Electrophoretic profile of rPCN_exon4 and rPCN_full.

<p><b>Panel A:</b> Induction time/response rPCN_exon4 detected in the lysates of <i>E. coli.</i> The time elapsed since IPTG induction is shown in hours. <b>Panel B:</b> The bound material to glutathione-sepharose was separated by 12% SDS-PAGE Coomassie blue staining revealed a single band (lane 1), which was recognized by specific antibodies against GST (lane 2) and PCNprep (lane 3). <b>Panel C:</b> rPCN_full was purified and evaluated for its ability to bind to <i>N</i>-acetylglucosamine. The bound material was separated by 10% SDS-PAGE under reducing conditions, and then stained with Coomassie blue. Lane 1, material before refolding; Lane 2, material after refolding (a single band was detected with an apparent molecular mass of 28 kDa). Molecular markers were a mixture of pre-stained proteins (Fermentas). <b>Panel D:</b> The rPCN_full band was recognized by an anti-paracoccin antibodies (lane 2) and not by antibodies from pre-immune sera.</p

Anti-rPCN_full antibody reactivity on the yeast cell surface.

<p>Fluorescence labeling with anti-rPCN_full (<b>Panel A–D</b>) was evenly distributed over the yeast cell surface, with more intense labeling in some budding regions. Panel D is the merge of panels B and C.</p

Biological and enzymatic properties of rPCN_exon4 and rPCN_full.

<p><b>Panel A:</b> production of TNF-α, <b>Panel B:</b> production of NO by induced murine macrophages following <i>in vitro</i> stimulation with PCNprep and rPCN_exon4 or PCNprep and rPCN_full. Cells were harvested from the peritoneal cavity of C57BL/6 mice and induced with thioglycollate. Adherent cells were incubated for 48 h with different recombinant proteins (0.25 mg/mL), medium (negative control), or LPS+IFNγ (positive control). The standard deviation was calculated based on tests performed in triplicate. The activity of the samples was compared to that of the medium alone. <b>Panel C:</b> The PCNprep, rPCN_exon4 and rPCN_full were assayed for NAGase activity. A colorimetric assay was performed in spectrophotometer set at λ = 405 nm. The standard deviation was calculated by analysis of experiments performed in triplicate. <b>Panel D:</b> Binding of rPCN_full to laminin. Different amounts of biotinylated recombinant protein were incubated with laminin (250 ng) coated in the microplate wells. The binding of the biotinylated protein was detected with a neutravidin-peroxidase conjugate and a chemiluminescent substrate. Luminescence readings are reported as relative luminescence units (RLU). <b>Panel E:</b> Inhibition of rPCN_full binding to laminin by sugars. Different concentrations of GlcNAc, d-glucose, and d-galactose were pre-incubated with the recombinant protein (100 ng), and the mixture was then added to the laminin-coated wells. The margin of error was calculated by analysis of triplicate experiments. Each sample with sugar was compared to a sample without sugar.</p

Cloning strategies for cloning the paracoccin ORF for expression.

<p>The left panel shows the standard strategy, with PCR amplification of the largest exon, restriction endonuclease digestion, and cloning into the expression vector pGEX-4T-1. The positions of the exons are displayed on the map of the gene. Genomic DNA template was extracted from <i>P. brasiliensis</i> strain Pb18. Agarose gel electrophoresis (mid-left) shows the corresponding band amplified by PCR. The exon 4 amplicon was cloned by <i>Bam</i>HI and <i>Eco</i>RI digestion. The right panel shows the strategy for synthesis of the predicted paracoccin sequence fused with the 5′-UTR elements for transcription in the vector pUC57. Green arrow, T7 promoter; black box, lacO (lac operator); and red box, the phage T7 trailer sequence for ribosome binding.</p

Effect of tunicamycin (TM) on the distribution of paracoccin in <i>P. brasiliensis</i> yeasts.

<p>Fluorescence labeling with anti-paracoccin IgY was evenly distributed over the yeast surface. In some budding regions, a more intense labeling was detected for untreated <i>P. brasiliensis</i> yeast cells (B, C, I and J) compared with those treated with TM (E, F, M and N), which were more centrally labeled. Panels H and L show fluorescence labeling chitin, while panels B, E, I and M show fluorescence labeling paracoccin. C is the merge of A and B; F is the merge of D and E; J is the merge of G, H and I; and N is the merge of K, L and M.</p

Effect of tunicamycin (TM) on binding of paracoccin and gp43 to laminin.

<p>Binding of paracoccin and gp43 to laminin was assayed by incubating different amounts of fully glycosylated and underglycosylated crude extracts (white bars and black bars, respectively) of <i>P. brasiliensis</i> in laminin-coated (0.5 µg/well) microplate wells, followed by incubation with biotinylated monoclonal antibody anti-gp43 (A) or with biotinylated IgY anti-paracoccin (B). The reactions were developed with neutravidin–peroxidase conjugate and H₂O₂/OPD substrate. Optical density (OD) readings at 450 nm show dose-dependent binding (A and B). Inhibition assays for gp43 (C) and paracoccin (D) binding to laminin were performed in the presence of 10 mM d-galactose (d-Gal) or N-acetyl-glucosamine (GlcNAc). Data are representative of 3 independent assays.</p

Partial kinetic characterization of N-acetyl-β-d-glucosaminidase activity (NAGase) produced by <i>P. brasiliensis</i> in the absence or presence of tunicamycin.

<p>Data are representative of 3 independent assays.</p

Effect of tunicamycin (TM) on the morphology of <i>P. brasiliensis</i> yeast cells.

<p><i>P. brasiliensis</i> was grown in YPD medium in the absence (Ctrl), or in the presence of 15 µg/mL TM for 72 h at 37°C and analyzed by optical microscopy (A and C) and phase-contrast microscopy (B and D).</p