Identification and purification of DNAse produced by fungi of the Paracoccidioides spp complex and their action on NETs (Neutrophil Extracellular Traps) released by human neutrophils in response to the fungus

Abstract

Paracoccidioidomycosis is a systemic mycosis whose etiological agents are the fungi of the S1 (Paracoccidioides brasiliensis), PS2 (Paracoccidioides americana), PS3 (Paracoccidioides restrepiensis), PS4 (Paracoccidioides venezuelensis) and Paracoccidioides lutzii, all belonging to the Paracoccidioides spp. It´s an endemic disease in Latin American countries, its distribution is heterogeneous and with the largest outbreaks being concentrated in some countries, such as Brazil, Venezuela and Colombia. Recently, evolutionary molecular studies suggest different mechanisms of pathogenicity and intracellular survival in all Paracoccidioides species, demonstrating that the genus Paracoccidioides presents important differences in its metabolic profiles, which should play a critical role during the host-pathogen interaction. Neutrophils, phagocytic cells of innate immunity, are the main responsible for defensive response called NETosis, that´s result in Neutrophil Extracellular Traps (NETs) production. The NETs are capable of trapping and destroying the fungus. However, studies already showed the capability of some fungi to produce certain proteins, such as DNAse or DNAse like protein, as a escape mechanisms from NETs. Since the production of extracellular DNAse is an important virulence factor already demonstrated for several pathogens, the objectives of the present study will be the DNAse production evaluation by different strains of fungi of the complex of species Paracoccidioides spp. For this, it will be used two experimental designs, in which first, different species of the Paracoccidioides genus complex will be evaluated and the activity of DNAse produced in the fungal cultures will be visualized on DNAse Test Agar plates. The enzymes will also be extracted, identified and quantified from these cultures using HPLC and Mass Spectrometry techniques, and the DNAse gene expression will be analyzed by the RT-PCR technique. Besides, we will also evaluate this enzymatic production during the interaction of fungi with human neutrophils in vitro, to identify the production, release and different patterns of NETs in response to the different species of the fungus in the human neutrophil cultures challenged, correlating with ability of these species to produce DNAse in these cultures. To do so, analysis of different NET patterns production during challenge will be made using Confocal Microscopy, Electronic Scanning Microscopy and High Content analysis. Quantification of NETs by adapted fluorimetric kit Quant-iT PicoGreen and quantification of the fungal DNAse gene encoded, by mRNA expression performed by RT-PCR, will be also performed. (AU)