The PbMDJ1 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 anti-recombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial and cell wall (alkaline beta-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.

FIG. 1.

(A) Schematic representation of PbMDJ1, highlighting the localization of the primers and probes used in this work. Introns were localized from nt 205 to 313 and nt 1552 to 1683. (B) Schematic representation of PbMdj1 showing the localization of conserved domains. Kyte-Doolittle hydrophilicity plots (Protean module; DNAstar Inc.) of fungal Mdj1-like sequences are as follows: Pb, P. brasiliensis (AF334811); Bd, B. dermatitidis (http://genome.wustl.edu/BLAST/blasto_client.cgi); Hc, H. capsulatum (http://genome.wustl.edu/BLAST/histo_client.cgi); Ci, C. immitis (http://www.broad.mit.edu/annotation/fungi/coccidioides_immitis); An, A. nidulans (EAA57980); Cn, C. neoformans (EAL21819); Ca, C. albicans (EAK92195); Sc, S. cerevisiae (CAA82189); Ec, Escherichia coli (BA000007). The boxes indicate the predicted mitochondrial target sequence (MT) and the J domain. The dots localize the Zn²⁺ finger domains. Horizontal bars indicate the location of mouse T-cell epitopes with a minimum of 12 amino acids, as predicted by the Sette major histocompatibility complex motif (Protean module; DNAstar Inc). Percentages of identity with PbMdj1, as calculated with the MegAlign module of the DNAstar program, are given at right.

FIG. 2.

Phylogenetic tree of fungal Mdj1 sequences according to the Clustal W program. Besides the fungal sequences indicated in the legend to Fig. 1, we included those of A. fumigatus (EAL93469), N. crassa (EAA32959), F. graminearum (EAA76137), S. pombe (CAB09769), C. glabrata (CAG60838), and Homo sapiens (AF061749). A phylogenetic tree of the correspondent Lon sequences showed similar distributions. On the right, schematic representations of the chromosomal organization and gene direction in the MDJ1/LON locus of the species that bear these genes in the same chromosome. The distances separating the genes (interrupted line) in N. crassa, F. graminearum, and C. glabrata are, respectively, 236,960 kb, 1,982,719 kb, and 19,091 kb.

FIG. 3.

(A) Coomassie blue-stained 10% SDS-PAGE gels showing insoluble total bacterial extracts from recombinant bacteria expressing PbMdj1 or PbLon (extract) and the respective proteins eluted from Ni-NTA columns with pH 4.5 buffer. (B) Titration of anti-rPbMdj1 and anti-rPbLon rabbit immune sera (1:500 to 1:16,000) in immunoblotting. As a substrate, 100 ng of rPbMdj1 (left) or rPbLon (right), as shown in panel A, was used. NC, negative control with preimmune serum. The same lack of reactivity was observed when testing insoluble bacterial components from recombinant bacteria with vector alone eluted at pH 4.5 from a Ni-NTA column. (C) Coomassie blue-stained 10% SDS-PAGE gel profiles of total cell (T) and mitochondrial (M) extracts (20 μg per lane) from a P. brasiliensis yeast culture growing at 36°C before and after heat shock at 42°C for 60 min. (D) Western blot of the gels shown in panel B, probed with the indicated rabbit sera at a concentration of 1:1,500. SDS-PAGE molecular markers are indicated (in kilodaltons) in panels A through D. (E) Northern blot analysis of total RNA isolated from P. brasiliensis cells growing at 36°C before and after heat shock at 42°C for 30 and 60 min, using PbMDJ1 and PbLON probes (upper panels). The band sizes are indicated (in kilobases). Lower panels show ethidium bromide-stained agarose gel replicas of the blotted gels in which the 18S and 28S rRNA components are visible.

FIG. 4.

Localization of PbMdj1 and PbLon by confocal microscopy of P. brasiliensis yeast cells. Red images are active mitochondria stained with MitoTracker Red. Green images are reactions with affinity-purified anti-rPbMdj1 or anti-rPbLon conjugated with FITC. Control reactions were carried out with preimmune IgG (panel C, same result for both preimmune sera). Merged images show colocalization of the labels (yellow-orange). Panel D shows two inner sections of budding cells reacting only with anti-rPbMdj1 (lower panel) or as a merged image (upper panel). Similar results were obtained with anti-rPbMdj1 IgG or affinity-purified antibodies, while only affinity-purified anti-PbLon showed interpretable images.

FIG. 5.

Ultrastructural localization of PbMdj1 in P. brasiliensis yeast cells incubated with IgG fractions (60 μg/ml) of either anti-rPbMdj1 (Athrough D) or preimmune control (E). Each panel shows a different cell. (A) Panoramic photomicrograph. (B and C) Details. Note labeling inside the mitochondria (M, arrows) and in the cell wall (CW). In panel C, labeling is also visible in the region between the cell membrane and the mitochondrion. (D) The bud neck is intensely labeled.

FIG. 6.

(A) FACS analysis of P. brasiliensis yeast cells incubated with 200 μg/ml of either anti-rPbMdj1 or preimmune IgG. (B) Coomassie blue-stained SDS-PAGE profile of an alkaline β-mercaptoethanol cell wall extract (β-ME, 40 μg), which was also Western blotted and assayed with anti-rPbMdj1, anti-PbLon, and preimmune (PI) IgG at a dilution of 1:1,500. A mitochondrial extract (Mito) was run in parallel and assayed with anti-rPbMdj1. Molecular masses are indicated (in kilodaltons).

FIG. 7.

Immunoblot reactivity of three PCM patients' sera with rPbMdj1 (note visible bands for patients 1 and 2). Controls were serum from a healthy individual (−) and anti-rPbMdj1 (+ [at 1:16,000]). Human sera were tested at a 1:200 dilution. The PCM sera reacted with gp43 with immunodiffusion titers of 1:64 and 1:32.