Mass spectrometric analyses of the 10- and 15-kDa SDS-PAGE band-derived proteins showing mass-to-charge ratio (m/z), average mass of peptide (Mr; calculated by MASCOT software), peptide sequences (identified by MASCOT software), BLAST search result (against ~15,000 genome-derived predicted proteins of P. insidiosum), and amino acid position of identified peptides.

<p>Mass spectrometric analyses of the 10- and 15-kDa SDS-PAGE band-derived proteins showing mass-to-charge ratio (m/z), average mass of peptide (Mr; calculated by MASCOT software), peptide sequences (identified by MASCOT software), BLAST search result (against ~15,000 genome-derived predicted proteins of P. insidiosum), and amino acid position of identified peptides.</p

Mass spectrometric analyses of ELI025 by LC-MS/MS.

<p>(<b>A</b>) MS spectra of the 10- and 15-kDa SDS-PAGE band-derived proteins. The arrows indicate peptides with the mass-to-charge ratio (m/z), including 458.3, 569.6, 686.9 and 853.9, that match the ELI025 protein. Peptide sequences corresponding to the ELI025-matched peaks are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118547#pone.0118547.t002" target="_blank">Table 2</a>; (<b>B</b>) MS/MS spectra of the 686.91 peaks from the 10- and 15-kDa SDS-PAGE band-derived proteins.</p

Cloning and expression of <i>ELI025</i>.

<p>(<b>A</b>) Plasmid DNA map of pET28b-ELI025 shows the cloning sites (<i>Nde-</i>I and <i>EcoR-</i>I) of <i>ELI025</i>. Expression of <i>ELI025</i> is under the control of the T7 promoter. The numbers in parentheses indicate a location of each plasmid component; (<b>B</b>) Protein structure of ELI025 shows a signal peptide (SP; amino acid position 1–20), an elicitin domain (amino acid position 25–110), three disulfide bonds (C1, cysteine position 27 and 91; C2, cysteine position 47 and 76; C3, cysteine position 71 and 110), two predicted N-linked glycosylation sties (N; amino acid position, 22 and 87), and three predicted O-linked glycosylation sties (O; amino acid position 49, 51, and 54).</p

Sequence alignment of core promoter regions of the <i>P. insidiosum ELI025</i> gene and various genes from several oomycetes and parasites.

<p>The <i>ELI025</i> sequences (accession number AB971191–3), used for the alignment, are derived from three different <i>P</i>. <i>insidiosum</i> strains. Conserved nucleotides are highlighted in grey. The underlined letters indicate the known transcriptional start site, and is indicated below as "+1". Two putative core promoter components, an initiator element (Inr; 5’-TCATTCC-3’; positions-2 to +5) and a flanking promoter region (FPR; 5’-CAACCTTCC-3’; positions +7 to +15), are found in the upstream region of all genes. (Abbreviation: NCBI, National Center for Biotechnology Information).</p

BLAST search of the ELI025 amino acid sequence against the genomes, transcriptomes, or proteomes of 18 oomycetes, 10 fungi, 4 algae, 3 diatoms, and one protozoan (the cut-off E-value ≤ 1 x 10–4).

<p>^a Broad institute genome database</p><p>^b Genome portal of the Department of Energy Joint Genome Institute</p><p>BLAST search of the ELI025 amino acid sequence against the genomes, transcriptomes, or proteomes of 18 oomycetes, 10 fungi, 4 algae, 3 diatoms, and one protozoan (the cut-off E-value ≤ 1 x 10–4).</p

Cellular location of ELI025.

<p>Infected arterial tissue from a pythiosis patient was sequentially stained with rabbit anti-rELI025 serum, as the primary antibody, and then mouse anti-rabbit IgG antibody conjugated with horseradish-peroxidase, as the secondary antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118547#sec002" target="_blank">Materials and Methods</a>). Images of the hyphae and location of ELI025 (indicated by arrows) were captured with a bright-field microscope. The scale bar represents 10 μm.</p

Immunoreactivity of the recombinant protein rELI025 and crude protein extracts of <i>P. insidiosum</i>.

<p>Crude proteins (i.e., SABH and CFA) extracted from three different strains of <i>P</i>. <i>insidiosum</i> (Pi-S, MCC18, and P01) and rELI025 are separated in a SDS-PAGE gel <b>(A)</b>. The separated proteins are analyzed by Western blot, using the rabbit anti-rELI025 antibodies <b>(B)</b>, or sera from patients with pythiosis <b>(C)</b>, as probe. The black arrow head indicates the 12.4 kDa band of rELI025. The white arrow heads indicate the 10- and 15-kDa bands of native ELI025. The numbers represent protein molecular weights standards, in kDa. (Abbreviations: SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; CFA, culture filtrate antigen; SABH, soluble antigen from broken hyphae; rELI025, recombinant ELI025).</p

The Dynamic Genome and Transcriptome of the Human Fungal Pathogen <i>Blastomyces</i> and Close Relative <i>Emmonsia</i>

<div><p>Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, <i>Blastomyces</i>, and two species of the related genus <i>Emmonsia</i>, typically pathogens of small mammals. Compared to related species, <i>Blastomyces</i> genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for <i>gypsy</i> elements, are variable in total size between isolates, and are least expanded in the avirulent <i>B</i>. <i>dermatitidis</i> strain ER-3 as compared with the virulent <i>B</i>. <i>gilchristii</i> strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the <i>Blastomyces</i> lineage. While gene content is highly conserved between <i>Blastomyces</i> and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of <i>B</i>. <i>dermatitidis</i> during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in <i>Blastomyces</i>, and upregulation of dioxygenases <i>in vivo</i> suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively <i>in vivo</i>, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in <i>in vitro</i> studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of <i>Blastomyces</i> to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in <i>Blastomyces</i>.</p></div

Correspondence of GC content and synteny for <i>Blastomyces</i>.

<p>Comparison of GC content (top panel) and genome synteny (lower panel) for a 5.2 Mb region of <i>B</i>. <i>dermatitidis</i> strain ER-3 (scaffold (sc) 1, coordinates from 4.5 to 9.7 Mb) and corresponding syntenic regions of <i>B</i>. <i>gilchristii</i> strain SLH14081 and <i>Histoplasma capsulatum</i> strain WU24. Location of genes (blue boxes) and gypsy elements (green boxes) are depicted across each genomic region. Orthologs between genomes are connected in pink, which are organized into syntenic regions that are disrupted by GC-poor regions in both <i>Blastomyces</i> genomes.</p

<i>Blastomyces</i> transcripts most significantly induced during macrophage infection.

<p><i>Blastomyces</i> transcripts most significantly induced during macrophage infection.</p