| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1116-1 | ||||
Resumo:Dimorphic and thermo-dependent fungi of the genus Paracoccidioides spp. are causative agents of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America with high prevalence in Brazil. In the environment (25°C), the fungus is present in the mycelium form, and after inhaling by the mammalian host (37°C), the fungus changes its form to yeast. In dimorphic fungi, a Dimorphism Regulatory Histidine Kinase (DRK1) is expressed mainly in the yeast phase. Recently, our group demonstrated that fludioxonil (a specific histidine kinase inhibitor) was able to inhibit the dimorphism of Paracoccidioides brasiliensis. Histidine kinases have great importance in the regulation of virulence and cell survival, and the investigation of signal transduction pathways associated with these kinases can help discover potential new pharmacological targets and, consequently, the development of drugs with antifungal activity since histidine kinases are present in prokaryotes, plants, bacteria, and fungi but absent in mammalian cells. Thus, this study aimed to evaluate the proteomic profile of the fungus after treatment with fludioxonil. Common proteomics approaches in medical mycology have been made with DDA methods in different instruments. The proteins inferred from peptides cover roughly 25% of the database, that is, around 2,000 proteins. Here we present a high-coverage proteome of the P. brasiliensis for the first time. We used Data Independent Acquisition (DIA) method in an Exploris 480 mass spectrometer coupled to a Vanquish Neo chromatography system combined with DIA-NN v1.8.1 search engine, which allowed us to find, on average 4,000 proteins per run. We compared two search strategies: one using a project-specific library produced by gas-phase fractionation and another using library-free search. Both strategies produced comparable results, with the project-specific library showing small benefits in data completeness. For simplicity, we decided to proceed with a library-free pipeline. Our investigation using sparse Partial Least Squares Discriminant Analysis proved useful for discriminating between yeasts treated or not with fludioxonil (25 μg/mL) using the protein abundance distribution. Refining the sPLS-DA enables us to identify the proteins responsible for the classification. Our preliminary data show that: (I) Data Independent Acquisition mass spectrometry allows, for the first time, to investigate the deep proteome of dimorphic fungi, covering 50% of the whole proteome in a single experiment; (II) Project-specific or library-free approaches have similar performances; (III) sPLS-DA enable to discriminate between fludioxonil treated cells and controls by using protein abundance information; and (IV) Refining the sPLS-DA to find the variable importance in projection allow to find the proteins most important to discriminate between groups. Palavras-chave: dimorphic fungi, Paracoccidioides brasiliensis, Proteomics, DIA, fludioxonil Agência de fomento:CAPES e FAPESP | |||||