The Pb MDJ1 Gene Belongs to a Conserved MDJ1/LON Locus in Thermodimorphic Pathogenic Fungi and Encodes a Heat Shock Protein That Localizes to both the Mitochondria and Cell Wall of Paracoccidioides brasiliensis

Abstract

J-domain (DnaJ) proteins, of the Hsp40 family, are essential cofactors of their cognate Hsp70 chaperones, besides acting as independent chaperones. In the present study, we have demonstrated the presence of Mdj1, a mitochondrial DnaJ member, not only in the mitochondria, where it is apparently sorted, but also in the cell wall of Paracoccidioides brasiliensis, a thermodimorphic pathogenic fungus. The molecule (PbMdj1) was localized to fungal yeast cells using both confocal and electron microscopy and also flow cytometry. The antirecombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial and cell wall (alkaline ␤-mercaptoethanol extract) component, compatible with the predicted size of the protein devoid of its matrix peptide-targeting signal. Labeling was abundant throughout the cell wall and especially in the budding regions; however, anti-PbMdj1 did not affect fungal growth in the concentrations tested in vitro, possibly due to the poor access of the antibodies to their target in growing cells. Labeled mitochondria stood preferentially close to the plasma membrane, and gold particles were detected in the thin space between them, toward the cell surface. We show that Mdj1 and the mitochondrial proteinase Lon homologues are heat shock proteins in P. brasiliensis and that their gene organizations are conserved among thermodimorphic fungi and Aspergillus, where the genes are adjacent and have a common 5 region. This is the first time a DnaJ member has been observed on the cell surface, where its function is speculative.

Extracellular Vesicles Produced by Cryptococcus neoformans Contain Protein Components Associated with Virulence

“…Glyceraldehyde-3-phosphate dehydrogenase, a major protein component of the glycolytic pathway, is present in the cell wall of Paracoccidioides brasiliensis, where it participates in the pathogenic processes mediating the adhesion of yeast cells to host cells and the extracellular matrix (2). In the same model, the mitochondrial protein Mdj1 was detected not only in the mitochondria, where it is apparently sorted, but also in the cell wall (4). Proteomic analysis of the C. neoformans vesicles revealed a complex protein composition that included chaperone and membrane, cytoplasmic, and even nuclear and mitochondrial proteins.…”

“…In accordance with this supposition, treatment of C. neoformans with brefeldin A, an inhibitor of the Golgi apparatus-derived transport of molecules, results in a significant inhibition of capsule expression (23). Fungal proteins frequently have more than a single function and are found in different cellular locations (2,4,16,37). For example, histones have been described as being present at the cell wall of Histoplasma capsulatum, where they are targeted by antifungal antibodies (37).…”

Paracoccidioides brasiliensis Enolase Is a Surface Protein That Binds Plasminogen and Mediates Interaction of Yeast Forms with Host Cells

“…The nuclear histone-like protein H2B, for instance, is also found at the cell wall of Histoplasma capsulatum, where it functions as a target for protective antibodies (36). In P. brasiliensis, the mitochondrial protein Mdj1p and the cytosolic enzymes GAPDH and TPI were also characterized as cell wall components (4,5,41). This multiplicity in cellular distribution and functions is also common to enolase because this protein functions in sugar metabolism but is also present at the cell surface (30) and in secretory vesicles that reach the extracellular space (45).…”

The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

“…For transmission electron microscopy (TEM) analysis of P. brasiliensis yeasts, cells from logarithmic-phase cultures grown in liquid mYPD medium were processed in fixative solutions and embedded in hydrophobic Spurr resin exactly as described by Batista et al (9). For immunogold labeling, both P. brasiliensis Pb18 cells and vesicle pellets obtained after centrifugation at 100,000 ϫ g were processed as described by Rodrigues et al (33).…”