FIG 1.

MsbA conditional deletion strain. (A) Growth of CFT073 wild type (WT) versus CFT073 msbA-cKO with or without arabinose (ara) to induce MsbA expression, as measured by OD₆₀₀ (left) and CFU count (right). Data are shown from a representative experiment. (B) Western blots of MsbA protein expression over time. Loading was normalized by the OD₆₀₀ value, and MsbA protein was detected using a rabbit polyclonal antibody generated in-house. (C) Confocal images of the CFT073 wild type versus the CFT073 msbA-cKO strain grown in the absence of arabinose for the indicated amount of time. Membrane is stained with Nile red, and DNA is stained with DAPI (blue). (D) TEM images of the CFT073 wild type versus the CFT073 msbA-cKO strain grown in the absence of arabinose for the indicated amount of time. Examples of vesicle-like membrane accumulation are indicated with black arrows, and parallel membrane stacks are indicated with white arrows. Lysed cells at the 4-h time point are indicated by open arrows. (E) CFT073 wild-type versus CFT073 msbA-cKO CFU counts isolated from mouse livers and spleens 30 min or 24 h following intravenous injection with 10⁶ CFU in neutropenic mice. Ten mice were injected with each strain, and five were sacrificed at each time point. Two mice injected with the wild-type CFT073 died prior to the 24-h time point, so CFU counts are plotted for the remaining three mice.