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GONÇALES, R. A. Paracoccin: a major chitinase for the pathobiology and virulence of Paracoccidioides brasiliensis. 2018. 154 f. Thesis (Doctorade Scientiae degree) – School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 2018. Species of the genus Paracoccidioides spp are thermodymorphic fungi that cause a systemic disease, which is endemic in several regions of the Latin America. The infected individual develops a specific response that, when associated with the high production of TNF-α and IFNγ, favors resistance to the fungus. Components of some pathogenic fungi were characterized by gene knockdown techniques as important the for fungal virulence. Our group has identified a component of P. brasiliensis, named Paracoccin (PCN); it is a bifunctional protein with an enzymatic domain, endowed with chitinase activity and a lectin domain, which binds GlcNAc and chitin, a GlcNAc polymers. PCN has the following properties: (a) contributes to the fungus growth; (b) promotes the yeast adhesion to the extracellular matrix, by binding to laminina glycans; (c) interacts with TLR2 and TLR4 N-glycans, which triggers cell activation; (d) stimulates macrophages to produce proinflammatory mediators, such as IL-12, TNF-α and NO; (e) promotes the M1 polarization of macrophages; (f) induces the neutrophils fungicidal activity, NETs formation, and suppression of neutrophils apoptosis, which are depending events on the de novo protein synthesis by neutrophils. Given the relevant biological activities exerted by PCN, we have performed recently the silencing of the gene that codes for this protein through a system that uses RNA anti-sense and Agrobacterium tumefaciens mediated transformation (ATMT). Once having the PCN gene silenced, yeast lost the ability of doing the transition to mycelium and decreased its resistance to macrophages fungicidal activities. Mice infection with PCN-silenced yeasts, compared to the infected with WT yeasts, exhibited a milder pulmonary disease with reduced fungal burden and low mortality rate. These observations suggest that PCN acts as a P. brasiliensis virulence factor that affects the pathogenesis of the fungal infection. In the present study, we expanded the molecular tools for the fungus manipulation and enabled the overexpression of PCN in P. brasiliensis yeasts, aiming to elucidate the PCN role in the fungus virulence and the infection pathogenesis, as well as determining the responsible mechanisms for the PCN activities. Inoculation of the PCN overexpressing yeasts (ov-PCN) into mice caused a very severe lung disease, compared to the mild and moderate diseases caused by PCN-silenced and WT yeasts, respectively. Then our efforts became dedicated to the search of mechanisms through which PCN influences the course of the experimental fungal disease. In an attempt to identify the role of the PCN chitinase domain, we harvested the supernatant of the ov-PCN and WT yeasts cultures. Chitin particles contained in the supernatants have been captured by affinity to the immobilized WGA (wheat germ agglutinin) lectin. By measuring through electron microscopy and application of the ImageJ program the area of the isolated chitin particles, we verified that the overexpression of PCN resulted in a more efficient cleavage of whole chitin molecules contained in the yeast cell wall, since only very small particles (median of the measurements = 2 nm) were detected, while the the chitin particles areas obtained from WT-yeasts provided a median 3 fold higher (6 nm). Then, the preparations of chitin particles were taken to stimulate macrophages to produce cytokines. The particles obtained from ov-PCN have stimulated preponderantly the secretion of the anti-inflammatory cytokine IL-10, whereas the macrophages stimulated with WT yeast particles have produced higher concentrations of TNF-α and IL-1β, which are known proinflammatory cytokines. These results allowed the identification of an important mechanism for the association of PCN overexpression to the occurrence of very severe pulmonary disease: the anti-inflammatory microenvironment created by the macrophages stimulation with PCN leads to the development of a non-protective Th2-type immune response and the more severe pulmonary injury. A second mechanism was identified as implicated in the severity of the lung disease associated to PCN overexpression. We compared the sensitivity of ov-PCN and WT yeasts to macrophages effector functions. PCN overexpressing yeasts were better internalized by macrophages and more resistant to the fungicidal activity of these cells, events that contributes for the high pulmonary fungal load verified in mice infected with ov-PCN yeasts. The study demonstrates that different levels of a chitinase (PCN) expression and enzymatic activity lead yeasts to change their sensitivity to macrophages antifungal activities as well as to different grades of chitin cleavage. The cleavage, in its turn, leads to changes in the structure of the fungal cell wall and generation of chitin fragments, whose sizes and concentrations influence the cytokines production by macrophages. Under the influence of pro-inflammatory or anti-inflammatory cytokines released by macrophages, the mounted adaptative responses can be decisive in conferring susceptibility or resistance to the P. brasiliensis infection. This study provides an important advance in the knowledge on the role of a chitinase in the host antifungal response.

Paracoccina: uma quitinase importante para a patobiologia e virulência de Paracoccidioides brasiliensis