Avaliação das propriedades adesivas e imunogênicas da enolase de Sporothrix spp.
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Data
2017-08-25
Autores
Loesch, Maria Luiza de Aguiar [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A esporotricose é uma micose subcutânea aguda ou crônica causada por fungos termodimórficos do complexo de espécies de Sporothrix schenckii e pode acometer tanto animais como seres humanos. A doença tem distribuição mundial e no Brasil se tornou endêmica, sendo S. schenckii e S. brasiliensis as duas espécies patogênicas maisrelacionadasà transmissão zoonóticaassociada principalmente ao gato. O tratamento convencional com antifúngicos é demorado e tem muitos efeitos adversos, especialmente em pacientes com imunocomprometidos. Logo, nos últimos anos tem-se focado a atenção sobre os componentes da parede celular de espécies do complexo Sporothrixque possam estar envolvidos na sua virulência, para serem utilizados como potenciais alvos antigênicos nodiagnóstico e terapêutica contra a esporotricose. Recentemente, nosso grupo de trabalho demonstrou que um soro obtido em camundongos imunizados com um extrato de proteínas da superfície celular (PSC) de S. schenckii foi imunorreativo contra a enolase contida nesse extrato e uma outra proteína de função desconhecida. A capacidade desse soro de potencializar a fagocitose e inibir a adesão do fungo aos fibroblastos in vitro, e ainda a proteção por transferência passiva em animais infectados pelo fungo foi atribuída à sua atividade ligante de enolase.Diante disto, o objetivo deste estudo foi avaliar o efeito da enolase na adesão de S. schenckii ATCC 16345 e S. brasiliensis (Ss250 e Ss256) a fibroblastos assim como suas propriedades imunogênicas. A enolase de S. schenckii(rEno) foi obtida por via recombinante em Escherichia coli com alto grau de pureza, conferido por cromatografia de afinidade e exclusão molecular. O immunoblottig revelou a especificidade do soro anti-rEno e mostrou a reatividade desse soro com uma banda de proteína próxima a 50 kDa presente na PSC de Ss250 e Ss256. A enolase presente nessa banda e suas possíveis isoformas foram confirmadas por espetrometria de massas e eletroforese bidimensional, respectivamente. O alto título de anticorpos induzidos em camundongos contra a enolase em formulação ou não com o adjuvante de Freund revelou a imunogenicidade desta proteína. O soro anti-rEno foi capaz de reduzir a adesão das leveduras de S. schenckii e S. brasiliensisa fibroblastos assim como potencializar a fagocitose das mesmas por macrófagos peritoneais. Esses resultados, unidos à capacidade de ligação da enolase a fibronectina e ao plasminogênio sugeremque a enolase de Sporothrix spp além de ser imunogênica pode estar envolvida na virulência do fungo, tornando-a um alvo relevante de estratégias vacinais e/ou terapêuticas na proteção contra a esporotricose.
Sporotrichosis is an acute or chronic subcutaneous mycosis caused by thermodimorfic fungi from the Sporothrix schenckii species complex that can affect both humans and other animals. The disease is amply distributed worldwide and endemic in Brazil, with S. schenckii and S. brasiliensis being the two pathogenic species most associated to the zoonotic transmission which, in turn, involves mainly the domestic cat. The conventional antifungal therapy takes long and has adverse effects, especially in immunocompromised patients. In light of this, attention has been focused in recent years on the cell wall components of species from the Sporothrix complex that could be involved in their virulence, so that they could be used as potential antigenic targets in the diagnosis and therapy of sporotrichosis. Recently, we showed that a serum obtained from mice which had been immunized with a S. schenckii cell surface protein (CSP) extract reacted with enolase, found in the extract, and another protein of unknown function. The capacity of this serum to enhance phagocytosis and inhibit adhesion of the fungus to fibroblasts in vitro, as well as the protection afforded upon its passive transference into infected mice, was attributed to its enolase-binding activity. In face of the above, this study aimed to assess both the immunogenic properties and the role played by enolase in S. schenckii ATCC 16345 and S. brasiliensis (Ss250 and Ss256) adhesion to fibroblasts. The S. schenckii enolase (rEno) was obtained through the recombinant route in Escherichia coli in a highly purified form, as determined by both affinity and molecular exclusion chromatography. The specificity of the anti-sEno serum and its reactivity with a ~50 kDa protein band among the CSP from Ss250 and Ss256 was confirmed by immunoblotting. The presence of enolase in this band and the existence of possible isoforms were shown by mass spectrometry and two-dimensional electrophoresis, respectively. Moreover, the high anti-rEno titers induced in mice upon immunization with rEno alone or formulated with Freund’s adjuvant showed that enolase is immunogenic. Furthermore, the anti-rEno serum was able to decrease the adhesion of S. schenckii and S. brasiliensis yeasts to fibroblasts, as well as to enhance their phagocytosis by peritoneal macrophages. These results, together with the fibronectin- and plasminogen-binding activity of enolase, suggest that the Sporothrix sppenolase, besides being immunogenic, may have a role in the virulence of these fungi, making it a relevant target for therapeutic and/or vaccine strategies against sporotrichosis.
Sporotrichosis is an acute or chronic subcutaneous mycosis caused by thermodimorfic fungi from the Sporothrix schenckii species complex that can affect both humans and other animals. The disease is amply distributed worldwide and endemic in Brazil, with S. schenckii and S. brasiliensis being the two pathogenic species most associated to the zoonotic transmission which, in turn, involves mainly the domestic cat. The conventional antifungal therapy takes long and has adverse effects, especially in immunocompromised patients. In light of this, attention has been focused in recent years on the cell wall components of species from the Sporothrix complex that could be involved in their virulence, so that they could be used as potential antigenic targets in the diagnosis and therapy of sporotrichosis. Recently, we showed that a serum obtained from mice which had been immunized with a S. schenckii cell surface protein (CSP) extract reacted with enolase, found in the extract, and another protein of unknown function. The capacity of this serum to enhance phagocytosis and inhibit adhesion of the fungus to fibroblasts in vitro, as well as the protection afforded upon its passive transference into infected mice, was attributed to its enolase-binding activity. In face of the above, this study aimed to assess both the immunogenic properties and the role played by enolase in S. schenckii ATCC 16345 and S. brasiliensis (Ss250 and Ss256) adhesion to fibroblasts. The S. schenckii enolase (rEno) was obtained through the recombinant route in Escherichia coli in a highly purified form, as determined by both affinity and molecular exclusion chromatography. The specificity of the anti-sEno serum and its reactivity with a ~50 kDa protein band among the CSP from Ss250 and Ss256 was confirmed by immunoblotting. The presence of enolase in this band and the existence of possible isoforms were shown by mass spectrometry and two-dimensional electrophoresis, respectively. Moreover, the high anti-rEno titers induced in mice upon immunization with rEno alone or formulated with Freund’s adjuvant showed that enolase is immunogenic. Furthermore, the anti-rEno serum was able to decrease the adhesion of S. schenckii and S. brasiliensis yeasts to fibroblasts, as well as to enhance their phagocytosis by peritoneal macrophages. These results, together with the fibronectin- and plasminogen-binding activity of enolase, suggest that the Sporothrix sppenolase, besides being immunogenic, may have a role in the virulence of these fungi, making it a relevant target for therapeutic and/or vaccine strategies against sporotrichosis.
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Palavras-chave
Sporothrix, Enolase, Adesão, Adhesion