Figure 7.

O-Glycosylation of DMBT1 does not contribute to inhibition of bacterial twitching motility. (A) Effect of DMBT1 digested by an O-deglycosylation enzyme mix on twitching velocity of P. aeruginosa PAO1 after 4 h. DMBT1 (100 ng/µL) was digested by a mixture of O-deglycosylation enzymes (O-glycosidase, Neuraminidase, Galactosidase and N-acetyl-glucosaminidase) at 37 °C for 2 days in native glycobuffer (50 mM sodium phosphate, pH 7.5) to remove complex O-glycosylation of DMBT1. PBS and DMBT1 diluted with same volume of glycobuffer served as controls. (B) Effects of DMBT1 digested by the O-deglycosylation enzyme mix on colony size of P. aeruginosa PAO1 after 4 h and 24 h. (C) Effects of differently digested DMBT1 on colony size of P. aeruginosa PAO1 after 4 h and 24 h. DMBT1 (100 ng/µL) was digested by Neuraminidase (desialylation glycosidase), Galactosidase or N-acetylglucosaminidase (both exoglycosidases) at 37 °C for 2 h in glycobuffer (50 mM sodium acetate, 0.5 mM CaCl₂, pH 5.5) to remove sialic acid residues and other complex glycosylation of DMBT1. PBS and DMBT1 diluted with same volume of glycobuffer served as controls. Data shown as the mean ± SEM per sample from three independent experiments. Significance was determined using one-way ANOVA (panel A) or two-way ANOVA (panel B) each with Tukey’s multiple comparisons, ****P < 0.0001; ns, not significant.