FIGURE 5.

Acinetobacter baumannii secretes a TLR2 activating lipid that triggers canonical NF-κB signaling. (A) Wildtype THP1-XBlue reporter cells or THP1-XBlue IRAK4^–/– reporter cells were infected with wildtype A. baumannii (Ab19606) or treated its culture filtrate. TSB medium was used to grow the bacterial cultures and fresh TSB medium treatment served as the negative control. Levels of SEAP were assessed after 24 h from the beginning of the treatment. The experiments were done in triplicates. Error bars represent standard deviation. One-way ANOVA with Tukey’s multiple comparisons test. (B,C) HEK293 reporter cells stably expressing TLR2 (B) or TLR4 (C) were infected with wildtype A. baumannii (Ab19606) or ΔlpxA at MOI 10 or were treated with the respective culture filtrates. Levels of SEAP were assessed after 24 h of infection or culture filtrate treatment. TSB medium was used to grow the bacterial cultures and fresh TSB medium treatment served as the negative control. Pam2CSK4 and LPS served as additional controls. The experiments were done in triplicates. Error bars represent standard deviation. One-way ANOVA with Tukey’s multiple comparisons test. (D) THP1 macrophages treated with or without the TLR2 inhibitor C29 were exposed to ΔlpxA culture filtrate. Western blot was performed to check the phosphorylation of NF-κB p65. (E) Schematic representation depicting the secretion of the bioactive lipid by A. baumannii that activates inflammatory signaling (created with BioRender.com). All the experiments were performed three times independently. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.