Fig. 1: Binding of C-type DC-SIGN, MR and Dectin-2 to P. aeruginosa biofilms and planktonic cells. | npj Biofilms and Microbiomes

Fig. 1: Binding of C-type DC-SIGN, MR and Dectin-2 to P. aeruginosa biofilms and planktonic cells.

From: Carbohydrates from Pseudomonas aeruginosa biofilms interact with immune C-type lectins and interfere with their receptor function

Fig. 1

a PAO1, ΔwspF (Psl + /Pel+), ΔwspF Δpel (Psl + /Pel-), ΔwspF Δpsl (Psl-/Pel+) and ΔwspF Δpsl Δpel (Psl-/Pel-) were grown in 96-well plates for 24 h in X-Vivo-15 and biofilm formation was analysed using the crystal violet assay. Data analysed using One-way ANOVA and corrected for multiple comparisons using Dunnett’s multiple comparison test. N = 6-7 in triplicate. ** 0.001; **** <0.0001. b and c. PAO1, ΔwspF, ΔwspF Δpel or ΔwspF Δpsl biofilms were generated in 96-well plates for 24 h, fixed and incubated with DC-SIGN-Fc (B) or MR-CTLD4-7-Fc or Dectin-2-Fc (C) followed by anti-human Fc secondary antibody conjugated to alkaline phosphatase. Cultures of ΔwspF Δpsl Δpel were used as controls. Analysis performed using Two-way ANOVA corrected for multiple comparisons using the Dunnett’s multiple comparisons test. N = 3 in triplicate. In B * 0.0324 (for ΔwspF Δpsl); 0.0413 for ΔwspF ΔpslΔpel; ** 0.007; *** 0.0007; **** <0.0001. In C **** <0.0001. d Planktonic cultures of P. aeruginosa PAO1 and the different mutants were collected, fixed and used to coat wells of MaxiSorp plates. Wells were incubated with MR-CTLD4-7-Fc or DC-SIGN-Fc followed by anti-human Fc secondary antibody conjugated to alkaline phosphatase. DC-SIGN, but not MR, bound planktonic bacteria and binding was independent of the presence of Psl and/or Pel. Two-way ANOVA corrected for multiple comparisons using the Dunnett’s multiple comparisons test. N = 4 in triplicate. *, 0.0412 for PAO1 and 0.0148 for ΔwspF; ****, <0.0001.e. DC-SIGN binding to planktonic bacteria was dependent on the presence of CPA LPS which is absent in the Δrmd and ΔwbpL mutants. N = 3 in triplicate. *, 0.0111; ****, <0.0001. Right panel: Adherence of planktonic cells to the wells was confirmed by ELISA using an antibody against P. aeruginosa (Anti-PA). N = 3 in triplicate. *, 0.0161. Analysis performed using Two-way ANOVA corrected for multiple comparisons using the Tukey’s multiple comparisons test. Graphs show mean +/− SEM. Specificity controls for these assays are shown in Fig. S1. In all cases, reading were taken within 20 min development and all strains in each graph were tested simultaneously.

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