FIG 3.
Temperature dependency of lipid A profiles and expression of relevant genes. (A to D) MALDI-TOF MS scans of lipid A show that 1026b lipid A acylation is decreased in response to temperature while 576a lipid A acylation remains the same. It was also found that 576a lipid A hydroxylation was decreased in comparison to that of 1026b. 1026b 23°C lipid A, blue scan; 1026b 37°C lipid A, red scan; 576a 23°C, orange scan; 576a 37°C, black scan. *, –OH peaks; −228, mass of one C14:0 acyl chain; −80, mass of one phosphate group. r. int (%), percent relative intensity. (E to G) Gene expression analysis agrees with the observations shown in panels A to D. Scans are the normalized average of at least two LPS preparations. (E) In the 1026b derivative Bp82, lpxO and pagL had the highest expression in planktonic cells at 37°C, followed by plated bacteria at 37°C, and then plated bacteria at 23°C (with the lowest). The 576a derivative 576mn showed no significant differential gene expression of these two genes. lpxO expression in 576mn was 50% that of Bp82 when both were planktonic cells grown at 37°C, while pagL expression was significantly lower (∼60% lower). At 23°C, there was little difference between the two, but at 37°C, lpxO expression in 576mn was nearly half that in Bp82. Data shown are the standard deviation of ratios resulting from three independent experiments carried out in technical duplicate. Significance was determined using REST software by propagation of efficiencies. (F) TNF-α release from RAW264.7 monolayers following treatment with 10 ng/ml of endotoxin-normalized LPS for 24 h. Significance was determined by one-way ANOVA to 1026b 37°C LPS treatment. neg CT, negative control. (G) Cytotoxicity caused by intracellular triggering of the caspase-11 pyroptotic pathway by 1026b 37°C LPS was significantly lower than that of Salmonella LPS and 576a 37°C LPS. White bars indicate LPS primed and incubated with the indicated LPS but in the absence of Lipofectamine (−lipo), while gray bars represent treatments equivalent to those indicated by the white bars but in the presence of Lipofectamine (+lipo). Significance in inflammasome activation experiments was determined by a one-way ANOVA comparison to Salmonella LPS treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data shown are the mean and standard deviation of a representative result of three independent experiments carried out in triplicate.