FIG 5.
The B. pseudomallei lpxO mutant is unable to produce hydroxylated lipid A. (A) MALDI-TOF MS scan in negative-ion mode of 1026b wild-type (WT) lipid A (green scan) compared to that of the lpxO mutant (orange scan). Asterisks indicate –OH group peaks, and arrows indicate –OH peaks missing from the lpxO mutant lipid A scan. Horizontal arrows indicate 228 mass differences indicative of C14:0-OH loss and an 80 mass difference indicative of a phosphate group. (B) The negative-ion mode B. pseudomallei lpxO mutant scan (orange scan) compared to the B. thailandensis wild type (green scan). (C) The positive-ion mode scan of the 1026b wild type (green) compared to that of the lpxO mutant (orange) from 900 to 1,100 m/z, with the only prominent peak of the sodium adduct of the triacylated oxonium ion depicted by the diagram. Mass differences of the –OH are indicated by −16 and the asterisk. The –OH peak indicated by the arrow is absent in the lpxO mutant. (D) Cytotoxicity measurements following LPS transfection into Salmonella LPS-primed RAW264.7 murine macrophages. White bars represent no-Lipofectamine controls, and gray bars represent exactly the same treatment but with Lipofectamine. Data are the mean and standard deviation of the results of one representative experiment carried out in triplicate. Significance was determined by one-way ANOVA to 1026b WT LPS or as indicated by a horizontal bar. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant.