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Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape

Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are respo...

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Autores principales: Zonta, Yohan Ricci, Dezen, Ana Laura Ortega, Della Coletta, Amanda Manoel, Yu, Kaio Shu Tsyr, Carvalho, Larissa, dos Santos, Leandro Alves, Deprá, Igor de Carvalho, Kratofil, Rachel M., Willson, Michelle Elizabeth, Zbytnuik, Lori, Kubes, Paul, Ximenes, Valdecir Farias, Dias-Melicio, Luciane Alarcão
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902888/
https://www.ncbi.nlm.nih.gov/pubmed/33643928
http://dx.doi.org/10.3389/fcimb.2020.592022
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author Zonta, Yohan Ricci
Dezen, Ana Laura Ortega
Della Coletta, Amanda Manoel
Yu, Kaio Shu Tsyr
Carvalho, Larissa
dos Santos, Leandro Alves
Deprá, Igor de Carvalho
Kratofil, Rachel M.
Willson, Michelle Elizabeth
Zbytnuik, Lori
Kubes, Paul
Ximenes, Valdecir Farias
Dias-Melicio, Luciane Alarcão
author_facet Zonta, Yohan Ricci
Dezen, Ana Laura Ortega
Della Coletta, Amanda Manoel
Yu, Kaio Shu Tsyr
Carvalho, Larissa
dos Santos, Leandro Alves
Deprá, Igor de Carvalho
Kratofil, Rachel M.
Willson, Michelle Elizabeth
Zbytnuik, Lori
Kubes, Paul
Ximenes, Valdecir Farias
Dias-Melicio, Luciane Alarcão
author_sort Zonta, Yohan Ricci
collection PubMed
description Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.
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spelling pubmed-79028882021-02-25 Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape Zonta, Yohan Ricci Dezen, Ana Laura Ortega Della Coletta, Amanda Manoel Yu, Kaio Shu Tsyr Carvalho, Larissa dos Santos, Leandro Alves Deprá, Igor de Carvalho Kratofil, Rachel M. Willson, Michelle Elizabeth Zbytnuik, Lori Kubes, Paul Ximenes, Valdecir Farias Dias-Melicio, Luciane Alarcão Front Cell Infect Microbiol Cellular and Infection Microbiology Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action. Frontiers Media S.A. 2021-02-10 /pmc/articles/PMC7902888/ /pubmed/33643928 http://dx.doi.org/10.3389/fcimb.2020.592022 Text en Copyright © 2021 Zonta, Dezen, Della Coletta, Yu, Carvalho, Santos, Deprá, Kratofil, Willson, Zbytnuik, Kubes, Ximenes and Dias-Melicio http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Zonta, Yohan Ricci
Dezen, Ana Laura Ortega
Della Coletta, Amanda Manoel
Yu, Kaio Shu Tsyr
Carvalho, Larissa
dos Santos, Leandro Alves
Deprá, Igor de Carvalho
Kratofil, Rachel M.
Willson, Michelle Elizabeth
Zbytnuik, Lori
Kubes, Paul
Ximenes, Valdecir Farias
Dias-Melicio, Luciane Alarcão
Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape
title Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape
title_full Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape
title_fullStr Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape
title_full_unstemmed Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape
title_short Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape
title_sort paracoccidioides brasiliensis releases a dnase-like protein that degrades nets and allows for fungal escape
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902888/
https://www.ncbi.nlm.nih.gov/pubmed/33643928
http://dx.doi.org/10.3389/fcimb.2020.592022
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