FIG 2.

Crp127 requires O-acetylation, but not xylosylation, of GXM for epitope recognition. The ability of Crp127 to recognize mutants with specific defects in GXM modification was quantified via flow cytometry. (A to C) Scatter plots (top row) and representative histograms (bottom row) for KN99α uxs1Δ, JEC155 uxs1Δ, and the corresponding wild-type strains (A); JEC156 cas1Δ and wild-type JEC155 (B); and KN99α cas3Δ, KN99α cas31Δ, and wild-type KN99α (C). (D to F) JEC155 uxs1Δ (D), JEC156 cas1Δ (E), KN99α cas31Δ (F), and the corresponding wild-type strains were labeled for chitin, and Crp127 was imaged via confocal microscopy. (G and H) Binding of Crp127 to chemically deacetylated (DeAc) cells of H99 and B3501 was quantified via flow cytometry (G), with a representative histogram presented for H99 (H). (I) Untreated (top) and chemically deacetylated (bottom) H99 cells were labeled for chitin and Crp127 and imaged via confocal microscopy. (J) Binding of 18B7 to chemically deacetylated cells of H99 and B3501 quantified via flow cytometry. (K) Representative cells from the above-described strains labeled for chitin (blue) and O-acetyl-independent mAb F12D2 (far red). For scatter plots, corrected MFI values were calculated by subtracting the MFI value of isotype control cells from the MFI value of the Crp127- or 18B7-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 KN99α uxs1Δ, JEC155 uxs1Δ, and JEC156 cas1Δ and their corresponding wild-type strains as well as between untreated and chemically deacetylated cells of the same strain (n = 3). Dunnett’s multiple-comparison test was used to test for statistically significant differences between the KN99α Δcas3 and KN99α Δcas31 mutants and the KN99α wild-type strain (n = 3) (ns, not significant [P > 0.05]; **, P < 0.01). Histograms show a representative distribution of Crp127 or 18B7 binding for one or all of the strains in the scatter plot above, with a 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. Representative maximum-intensity projections were merged for transmitted light and Crp127 (far red) (left panels) and Crp127 and chitin (blue) (right panels). Bars, 5 μm.