Automated Genome-Wide Screen for Increased β-Glucan Exposure

<div><p>We screened the genome-wide set of S. cerevisiae knockout mutants by staining with anti-β-glucan antibody and quantifying β-glucan exposure using the Cellomics system.</p><p>(A) Images of yeast were taken at 20×, and object-finding software used the ConA fluorescence signal to identify cells (green outline); the software then applied a mask (blue outline) and quantified the average level of fluorescence from the anti-β-glucan channel for each cell. Wild-type yeast (left photomicrographs) show little or no fluorescence from the anti-β-glucan channel, whereas the unmasked <i>vrp1Δ</i> mutant shows high levels of fluorescence from this channel.</p><p>(B) Most of the mutants found with increased β-glucan exposure also showed greater binding to Dectin-CRD and increased TNFα elicitation from RAW 264.7 macrophages. Of the 76 mutants identified with increased anti-β-glucan binding (encircled with the black line), 48 (encircled with the dotted line) hyperelicited TNFα from macrophages and 65 (encircled with the red line) showed increased binding to the labeled Dectin-CRD. Numbers within the black oval show the intersections of each of these groups (e.g., 44 had increased anti-β-glucan binding and increased Dectin-CRD binding and increased TNFα elicitation from macrophages).</p><p>(C–E) To exemplify the methodology used, we chose the <i>gas1Δ</i> mutant, which has intermediate β-glucan exposure and TNFα elicitation. (C) Live wild-type (upper left image) or <i>gas1Δ</i> mutant (lower left image) S. cerevisiae were stained with anti-β-glucan antibody. To show specificity of the binding, <i>gas1Δ</i> cells were stained omitting primary antibody (upper right image) or antibody was preincubated in 100 μg/ml soluble glucan (laminarin) before and during the staining (lower right image). (D) Live wild-type or <i>gas1Δ</i> mutant cells were stained with Alexa Fluor-labeled Dectin-CRD. (E) Live wild-type or <i>gas1Δ</i> mutant cells were exposed to RAW264.7 macrophages at a ratio of 5:1 (yeast:macrophage), and supernatants were collected after 6 h and measured for TNFα levels.</p>