FIG 4.

Attenuation of C. rodentium ΔarcA strain. (A) Localization of C. rodentium wild-type and ΔarcA strains in distal colon samples of day 9 infected susceptible mice. Sections were stained with DAPI (blue) and anti-Citrobacter LPS (green). Panels on the right show a zoomed-in view of the boxed areas in the panels on the left. Scale bars, 100 μm. (B) In vitro adherence assay. HeLa cells were infected with C. rodentium wild-type and ΔarcA strains for 8 h. After extensive washing, the samples were fixed and stained with DAPI and anti-Citrobacter LPS. The total number of bacteria per HeLa cell was counted, and adherence was expressed as a percentage of wild-type adherence levels. An unpaired t test was used to determine statistical significance (**, P < 0.01). (C) Total secreted proteins of the C. rodentium wild-type (lane 1) and ΔarcA (lane 2) strain were prepared, separated on a 10% SDS-PAGE gel, and stained with Coomassie blue. The positions of two of the major T3SS-secreted proteins, EspA and EspB, are indicated with arrows to the right of the gel. (D and E) Expression levels of the espA (D) and espB (E) genes by C. rodentium wild-type and ΔarcA strains were measured by real-time RT-PCR. Data were normalized to rpoD expression levels. Statistical significance was assessed by performing an unpaired t test (****, P < 0.0001; **, P < 0.01).