Ação de DNAse produzida por Paracoccidioides brasiliensis sobre NETs (Neutrophil Extracellular Traps) liberadas por neutrófilos humanos em resposta ao fungo
Data
2020-02-27
Autores
Zonta, Yohan Ricci [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A paracoccidioidomicose é uma doença fúngica sistêmica, considerada endêmica na América Latina, principalmente no Brasil, Venezuela, Argentina e Colômbia. Seus agentes etiológicos, fungos do complexo Paracoccidioides, possuem habitat geográfico restrito, conídios como forma infectante e características termodimórficas. A infecção ocorre por inalação dos conídios infectantes, levando a uma resposta imune grave nos pulmões e, dependendo da forma clínica, disseminação para outros órgãos. Os neutrófilos (PMNs) são responsáveis por uma importante resposta de defesa, liberando NETs (armadilhas extracelulares de neutrófilos), que podem aprisionar e possivelmente destruir as leveduras. No entanto, foi descrito que alguns patógenos são capazes de escapar dessas estruturas de DNA liberando DNAse como fator de virulência. Como diferentes padrões de NETs foram identificados em culturas de PMNs desafiadas com duas cepas de Paracoccidioides brasiliensis, procuramos avaliar se essas diferenças na conformação de NETs estariam relacionadas à capacidade de tais cepas produzirem DNAse, ou uma proteína com atividade semelhante à DNAse, que teria a capacidade de degradar as NETs. Assim, o objetivo geral deste trabalho foi identificar se diferentes padrões de NETs liberados por PMNs humanos desafiados com cepas Pb18 (virulenta) e Pb265 (avirulenta) seriam correlacionados com a capacidade dos fungos em produzir DNAse. Para este fim, os PMNs de indivíduos saudáveis foram isolados e desafiados in vitro com ambas as cepas de P. brasiliensis. A produção, liberação e conformação de NETs em resposta aos fungos foram avaliadas por Microscopia Confocal, Microscopia de Varredura e Imunoensaio para quantificação de NETs; a identificação de DNAse fúngica foi avaliada por DNAse TEST Agar e a expressão relativa de genes que codificariam proteínas hipotéticas identificadas no genoma dos fungos, foi investigada por RT-qPCR. Foi possível verificar a liberação de NETs pelos PMNs, mostrando diferentes conformações de NETs quando em contato com ambas as cepas do fungo. A cepa Pb18 induziu a liberação de NETs mais frouxas, maiores e com aspecto de uma rede aberta em comparação à cepa Pb265, que induziu a liberação de redes mais densas e compactas. Isto se dá, possivelmente, pela liberação de uma proteína semelhante à DNAse pela cepa virulenta, fato confirmado pelo DNAse Test Agar que demonstrou a capacidade de produção de enzima que causou a degradação do DNA do meio pela cepa virulenta, enquanto a cepa avirulenta não foi capaz de degradá-lo. Por fim, conseguimos identificar que os genes PADG_08528 e PADG_11161, que poderiam ser responsáveis por uma proteína DNAse-like hipotética estavam sendo expressos nas culturas desafiadas com os fungos, sendo o gene PADG_08528 altamente expresso pela cepa virulenta, sugerindo que esse gene poderia ter uma contribuição maior para essa produção. Portanto, a cepa virulenta pode estar produzindo DNAse como um importante mecanismo de escape do fungo para driblar ação das NETs, subvertendo assim esse importante mecanismo efetor de neutrófilos.
Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America, mainly in Brazil, Venezuela, and Colombia. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographical habitat, conidia as an infectious form and thermodimorphic characteristics. The infection occurs by inhalation of infective conidia, leading to a severe immune response in the lungs and, depending on the clinical type, spread to other organs. Neutrophils (PMNs) are responsible for an important defense response, releasing NETs (Neutrophil Extracellular Traps), which can envelop and destroy yeasts. However, it has been reported that some pathogens can escape from these DNA structures by releasing DNAse as a virulence factor. As different NET patterns were released in PMN cultures challenged with different Paracoccidioides brasiliensis strains, we sought to assess whether these differences in NETs would be related to the ability of such strains to produce DNAse, or a protein with activity similar to DNAse, which would have the ability to degrade NETs. Thus, the general objective of this study was to identify different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) strains and correlate this with the capacity of fungi to release DNAse. To this end, isolated PMNs were isolated and challenged in vitro with both strains of P. brasiliensis. The production, release and configuration of NETs in response to fungi were evaluated by Confocal Microscopy, Scanning Microscopy and Immunoassay to quantify NETs; the identification of fungal DNAse was evaluated by DNAse TEST Agar and relative expression of genes encoding hypothetical proteins identified in the fungal genome, was investigated by RT-qPCR. It was possible to verify the release of NETs by PMNs, showing different forms of NETs when coming into contact with different strains of the fungus. A Pb18 strain induced the release of looser, larger NETs with the appearance of an open network compared to the Pb265 strain, which induced the release of denser and more compact NETs. Thus, it was identified that a virulent strain is possibly inducing looser and more degraded NETs, probably by releasing a DNAse-like protein, a fact confirmed by the DNAse Test Agar, which demonstrates the ability to produce enzymes that cause the degradation of DNA from the medium by the virulent strain, while an avirulent strain was not able to degrade it. Finally, we identified the expression of genes PADG_08528 and PADG_11161, probably involved with the production of hypothetical protein similar to DNAse, being the gene PADG_08528 highly expressed in the virulent strain, suggesting that this gene could have a greater contribution to this production. Therefore, the virulent strain may be producing DNAse as an important escape mechanism of the fungus to circumvent the action of NETs, thus subverting this important neutrophil effector mechanism.
Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America, mainly in Brazil, Venezuela, and Colombia. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographical habitat, conidia as an infectious form and thermodimorphic characteristics. The infection occurs by inhalation of infective conidia, leading to a severe immune response in the lungs and, depending on the clinical type, spread to other organs. Neutrophils (PMNs) are responsible for an important defense response, releasing NETs (Neutrophil Extracellular Traps), which can envelop and destroy yeasts. However, it has been reported that some pathogens can escape from these DNA structures by releasing DNAse as a virulence factor. As different NET patterns were released in PMN cultures challenged with different Paracoccidioides brasiliensis strains, we sought to assess whether these differences in NETs would be related to the ability of such strains to produce DNAse, or a protein with activity similar to DNAse, which would have the ability to degrade NETs. Thus, the general objective of this study was to identify different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) strains and correlate this with the capacity of fungi to release DNAse. To this end, isolated PMNs were isolated and challenged in vitro with both strains of P. brasiliensis. The production, release and configuration of NETs in response to fungi were evaluated by Confocal Microscopy, Scanning Microscopy and Immunoassay to quantify NETs; the identification of fungal DNAse was evaluated by DNAse TEST Agar and relative expression of genes encoding hypothetical proteins identified in the fungal genome, was investigated by RT-qPCR. It was possible to verify the release of NETs by PMNs, showing different forms of NETs when coming into contact with different strains of the fungus. A Pb18 strain induced the release of looser, larger NETs with the appearance of an open network compared to the Pb265 strain, which induced the release of denser and more compact NETs. Thus, it was identified that a virulent strain is possibly inducing looser and more degraded NETs, probably by releasing a DNAse-like protein, a fact confirmed by the DNAse Test Agar, which demonstrates the ability to produce enzymes that cause the degradation of DNA from the medium by the virulent strain, while an avirulent strain was not able to degrade it. Finally, we identified the expression of genes PADG_08528 and PADG_11161, probably involved with the production of hypothetical protein similar to DNAse, being the gene PADG_08528 highly expressed in the virulent strain, suggesting that this gene could have a greater contribution to this production. Therefore, the virulent strain may be producing DNAse as an important escape mechanism of the fungus to circumvent the action of NETs, thus subverting this important neutrophil effector mechanism.
Descrição
Palavras-chave
Paracoccidioidomicose, Paracoccidioides brasiliensis, Neutrófilos, NETs, DNAse, Paracoccidioidomycosis, Neutrophils