RESUMO
BACKGROUND Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. OBJECTIVES We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). METHODS Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. FINDINGS Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. MAIN CONCLUSIONS The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.
RESUMO
Introducción: El oxígeno singulete es una especie reactiva que se obtiene mediante transferencia energética usando un fotosensibilizador. Su cuantificación directa requiere de instrumentación costosa, por lo cual es necesario recurrir a métodos indirectos que tengan suficiente selectividad y bajo costo. Estos procedimientos se basan en la interceptación química del oxígeno singulete produciendo una especie que se pueda detectar por métodos analíticos convencionales. En este artículo se describe la utilización del 9-[(E)-2-feniletenil] antraceno 1 (PEA) y del 9-[(E)-2-(naftalen-2-il) etenil]antraceno 2 (NEA), como alternativas viables y económicas para la cuantificación indirecta del oxígeno singulete, en medios acuosos. Su ventaja radica en la fácil detección de la desactivación de su fluorescencia una vez son oxidados por el oxígeno singulete. Materiales y Métodos: Los compuestos se sintetizaron y caracterizaron siguiendo procedimientos previamente reportados. Su capacidad para atrapar oxígeno singulete se determinó siguiendo su oxidación fotosensibilizada en solución de H2O/THF y en parásitos de Leishmania tarentolae, empleando azul de metileno o rosa bengala como fotosensibilizadores. Las muestras experimentales se iluminaron con una lámpara de emisión de luz visible, y se utilizaron métodos espectroscópicos (absorción UV-Vis, fluorescencia, RMN-1H) y espectrometría de masas para monitorear el atrapamiento y fotooxidación. Resultados y Discusión: Las pruebas espectroscópicas demostraron la capacidad que tienen los compuestos PEA 1 y NEA 2 para atrapar oxígeno singulete en solución acuosa y dentro de parásitos de L. tarentolae. Estudios de viabilidad parasitaria demuestran que PEA 1 es citotóxico en la oscuridad y cuando los cultivos son expuestos a la luz, mientras que NEA 2 no es citotóxico en la oscuridad, pero sí lo es cuando el cultivo es expuesto a la luz. En conclusión, los compuestos estudiados pueden servir como sondas para detectar y medir la producción de oxígeno singulete en medio acuoso y potencialmente en cultivos celulares, aunque es recomendable evaluar su actividad citotóxica en la oscuridad y bajo iluminación en estos casos.
Introduction: Singlet oxygen is a reactive species obtained via energy transfer using a photosensitizer. Its direct quantification requires expensive instrumentation, so it is necessary to use indirect methods having sufficient selectivity and low cost. These procedures are based on the chemical interception of singlet oxygen producing a species that can be detected using conventional analytical methods. This article describes the utilization of 9-[(E)-2-phenylethenyl]anthracene 1 (PEA) and 9-[(E)-2-(naphtalen-2-yl)ethenyl]anthracene 2 (NEA) as suitable and economic alternatives for the indirect quantification of singlet oxygen in aqueous media. Their advantage is the easy detection of their fluorescence once they are oxidized by singlet oxygen. Materials and Methods: Compounds were synthesized and characterized following procedures previously reported. Their capacity to trap singlet oxygen was determined by monitoring their photosensitized oxidation in either a H2O/THF solution or within Leishmania tarentolae parasites, utilizing methylene blue or rose bengal as photosensitizers. Experimental samples were illuminated with a lamp emitting visible light, while spectroscopical techniques (absorption, fluorescence, 1 H-NMR) and mass spectrometry were used to monitor trapping and photooxidation. Results and Discussion: Spectroscopical evidence demonstrates that both PEA 1 and NEA 2 are capable of trapping singlet oxygen in both aqueous media and within L. tarentolae parasites. Viability studies demonstrate that PEA 1 is cytotoxic in the dark and when parasite cultures were exposed to light, while NEA 2 does not show dark cytotoxicity, but is toxic when cultures were exposed to light. It can be concluded that both compounds under study may be utilized as probes to detect and quantify the production of singlet oxygen in aqueous media and potentially in cell cultures, although it is recommended to evaluate their cytotoxic activity both in the dark and upon light exposure in these cases.
RESUMO
Espécies do gênero Leishmania (Kinetoplastida, Trypanosomatidae) são agentescausadores de leishmanioses, que representam um complexo de doenças com variado espectroclínico e diversidade epidemiológica que, em alguns países, constituem-se em grave problema de saúde pública. A origem e evolução das leishmanioses têm estado em debate em seus aspectos clínicos e parasitológicos. Com a introdução da paleoparasitologia baseada na aplicação de diferentes técnicas, tornou-se possível a recuperação de vestígios do parasito,assim como o diagnóstico de infecções em humanos e em outros hospedeiros no passado. Os objetivos desta pesquisa foram diagnosticar parasitos do gênero Leishmania spp., em materiais arqueológicos da América do Sul por meio de técnicas moleculares e imunológicas. Estes são os primeiros registros de recuperação de DNA de Leishmania sp. em material arqueológico do Brasil, assim como também o primeiro registro deL. tarentolae em material antigo. Dois pares de primers que amplificam outras regiões de L. tarentolae foram desenhados para o estudo no intuito de averiguar se outros trechos de DNA desta espécie também teriam se preservado no material, tendo os mesmos não amplificado.Duas explicações são possíveis: devido a natureza fragmentada do DNA antigo o mesmo não se conservou de maneira homogenia no material, ou pode se tratar de um infectron, o que também é um dado relevante pois é uma assinatura da relação estabelecida entre parasito e hospedeiro...
Species of the genus Leishmania (Kinetoplastida, Trypanosomatidae) are causativeagents of leishmaniasis, which represents a complex disease with varied clinical spectrum and epidemiological diversity that, in some countries, constitute a serious public health problem. The origin and evolution of leishmaniasis has been under discussion in clinical and parasitological aspects. By the introduction of paleoparasitology based on the application of different techniques, as well as the diagnosis of infections in humans and other hosts in the past. Theaims of this research were: diagnosis by molecular PCR technique parasites of the genus Leishmaniain archaeological materials of South America, with the comparison of the sequences obtained from the GenBank for both recent and archaeological material, diagnosis through serological techniques: rapid test DPP and Western Blot, anti-Leishmania antibodies in the samples submitted to PCR, with subsequent comparison of these serological techniques with the previous lyused molecular technique and thus contribute to the knowledge of paleoparasitology of leishmaniasis, giving contributing to studies on the origin and evolution of Leishmania genus...