Determination of the peroxisomal proteome of pathogenic stage <i>Histoplasma capsulatum</i>

ABSTRACT During infection, Histoplasma capsulatum exists primarily as an intracellular pathogen of phagocytes, where it overcomes host defense mechanisms and replicates within the phagosome. This intracellular proliferation of Histoplasma yeasts requires the function of the fungal peroxisome for multiple, incompletely defined roles, with early pathogenesis functions dependent on the proper trafficking of peroxisomal matrix proteins carrying a type 1 peroxisome-targeting signal (PTS1). To better understand the potential roles of peroxisomes during Histoplasma infection, we identified the constituents of the peroxisomal proteome of pathogenic-phase Histoplasma yeasts through proximity labeling using a peroxisome-targeted TurboID biotin ligase. Comparative proteomics using peroxisome- or cytosol-localized TurboID identified 90 high-confidence peroxisomal proteins. To validate the peroxisomal proteome, a subset of nine putative peroxisomal proteins was localized to the peroxisome using fluorescent protein fusions. Comparison of peroxisomal proteomes for Histoplasma yeasts lacking the Pex5 or Pex7 cytosolic PTS1 or PTS2 receptors assigned proteins to either the PTS1- or PTS2-dependent peroxisomal import pathways, and refined the Histoplasma PTS1 tripeptide consensus sequence. The peroxisomal proteome of Histoplasma yeast during macrophage infection was ultimately determined to provide relevance to Histoplasma ’s primary pathogenic niche. The identities of the proteins comprising the peroxisomal proteome include several enzymes for canonical peroxisome-localized biochemical pathways (e.g., fatty acid utilization and siderophore biosynthesis), but also represent several potential novel functions for the organelle. The defined peroxisomal proteome of pathogenic-phase Histoplasma yeasts, particularly the set of proteins imported via the PTS1 import pathway, thus establishes the important foundation for understanding how peroxisomes promote intracellular pathogenesis. IMPORTANCE Peroxisomes are eukaryotic organelles that serve as a subcellular compartment for diverse metabolic and biosynthetic reactions, some of which play crucial roles in supporting the intracellular pathogenesis of the fungal pathogen Histoplasma capsulatum . In this study, we used proximity labeling with the biotin ligase TurboID to capture peroxisomal matrix proteins, enabling the empirical derivation of the peroxisomal proteome of pathogenic-phase Histoplasma yeasts, including during Histoplasma infection of mammalian cells. This represents the first organelle-specific proteome definition in Histoplasma and highlights the utility of proximity-based biotinylation and comparative proteomics for understanding how subcellular compartments and their functions contribute to intracellular pathogenesis.