| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1201-1 | ||||
Resumo:Paracoccidioidomycosis (PCM) is caused by the dimorphic fungi Paracoccidioides spp., an microrganism that have ability to adapt to dramatic environmental changes inside the host. The morphological switch from mycelium to yeast form is obligatory for the establishing of the pathogenicity in Paracoccidioides brasiliensis. Two‑component signaling systems are used by microorganisms to sense and respond to external environmental changes. Fungi possess a large family of histidine kinases (HKs) classified into 11 distinct groups. Only Group III (HK3) homologs in some fungi can sense osmotic and/or oxidative cues upstream of the signaling MAPK Hog1 cascade. HK3 represent an appealing new therapeutic drug target because they are widely expressed in fungi but absent from humans. Drk1 is a HK3, which functions as a global regulator of dimorphism and virulence in Blastomyces dermatitidis and Histoplasma capsulatum. Recently, our group characterized the PbDRK1 gene expression (only HK3 present in the fungus) in P. brasiliensis, and we demonstrated its role in dimorphism using a specific HK3 inhibitor (fludioxonil - iDrk). We observed that the fungus treated with iDrk remained in the mycelial form even when cultivated at 37°C. This study aimed to evaluate Hog1 activation in the presence of a Drk1 inhibitor (fludioxonil) after different stresses. First, to reveal the sublocation of PbDrk1 and Hog1 in P. brasiliensis after stress immunofluorescence assays were performed. Laser scanning confocal microscopy (LSCM) demonstrated that PbDrk1 appeared abundantly at the periphery of yeast cells. Then, we investigated the phosphorylation profile of Hog1. P. brasiliensis yeast cells were subjected to different stresses (NaCl, CR, CFW, and H2O2) in the presence or absence of iDrk1 incubated for 15 and 30 min. Next, protein extraction and SDS-PAGE were performed, followed by Western blotting. The antibodies used were phospho-Hog1 (Thr180/Tyr 182), and the total Hog1 control was obtained from the Hog1 antibody. We observed that after incubation with NaCl, there was an increase in Hog1 phosphorylation levels. On the other hand, when yeasts were incubated with NaCl and iDrk1, a reduction in the phospho-Hog1 levels was observed. Finnaly, P. brasiliensis yeasts stimulated with cell wall stressors (CR and CFW) in the presence of iDrk1 showed a reduction in phospho-Hog1 levels. This evidence strongly suggests a connection between the Hog1 and Drk1 pathway, indicating that PbDrk1 is probably not exclusively related to the pathway that regulates dimorphism. Palavras-chave: dimorphic fungi, Paracoccidioides , MAP kinase Hog1, PbDrk1 Agência de fomento:CNPq, FAPESP | |||||