DNAse action produced by Paracoccidioides brasiliensis on NETs (Neutrophil Extracellular Traps) released by human neutrophils in response to the fungus

Author(s)

Yohan Ricci Zonta

• Document type: Master's Dissertation
• Press: Botucatu. 2020-03-10.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina. Botucatu
• Defense date: 2020-02-27
• Advisor: Luciane Alarcão Dias Melicio

Abstract

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. (AU)

• FAPESP's process: 17/26230-3 - Action of DNase produced by Paracoccidioides Brasiliensis on nets (neutrophil extracellular traps) released by human neutrophils in response to the fungus
• Grantee: Yohan Ricci Zonta
• Support Opportunities: Scholarships in Brazil - Master