Transbilayer dynamics of phospholipids in the plasma membrane of the <em>Leishmania</em> genus

Protozoans of the Leishmania genus are the etiological agents of a wide spectrum of diseases commonly known as leishmaniases. Lipid organization of the plasma membrane of the parasite may mimic the lipid organization of mammalian apoptotic cells and play a role in phagocytosis and parasite survival in the mammal host. Here, we analyzed the phospholipid dynamics in the plasma membrane of both the L. (Leishmania) and the L. (Viannia) subgenera. We found that the activity and substrate specificity of the inward translocation machinery varied between Leishmania species. The differences in activity of inward phospholipid transport correlated with the different sensitivities of the various species towards the alkyl-phospholipid analogue miltefosine. Furthermore, all species exhibited a phospholipid scramblase activity in their plasma membranes upon stimulation with calcium ionophores. However, binding of annexin V to the parasite surface was only detected for a subpopulation of parasites during the stationary growth phase and only marginally enhanced by scramblase activation

Toll-Like Receptor and miRNA-let-7e Expression Alter the Inflammatory Response in Leishmania amazonensis-Infected Macrophages

Parasite recognition by Toll-like receptors (TLRs) contributes to macrophage activation and subsequent control of Leishmania infection through the coordinated production of pro-inflammatory and microbicidal effector molecules. The modulation of microRNA (miRNA) expression by Leishmania infection potentially mediates the post-transcriptional regulation of the expression of genes involved in leishmanicidal activity. Here, the contribution of TLR signaling to the miRNA profile and gene expression was evaluated in Leishmania amazonensis-infected murine macrophages. The infectivity of L. amazonensis was higher in murine bone marrow-derived macrophages from mice knockout for myeloid differentiation factor 88 (MyD88−/−), TLR2 (TLR2−/−), or TLR4 (TLR4−/−) than wild type C57BL/6 (WT). L. amazonensis infection of WT macrophages modulated the expression of 32% of the miRNAs analyzed, while 50% were upregulated. The absence of MyD88, TLR2, and TLR4 altered the percentage of miRNAs modulated during L. amazonensis infection, including the downregulation of let-7e expression. Moreover, the absence of signals mediated by MyD88, TLR2, or TLR4 reduced nitric oxide synthase 2 (Nos2) mRNA expression during infection. Indeed, the inhibition of let-7e increased levels of the Nos2 mRNA and NOS2 (or iNOS) protein and nitric oxide (NO) production in L. amazonensis-infected macrophages (4–24 h). The absence of TLR2 and inhibition of let-7e increased the expression of the arginase 1 (Arg1) mRNA but did not alter the protein level during infection. However, higher levels of the L-arginine transporters Cat2B and Cat1 were detected in the absence of Myd88 signaling during infection but were not altered following let-7e inhibition. The inhibition of let-7e impacted the global expression of genes in the TLR pathway by upregulating the expression of recognition and adaptors molecules, such as Tlr6, Tlr9, Ly96, Tirap, Traf 6, Ticam1, Tollip, Casp8, Map3k1, Mapk8, Nfkbib, Nfkbil1, Ppara, Mapk8ip3, Hspd1, and Ube2n, as well as immunomodulators, such as Ptgs2/Cox2, Csf 2, Csf 3, Ifnb1, Il6ra, and Ilr1, impacting NOS2 expression, NO production and parasite infectiveness. In conclusion, L. amazonensis infection alters the TLR signaling pathways by modulating the expression of miRNAs in macrophages to subvert the host immune responses

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

Abstract In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg 2 L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg 2 mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosoltargeted ARG lacking the glycosomal SKL targeting sequence (argDSKL) restored growth but failed to restore infectivity. Further study showed that the ARGDSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration

Leishmania Promastigotes Lack Phosphatidylserine but Bind Annexin V upon Permeabilization or Miltefosine Treatment

The protozoan parasite Leishmania is an intracellular pathogen infecting and replicating inside vertebrate host macrophages. A recent model suggests that promastigote and amastigote forms of the parasite mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger their phagocytic uptake into host macrophages. PS presentation at the cell surface is typically analyzed using fluorescence-labeled annexin V. Here we show that Leishmania promastigotes can be stained by fluorescence-labeled annexin V upon permeabilization or miltefosine treatment. However, combined lipid analysis by thin-layer chromatography, mass spectrometry and 31 P nuclear magnetic resonance (NMR) spectroscopy revealed that Leishmania promastigotes lack any detectable amount of PS. Instead, we identified several other phospholipid classes such phosphatidic acid, phosphatidylethanolamine; phosphatidylglycerol and phosphatidylinositol as candidate lipids enabling annexin V staining.FAZIT (AW)Research Training Group 1121 of the German Research FoundationCarlsberg FoundationCenter for Synthetic Biology at Copenhagen UniversityUNIK research initiative of the Danish Ministry of Science, Technology and Innovatio

Visceral leishmaniasis caused by Leishmania (Leishmania) amazonensis associated with Hodgkin’s lymphoma

Visceral leishmaniasis (VL) is mainly caused by Leishmania (Leishmania) donovani and Leishmania (L.) infantum; however, other Leishmania species have been associated with VL. We report a case of a patient simultaneously diagnosed with VL caused by Leishmania (L.) amazonensis and Hodgkin’s lymphoma. After treatment with liposomal amphotericin B and chemotherapy, the patient presented a clinical cure. This case report reinforces the hypothesis that other Leishmania species can cause visceral lesions mainly related to immunosuppression

Melatonin and Leishmania amazonensis Infection Altered miR-294, miR-30e, and miR-302d Impacting on Tnf, Mcp-1, and Nos2 Expression

Leishmaniases are neglected diseases that cause a large spectrum of clinical manifestations, from cutaneous to visceral lesions. The initial steps of the inflammatory response involve the phagocytosis of Leishmania and the parasite replication inside the macrophage phagolysosome. Melatonin, the darkness-signaling hormone, is involved in modulation of macrophage activation during infectious diseases, controlling the inflammatory response against parasites. In this work, we showed that exogenous melatonin treatment of BALB/c macrophages reduced Leishmania amazonensis infection and modulated host microRNA (miRNA) expression profile, as well as cytokine production such as IL-6, MCP-1/CCL2, and, RANTES/CCL9. The role of one of the regulated miRNA (miR-294-3p) in L. amazonensis BALB/c infection was confirmed with miRNA inhibition assays, which led to increased expression levels of Tnf and Mcp-1/Ccl2 and diminished infectivity. Additionally, melatonin treatment or miR-30e-5p and miR-302d-3p inhibition increased nitric oxide synthase 2 (Nos2) mRNA expression levels and nitric oxide (NO) production, altering the macrophage activation state and reducing infection. Altogether, these data demonstrated the impact of melatonin treatment on the miRNA profile of BALB/c macrophage infected with L. amazonensis defining the infection outcome

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg− L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg− mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (argΔSKL) restored growth but failed to restore infectivity. Further study showed that the ARGΔSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration

Comparative analysis of molecular methods for detection and identification of Leishmania spp. and development of methodology for leishmaniasis diagnosis.

No Brasil, leishmanioses são causadas por 7 espécies de Leishmania. Assim, um diagnóstico diferencial se torna relevante. Este trabalho tem como objetivos avaliar condições de armazenamento de amostras, métodos de extração de DNA e desenvolver protocolos de diagnóstico de leishmanioses. O tratamento de amostras com tampão NET produziu os melhores resultados e os métodos de extração não influenciaram a qualidade dos testes. Com os alvos testados por PCR, os melhores resultados foram alcançados com a utilização do 18S e o emprego da técnica de nested PCR com esse alvo aumentou a sensibilidade de detecção. A utilização de PCR multiplex com alvos 18S e g6pd foi capaz de detectar parasitas e discriminar seu subgênero. A aplicação da técnica HRM sobre mutações do 18S foi precisa na discriminação de L. chagasi e mutações no gene g6pd permitiram a discriminação de L. amazonenses, L. braziliensis e subgênero L. (Viannia). Este trabalho propõe protocolos de conservação de amostras, extração de DNA e ensaios de identificação de Leishmania, testados em amostras padronizadas.In Brazil, leishmaniasis is caused by 7 Leishmania species. Thus, a differential diagnosis becomes relevant. This work aims are to evaluate samples storage conditions, methods of DNA extraction and develop diagnostic protocols for leishmaniasis. Treatment of samples with NET buffer produced the best results and extraction methods did not affect the quality of PCR tests. Regarding PCR targets tested, the best results were achieved using 18S gene and the use of nested PCR increased detection sensitivity. The use of multiplex PCR targeting 18S and g6pd genes enabled Leishmania detection and subgenus discrimination. The implementation of HRM technique on 18S mutations was accurate to discriminate L. chagasi and mutations in the g6pd allowed discrimination of L. amazonenses, L. braziliensis and L. (Viannia) subgenus. This work proposes sample conservation and DNA extraction protocols for Leishmania detection, tested on standard sampling protocols, and identification using HRM technique

Discrimination of organisms from the Leishmania genus by High Resolution Melting analysis (HRM)

Leishmanioses são doenças classificadas pela Organização Mundial da Saúde (OMS) como negligenciadas, o que as caracterizam como aquelas que prevalecem em condições de pobreza e representam significativo entrave ao desenvolvimento por contribuírem para desigualdade. Cerca de 350 milhões de pessoas vivem sob o risco de infecção em 98 países da África, Eurásia e Américas. Acometem o homem e outros animais sob um espectro clínico amplo, que varia de discretas lesões, de cura espontânea, a quadros de comprometimento sistêmico, potencialmente fatais. A manutenção do ciclo de transmissão está inserida em um sistema biológico complexo, com a participação de mais de 20 espécies de Leishmania e uma grande variedade de reservatórios e vetores, e o diagnóstico está entre as estratégias empregadas para o controle dessas doenças. A precisa identificação das espécies envolvidas no ciclo de transmissão permite a geração de dados importantes para mapeamentos ecoepidemiológicos e para o delineamento de estratégias terapêuticas e de controle. O material genético do parasita como alvo de detecção e identificação desses organismos é descrito na literatura científica em trabalhos que abordam diversas estratégias metodológicas. Entre as técnicas mais recentes estão as análises de dissociação em alta resolução (HRM- High Resolution Melting), descrita como uma estratégia eficiente para a discriminação de polimorfismos em fragmentos específicos de DNA gerados por PCR (Polymerase Chain Reaction). O presente trabalho teve como principal objetivo padronizar um protocolo de detecção e identificação de Leishmania capaz de discriminar o maior número possível de espécies com base em polimorfismos do gene hsp70, utilizando HRM como ferramenta metodológica. Sequências nucleotídicas de hsp70 disponíveis em banco de dados e obtidas no laboratório foram analisadas in silico e regiões polimórficas foram delimitadas. Das regiões delimitadas, três foram escolhidas por conterem polimorfismos que geraram fragmentos cujas temperaturas de dissociação simuladas são distintas entre espécies ou grupo de espécies. A exploração de perfis de dissociação dos três amplicons de hsp70 obtidos por PCR em tempo real revelou diferenças que permitiram a discriminação das espécies de Leishmania responsáveis por doenças nas Américas, África e Eurásia. Os testes foram padronizados com a utilização de DNA de cepas-referência de Leishmania e então aplicados a amostras de DNA obtidas de amostras clínicas, de campo ou experimentais, como isolados, biópsias humanas frescas ou fixadas, flebotomíneos e cães naturalmente infectados e camundongos experimentalmente infectados. Os resultados obtidos por HRM foram comparados aos obtidos previamente por outras metodologias como PCR convencional, RFLP (Restriction Fragment Length Polymorphism) ou sequenciamento, com confirmação da identidade do parasita nas amostras testadas. O protocolo descrito é relativamente barato, tecnicamente simples, passível de automatização, podendo ser uma alternativa para a detecção e identificação de Leishmania em amostras, em estudos diagnósticos e ecoepidemiológicos.According to the World Health Organization (WHO), the leishmaniases are classified as neglected diseases, since they are related to poverty and contribute to inequality. Approximately 350 million people are at risk of infection in 98 countries in Africa, Eurasia and Americas. They affect humans and other animals that, depending on the species, causes a wide spectrum of clinical manifestations, that range from discrete lesions of spontaneous healing to potentially fatal systemic disease. The maintenance of the transmission cycle is part of a complex biological system, in which there is the participation of more than 20 species of Leishmania and a large variety of reservoirs and vectors. Diagnosis is important for early control of the disease. A precise identification of the species involved in the transmission cycle allows the generation of important data for eco-epidemiological mapping and for therapeutic measures and control strategies. Several genes have been used as diagnostic targets and several molecular strategies have been used in diagnosis. High resolution melting (HRM) analysis is among the most recent techniques and it is described as an efficient strategy for discriminating polymorphisms in specific DNA fragments generated by PCR (Polymerase Chain Reaction). The main objective of this work was to standardize a protocol for detection and identification of Leishmania spp, capable of discriminating the largest possible number of species based on hsp70 gene polymorphisms through HRM methodological tool. Available nucleotide sequences of hsp70 in databases as well as the ones obtained in the laboratory were analyzed in silico and polymorphic regions were delimited. Three regions were chosen since they contained polymorphisms that generated distinct simulated dissociation temperatures among species or group of species. The analysis of dissociation profiles of the three amplicons of hsp70 obtained by real-time PCR revealed differences that allowed the discrimination of Leishmania species responsible for diseases in the Americas, Africa and Eurasia. The tests were standardized using DNA from Leishmania reference strains and then applied to DNA samples obtained from clinical or from field samples, such as isolates, fresh or fixed human biopsies, phlebotomines and dogs naturally infected, and experimentally infected mice. The results obtained by HRM were compared to those obtained previously by other methodologies such as conventional PCR, RFLP (Restriction Fragment Length Polymorphism) or sequencing, confirming the parasite identity in the samples tested. The described protocol is relatively inexpensive, technically simple, potentially automated, and may be an alternative for the detection and identification of Leishmania in biological samples, in diagnostic and eco-epidemiological studies

Discrimination of organisms from the Leishmania genus by High Resolution Melting analysis (HRM)

Leishmanioses são doenças classificadas pela Organização Mundial da Saúde (OMS) como negligenciadas, o que as caracterizam como aquelas que prevalecem em condições de pobreza e representam significativo entrave ao desenvolvimento por contribuírem para desigualdade. Cerca de 350 milhões de pessoas vivem sob o risco de infecção em 98 países da África, Eurásia e Américas. Acometem o homem e outros animais sob um espectro clínico amplo, que varia de discretas lesões, de cura espontânea, a quadros de comprometimento sistêmico, potencialmente fatais. A manutenção do ciclo de transmissão está inserida em um sistema biológico complexo, com a participação de mais de 20 espécies de Leishmania e uma grande variedade de reservatórios e vetores, e o diagnóstico está entre as estratégias empregadas para o controle dessas doenças. A precisa identificação das espécies envolvidas no ciclo de transmissão permite a geração de dados importantes para mapeamentos ecoepidemiológicos e para o delineamento de estratégias terapêuticas e de controle. O material genético do parasita como alvo de detecção e identificação desses organismos é descrito na literatura científica em trabalhos que abordam diversas estratégias metodológicas. Entre as técnicas mais recentes estão as análises de dissociação em alta resolução (HRM- High Resolution Melting), descrita como uma estratégia eficiente para a discriminação de polimorfismos em fragmentos específicos de DNA gerados por PCR (Polymerase Chain Reaction). O presente trabalho teve como principal objetivo padronizar um protocolo de detecção e identificação de Leishmania capaz de discriminar o maior número possível de espécies com base em polimorfismos do gene hsp70, utilizando HRM como ferramenta metodológica. Sequências nucleotídicas de hsp70 disponíveis em banco de dados e obtidas no laboratório foram analisadas in silico e regiões polimórficas foram delimitadas. Das regiões delimitadas, três foram escolhidas por conterem polimorfismos que geraram fragmentos cujas temperaturas de dissociação simuladas são distintas entre espécies ou grupo de espécies. A exploração de perfis de dissociação dos três amplicons de hsp70 obtidos por PCR em tempo real revelou diferenças que permitiram a discriminação das espécies de Leishmania responsáveis por doenças nas Américas, África e Eurásia. Os testes foram padronizados com a utilização de DNA de cepas-referência de Leishmania e então aplicados a amostras de DNA obtidas de amostras clínicas, de campo ou experimentais, como isolados, biópsias humanas frescas ou fixadas, flebotomíneos e cães naturalmente infectados e camundongos experimentalmente infectados. Os resultados obtidos por HRM foram comparados aos obtidos previamente por outras metodologias como PCR convencional, RFLP (Restriction Fragment Length Polymorphism) ou sequenciamento, com confirmação da identidade do parasita nas amostras testadas. O protocolo descrito é relativamente barato, tecnicamente simples, passível de automatização, podendo ser uma alternativa para a detecção e identificação de Leishmania em amostras, em estudos diagnósticos e ecoepidemiológicos.According to the World Health Organization (WHO), the leishmaniases are classified as neglected diseases, since they are related to poverty and contribute to inequality. Approximately 350 million people are at risk of infection in 98 countries in Africa, Eurasia and Americas. They affect humans and other animals that, depending on the species, causes a wide spectrum of clinical manifestations, that range from discrete lesions of spontaneous healing to potentially fatal systemic disease. The maintenance of the transmission cycle is part of a complex biological system, in which there is the participation of more than 20 species of Leishmania and a large variety of reservoirs and vectors. Diagnosis is important for early control of the disease. A precise identification of the species involved in the transmission cycle allows the generation of important data for eco-epidemiological mapping and for therapeutic measures and control strategies. Several genes have been used as diagnostic targets and several molecular strategies have been used in diagnosis. High resolution melting (HRM) analysis is among the most recent techniques and it is described as an efficient strategy for discriminating polymorphisms in specific DNA fragments generated by PCR (Polymerase Chain Reaction). The main objective of this work was to standardize a protocol for detection and identification of Leishmania spp, capable of discriminating the largest possible number of species based on hsp70 gene polymorphisms through HRM methodological tool. Available nucleotide sequences of hsp70 in databases as well as the ones obtained in the laboratory were analyzed in silico and polymorphic regions were delimited. Three regions were chosen since they contained polymorphisms that generated distinct simulated dissociation temperatures among species or group of species. The analysis of dissociation profiles of the three amplicons of hsp70 obtained by real-time PCR revealed differences that allowed the discrimination of Leishmania species responsible for diseases in the Americas, Africa and Eurasia. The tests were standardized using DNA from Leishmania reference strains and then applied to DNA samples obtained from clinical or from field samples, such as isolates, fresh or fixed human biopsies, phlebotomines and dogs naturally infected, and experimentally infected mice. The results obtained by HRM were compared to those obtained previously by other methodologies such as conventional PCR, RFLP (Restriction Fragment Length Polymorphism) or sequencing, confirming the parasite identity in the samples tested. The described protocol is relatively inexpensive, technically simple, potentially automated, and may be an alternative for the detection and identification of Leishmania in biological samples, in diagnostic and eco-epidemiological studies