In silico genomic, phylogenetic, and functional characterization of candidate DNase-like proteins in Paracoccidioides brasiliensis and Paracoccidioides lutzii: implications for neutrophil extracellular traps

Paracoccidioides brasiliensis and Paracoccidioides lutzii are thermodimorphic fungal pathogens causing paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Here, we investigated PADG_08285 and PADG_11161, two hypothetical proteins from Paracoccidioides associated with extracellular DNase activity and potential involvement in neutrophil extracellular trap (NET) degradation. Proteins were retrieved using Biopython Entrez, and P. lutzii orthologs were identified by local BLAST+ searches. Multiple sequence alignment and maximum-likelihood phylogenetic reconstruction were performed using MAFFT and IQ-TREE, respectively. Functional domains, physicochemical properties, localization, signal peptides, and structural models were assessed using InterPro, Biopython ProteinAnalysis, DeepLoc, SignalP 6.0, and AlphaFold. PADG_08285 and PADG_11161 were identified as one-to-one orthologs of A0A0A2UZ79 and C1H8L0, respectively. Phylogenetic analysis indicated that short- and long-form proteins diverged before Paracoccidioides speciation. Short-form proteins were classified as TatD family deoxyribonucleases and showed conservation of the predicted catalytic tetrad Glu121, His158, His184, and Asp232, whereas long-form proteins were assigned to the Exonuclease V family. Neither candidate possessed a canonical signal peptide. Our findings identify the short-form TatD protein as the stronger candidate for the reported extracellular DNase activity and support further experimental investigation of its potential role in NET degradation.