FIG 1.

Crp127 is an anti-GXM mAb. The ability of Crp127 to bind to GXM- and GXMGal-deficient mutants of C. gattii and C. neoformans was quantified via flow cytometry. (A to C) Scatter plots (top row) and representative histograms (bottom row) for R265 cap10Δ (A); B3501 cap67Δ (B); KN99α cap59Δ, KN99α uge1Δ, and KN99α cap59Δ uge1Δ (C); and their corresponding wild-type (WT) strains. For scatter plots, corrected median fluorescence intensity (MFI) values were calculated by subtracting the MFI value of isotype control cells from the MFI value of the Crp127-treated cells, with data points representing MFI values calculated from three biological replicates performed as independent experiments. Student’s t test was used to test for statistically significant differences between R265 cap10Δ and B3501 cap67Δ and their corresponding wild-type strains, while one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test was used to test for statistically significant differences between KN99α cap59Δ, KN99α uge1Δ cap59Δ, KN99α uge1Δ, and the wild-type strain KN99α (n = 3) (*, P < 0.05; **, P < 0.01). Histograms show a representative distribution of Crp127 binding for one or all of the strains in the scatter plot above, with the color-coded key provided for reference. Numerical values in the top left and right of each histogram correspond to the MFI value calculated from the strain labeled directly above. (D to F) R265 cap10Δ (D); B3501 cap67Δ (E); KN99α cap59Δ, KN99α uge1Δ, and KN99α uge1Δ cap59Δ (F); and their wild-type strains were labeled for chitin using calcofluor white (CFW) (blue) and Crp127 (goat Alexa 647-conjugated anti-mouse IgM μ-chain) (far red), and maximum-intensity projections were generated from confocal microscopy z-stacks. Presented are representative images merged for transmitted light and Crp127 (left panels) and Crp127 and chitin (right panels). Bars, 5 μm.