Figure 5.

The capsule of Bp 1026b mitigates the activation of TLR2 and TLR4. Bp 1026b (Wt) and Bp 1026b (ΔwcbR-wcbA, Δcapsule mutant) were grown for ~48 h on SBA at 37°C. (A) Colony morphology of Wt and Δcapsule mutant. (B) Bacterial cell suspension were made in PBS and used to inoculate HEK293-TLR2 or -TLR4 cells and activation of TLRs measured after 18 h. Samples were performed in triplicate, and results represent one of two repeats. Significant differences in activity between Wt and capsule mutant are shown above the results. (C) Western blot (WB) analysis of whole-cells (WC) of Bp 1026b Wt and Bp 1026b Δcapsule mutant probed with an anti-capsule monoclonal antibody (mAb) (AVA5). (D) WB analysis of LPS extract from Bp 1026b Wt and Bp 1026b Δcapsule mutant probed with an anti-LPS mAb (11G3-1). (E) Activation of TLR2 or TLR4 by LPS extract from Bp 1026b Wt and Bp 1026b Δcapsule mutant. Samples were analyzed in triplicate and results represent one of two independent repeats. Results of TLR activation (B, E) are presented as geometric mean with standard error of the mean. Media controls were PBS, and positive controls (Pos Con) were HKLM (2 x 10⁶ cells) for TLR2, and E. coli LPS (0.2 ng) for TLR4. Significant values compared to media control are shown: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; *****P ≤ 0.00001.