmacologia , Leishmania infantum/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Óxido Nítrico/metabolismo , Animais , Quimiocina CXCL10/metabolismo , Feminino , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Leishmania infantum/imunologia , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa/metabolismo
7.
Plant Sci ; 284: 117-126, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084864

RESUMO

Previously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression of CHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.


Assuntos
Antígenos de Protozoários/metabolismo , Proteínas de Choque Térmico/metabolismo , Leishmania infantum/metabolismo , Fotossíntese , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/metabolismo , Clorofila/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica , Temperatura Alta , Imunoprecipitação , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/parasitologia , Tabaco
8.
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
9.
Int J Mol Sci ; 20(8)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013713

RESUMO

Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the ß-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.


Assuntos
Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmaniose Visceral/parasitologia , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/imunologia , Técnicas de Visualização da Superfície Celular , Citocinas/metabolismo , Humanos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/tratamento farmacológico , Modelos Moleculares , Conformação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Nanomedicina Teranóstica , Tubulina (Proteína)/genética , Tubulina (Proteína)/imunologia
10.
Exp Parasitol ; 198: 39-45, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30716304

RESUMO

In recent years, several studies demonstrated the role of exosomes in intercellular communications, several Leishmania species belonging to subgenera Leishmania and Viannia have been demonstrated to release exosomes, and their role in parasite-macrophage interactions and in leishmaniasis development has been investigated. However, the release of exosomes by Leishmania infantum has not been studied so far. The aim of this study was to isolate and characterize L. infantum exosomes, and to investigate the biological activity of these exosomes in macrophage cultures. To this end, exosomes were collected from both amastigote and promastigote L. infantum conditioned medium by ultracentrifugation. Exosomes were then characterized by monitoring the presence of HSP70, HSP83/90 and acetylcholinesterase activity. Moreover, extracellular vesicles-tracking analysis revealed that promastigote and amastigote exosomes had mean diameter of 122 ±â€¯56 nm and 115 ±â€¯65 nm, respectively. Human monocytic cell line U937-derived macrophages treated with promastigote and amastigote exosomes showed an increase in motility and an overproduction of interleukin IL-10 and IL-18 reduction, involved in immune response. Since L. infantum exosomes demonstrated the capacity to modulate the chemotactic behaviour of the cells studied and cytokines production, they could contribute in the disease establishment and may be considered an appropriate candidate for a vaccine therapy in prophylaxis and treatment.


Assuntos
Quimiotaxia/fisiologia , Citocinas/metabolismo , Exossomos/metabolismo , Leishmania infantum/metabolismo , Citocinas/genética , Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-18/metabolismo , Células U937
11.
Nat Commun ; 10(1): 659, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737390

RESUMO

Many 2-Cys-peroxiredoxins (2-Cys-Prxs) are dual-function proteins, either acting as peroxidases under non-stress conditions or as chaperones during stress. The mechanism by which 2-Cys-Prxs switch functions remains to be defined. Our work focuses on Leishmania infantum mitochondrial 2-Cys-Prx, whose reduced, decameric subpopulation adopts chaperone function during heat shock, an activity that facilitates the transition from insects to warm-blooded host environments. Here, we have solved the cryo-EM structure of mTXNPx in complex with a thermally unfolded client protein, and revealed that the flexible N-termini of mTXNPx form a well-resolved central belt that contacts and encapsulates the unstructured client protein in the center of the decamer ring. In vivo and in vitro cross-linking studies provide further support for these interactions, and demonstrate that mTXNPx decamers undergo temperature-dependent structural rearrangements specifically at the dimer-dimer interfaces. These structural changes appear crucial for exposing chaperone-client binding sites that are buried in the peroxidase-active protein.


Assuntos
Cisteína/metabolismo , Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Microscopia Crioeletrônica , Leishmania infantum/metabolismo , Ligação Proteica , Dobramento de Proteína
12.
Artigo em Inglês | MEDLINE | ID: mdl-30297370

RESUMO

Drug repurposing affords the implementation of new treatments at a moderate cost and under a faster time-scale. Most of the clinical drugs against Leishmania share this origin. The antidepressant sertraline has been successfully assayed in a murine model of visceral leishmaniasis. Nevertheless, sertraline targets in Leishmania were poorly defined. In order to get a detailed insight into the leishmanicidal mechanism of sertraline on Leishmania infantum, unbiased multiplatform metabolomics and transmission electron microscopy were combined with a focused insight into the sertraline effects on the bioenergetics metabolism of the parasite. Sertraline induced respiration uncoupling, a significant decrease of intracellular ATP level, and oxidative stress in L. infantum promastigotes. Metabolomics evidenced an extended metabolic disarray caused by sertraline. This encompasses a remarkable variation of the levels of thiol-redox and polyamine biosynthetic intermediates, as well as a shortage of intracellular amino acids used as metabolic fuel by Leishmania Sertraline killed Leishmania through a multitarget mechanism of action, tackling essential metabolic pathways of the parasite. As such, sertraline is a valuable candidate for visceral leishmaniasis treatment under a drug repurposing strategy.


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/metabolismo , Sertralina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antidepressivos/farmacologia , Membrana Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos
13.
Mol Immunol ; 103: 7-20, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30173073

RESUMO

Leishmania elongation factor 2 (EF-2) has been previously identified as a TH1-stimulatory protein. In this study, we assayed the protective potential of the N-terminal domain of EF-2 (N-LiEF-2, 1-357 aa) that has been predicted to contain several overlapping MHC class I and II-restricted epitopes injected in the form of dendritic cell (DC)-based vaccine. Ex vivo pulsing of DCs with the recombinant N-LiEF-2 domain along with CpG oligodeoxynucleotides (ODNs) resulted in their functional differentiation. BALB/c vaccinated with CpG-triggered DCs pulsed with N-LiEF-2 were found to be the most immune-reactive in terms of induction of DTH responses, increased T cell proliferation and IL-2 production. Moreover, vaccination induced antigen-specific TH1 type immune response as evidenced by increased IFN-γ and TNFα levels followed by a significant increase of nitrite (NO) and reactive oxygen species (ROS) in splenocyte cultures. Vaccinated mice showed a pronounced decrease in parasite load in spleen and liver when challenged with L. infantum, increased expression of Stat1 and Tbx21 mRNA transcripts versus reduced expression of Foxp3 transcripts and were able to produce significantly elevated levels of IL-2, IFN-γ and TNFα but not IL-10 compared to non-vaccinated mice. Both antigen and parasite-specific CD4+ T and CD8+ T cells contributed to the IFN-γ production indicating that both subtypes contribute to the resistance to infection and correlated with robust nitrite generation, critical in controlling Leishmania infection. Together, these findings demonstrated the immunogenic as well as protective potential of the N-terminal domain of Leishmania EF-2 when given with CpG-triggered DCs representing a basis for the development of rationalized vaccine against leishmaniasis.


Assuntos
Células Dendríticas/imunologia , Imunidade Celular/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Animais , Antígenos de Protozoários/imunologia , Células Cultivadas , Células Dendríticas/parasitologia , Feminino , Imunidade Celular/efeitos dos fármacos , Interferon gama/imunologia , Interferon gama/metabolismo , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Leishmania infantum/imunologia , Leishmania infantum/metabolismo , Vacinas contra Leishmaniose/administração & dosagem , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/prevenção & controle , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Fator 2 de Elongação de Peptídeos/administração & dosagem , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/imunologia , Substâncias Protetoras/administração & dosagem , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/parasitologia
14.
Parasit Vectors ; 11(1): 355, 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921321

RESUMO

BACKGROUND: The Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa. METHODS: Using mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides. RESULTS: Metacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes. CONCLUSIONS: The data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.


Assuntos
Exossomos/metabolismo , Leishmania infantum/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Fatores de Virulência/metabolismo , Animais , Transporte Biológico , Cricetinae , Exossomos/química , Exossomos/genética , Humanos , Leishmania infantum/química , Leishmania infantum/genética , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/parasitologia , Masculino , Espectrometria de Massas , Mesocricetus , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Fatores de Virulência/química , Fatores de Virulência/genética
15.
PLoS One ; 13(3): e0193602, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29543820

RESUMO

The de novo crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1 (LiSir2rp1) has been solved at 1.99Å in complex with an acetyl-lysine peptide substrate. The structure is broadly commensurate with Hst2/SIRT2 proteins of yeast and human origin, reproducing many of the structural features common to these sirtuin deacetylases, including the characteristic small zinc-binding domain, and the larger Rossmann-fold domain involved in NAD+-binding interactions. The two domains are linked via a cofactor binding loop ordered in open conformation. The peptide substrate binds to the LiSir2rp1 protein via a cleft formed between the small and large domains, with the acetyl-lysine side chain inserting further into the resultant hydrophobic tunnel. Crystals were obtained only with recombinant LiSir2rp1 possessing an extensive internal deletion of a proteolytically-sensitive region unique to the sirtuins of kinetoplastid origin. Deletion of 51 internal amino acids (P253-E303) from LiSir2rp1 did not appear to alter peptide substrate interactions in deacetylation assays, but was indispensable to obtain crystals. Removal of this potentially flexible region, that otherwise extends from the classical structural elements of the Rossmann-fold, specifically the ß8-ß9 connector, appears to result in lower accumulation of the protein when expressed from episomal vectors in L. infantum SIR2rp1 single knockout promastigotes. The biological function of the large serine-rich insertion in kinetoplastid/trypanosomatid sirtuins, highlighted as a disordered region with strong potential for post-translational modification, remains unknown but may confer additional cellular functions that are distinct from their human counterparts. These unique molecular features, along with the resolution of the first kinetoplastid sirtuin deacetylase structure, present novel opportunities for drug design against a protein target previously established as essential to parasite survival and proliferation.


Assuntos
Histona Desacetilases do Grupo III/química , Histona Desacetilases do Grupo III/metabolismo , Leishmania infantum/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo
16.
RNA Biol ; 15(6): 739-755, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29569995

RESUMO

The Poly-A Binding Protein (PABP) is a conserved eukaryotic polypeptide involved in many aspects of mRNA metabolism. During translation initiation, PABP interacts with the translation initiation complex eIF4F and enhances the translation of polyadenylated mRNAs. Schematically, most PABPs can be divided into an N-terminal RNA-binding region, a non-conserved linker segment and the C-terminal MLLE domain. In pathogenic Leishmania protozoans, three PABP homologues have been identified, with the first one (PABP1) targeted by phosphorylation and shown to co-immunoprecipitate with an eIF4F-like complex (EIF4E4/EIF4G3) implicated in translation initiation. Here, PABP1 phosphorylation was shown to be linked to logarithmic cell growth, reminiscent of EIF4E4 phosphorylation, and coincides with polysomal association. Phosphorylation targets multiple serine-proline (SP) or threonine-proline (TP) residues within the PABP1 linker region. This is an essential protein, but phosphorylation is not needed for its association with polysomes or cell viability. Mutations which do impair PABP1 polysomal association and are required for viability do not prevent phosphorylation, although further mutations lead to a presumed inactive protein largely lacking phosphorylated isoforms. Co-immunoprecipitation experiments were carried out to investigate PABP1 function further, identifying several novel protein partners and the EIF4E4/EIF4G3 complex, but no other eIF4F-like complex or subunit. A novel, direct interaction between PABP1 and EIF4E4 was also investigated and found to be mediated by the PABP1 MLLE binding to PABP Interacting Motifs (PAM2) within the EIF4E4 N-terminus. The results shown here are consistent with phosphorylation of PABP1 being part of a novel pathway controlling its function and possibly translation in Leishmania.


Assuntos
Leishmania infantum/metabolismo , Iniciação Traducional da Cadeia Peptídica/fisiologia , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Protozoários/metabolismo , Motivos de Aminoácidos , Leishmania infantum/genética , Fosforilação/fisiologia , Proteínas de Ligação a Poli(A)/genética , Proteínas de Protozoários/genética
17.
Cell Immunol ; 323: 59-69, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29128045

RESUMO

Visceral leishmaniasis (VL) represents a serious public health problem, as Leishmania infantum is one of main disease causative agents in the Americas. In a previous immunoproteomic study, the prohibitin (PHB) protein was identified in L. infantum promastigote and amastigote extracts by antibodies in asymptomatic and symptomatic VL dog sera. This protein was found to be highly conserved between different Leishmania spp., but it presented a low identity with amino acid sequences of other organisms. The aim of the present study was to evaluate the cellular response induced by the recombinant PHB (rPHB) protein in BALB/c mice, as well as in PBMCs purified from untreated and treated VL patients, as well as to evaluate its protective efficacy against an infection by L. infantum promastigotes. Our data showed that there was a Th1 cellular response to rPHB, based on high levels of IFN-γ, IL-12, and GM-CSF in the immunized animals, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from individuals who had recovered from the disease. The protection was represented by significant reductions in the parasite load in the animals' spleen, liver, bone marrow, and draining lymph nodes, as compared to results found in the control groups. In addition, an anti-rPHB serology, using a canine and human serological panel, showed a high performance of this protein when diagnosing VL based on high sensitivity and specificity values, as compared to results found for the rA2 antigen and the soluble Leishmania antigenic extract. Our data suggest that PHB has a potential application for the diagnosis of canine and human VL through antibody detection, as well as an application as a vaccine candidate to protect against disease.


Assuntos
Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Proteínas Repressoras/imunologia , Animais , Antígenos de Protozoários/imunologia , Cães , Humanos , Leishmania infantum/imunologia , Leishmania infantum/metabolismo , Leishmaniose Visceral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Células Th1/imunologia , Vacinas/metabolismo
18.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 30(6): 625-629, 2018 Jul 26.
Artigo em Chinês | MEDLINE | ID: mdl-30891972

RESUMO

OBJECTIVE: To analyze the protein abundance differences between two Leishmania infantum strains isolated from different epidemiological types of visceral leishmaniasis in China by comparative proteomics method. METHODS: Tryptic digests of total proteins were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by label-free quantitative differential expression analysis. The MS data were analyzed with MaxQuant software (ver 1.3.0.5) against data base. RESULTS: This study resulted in the identification of 4 274 proteins across two strains (JIASHI-5 and SC6) . Of these, 1 219 differentially expressed proteins (ratio > 2.0 or < 0.5, P < 0.05) were identified. Considering the proteins differentially or uniquely expressed in the strains, 550 proteins were only found in the JIASHI-5 strain, and 174 proteins were only found in the SC6 strain. Totally 495 differentially proteins were expressed in the two groups, among which 328 proteins were down-regulated and 167 proteins were up-regulated in SC6 strain. Some of the identified differentially expressed proteins were demonstrated and they involved in energy metabolism, stress response, prolonging the lifetime of the infected host cell and survival and proliferation in virulent strains. CONCLUSIONS: This study reveals a group of differentially expressed proteins and the related biologic function that may lay the foundation for screening and identification of the key Leishmania molecules relative to pathogenicity.


Assuntos
Leishmania infantum , Leishmaniose Visceral , Proteoma , China/epidemiologia , Cromatografia Líquida , Humanos , Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Proteômica , Espectrometria de Massas em Tandem
19.
Molecules ; 22(12)2017 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-29186074

RESUMO

Proteins and glycolipids have been found to be decorated with phosphorylcholine (PC) both in protozoa and nematodes that parasitize humans and animals. PC epitopes can provoke various effects on immune cells leading to an immunomodulation of the host's immune system that allows long-term persistence of the parasites. So far, only a limited number of PC-modified proteins, mainly from nematodes, have been identified. Infections caused by Leishmania spp. (e.g., L. infantum in southern Europe) affect about 12 million people worldwide and are characterized by a wide spectrum of clinical forms in humans, ranging from cutaneous to fatal visceral leishmaniasis. To establish and maintain the infection, these protozoa are dependent on the secretion of effector molecules into the host for modulating their immune system. In this project, we analyzed the PC modification of L. infantum promastigotes by 2D-gel based proteomics. Western blot analysis with the PC-specific antibody TEPC-15 revealed one PC-substituted protein in this organism, identified as eEF1α. We could demonstrate that the binding of eEF1α to one of its downstream effectors is dependent on its PC-modification. In this study we provide evidence that in this parasite the modification of eEF1α with PC may be essential for its function as an important virulence factor.


Assuntos
Leishmania infantum/metabolismo , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/metabolismo , Fosforilcolina/química , Epitopos/química , Epitopos/imunologia , Imunomodulação/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Estrutura Molecular , Fator 1 de Elongação de Peptídeos/imunologia , Fosforilcolina/farmacologia
20.
Sci Rep ; 7(1): 10262, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860532

RESUMO

Human macrophage phagolysosome and sandfly midgut provide antagonistic ecological niches for Leishmania parasites to survive and proliferate. Parasites optimize their metabolism to utilize the available inadequate resources by adapting to those environments. Lately, a number of metabolomics studies have revived the interest to understand metabolic strategies utilized by the Leishmania parasite for optimal survival within its hosts. For the first time, we propose a reconstructed genome-scale metabolic model for Leishmania infantum JPCM5, the analyses of which not only captures observations reported by metabolomics studies in other Leishmania species but also divulges novel features of the L. infantum metabolome. Our results indicate that Leishmania metabolism is organized in such a way that the parasite can select appropriate alternatives to compensate for limited external substrates. A dynamic non-essential amino acid motif exists within the network that promotes a restricted redistribution of resources to yield required essential metabolites. Further, subcellular compartments regulate this metabolic re-routing by reinforcing the physiological coupling of specific reactions. This unique metabolic organization is robust against accidental errors and provides a wide array of choices for the parasite to achieve optimal survival.


Assuntos
Adaptação Biológica , Metabolismo Energético , Genoma de Protozoário , Genômica , Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmaniose Visceral/parasitologia , Biologia Computacional , Genômica/métodos , Espaço Intracelular/metabolismo , Estágios do Ciclo de Vida , Redes e Vias Metabólicas , Metaboloma , Metabolômica , Mitocôndrias/metabolismo
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