Figure 2.

Elevated expression of VirF₂₁ suppresses Shigella virulence at 37°C. (A) Western blot with VirF antibodies on extracts from a Shigella M90T ΔvirF mutant and M90T strains harbouring pControl (empty vector) or pVirF₂₁, a plasmid carrying the virF₂₁ coding sequence under an inducible pTaq promoter. The strains were grown in the presence of increasing concentration of IPTG (0, 0.1, 0.25, 1 mM) to induce VirF₂₁ expression. GroEL protein was detected and used as internal loading control. A loading control using the Stain free method is also shown. (B) (upper panel) % CR- colonies upon spreading of the indicated strains on CR plates containing increasing concentrations of IPTG (0, 0.1, 0.25, 1 mM) to induce VirF₂₁ expression. (bottom panel) % CR- colonies upon scraping and re-plating of the colonies obtained on the previous plates, onto new CR plates without IPTG selection. Data come from at least three replicates from two independent experiments. ∼200–600 colonies/replicate were examined for the CR phenotype. (C) Invasion efficiency of the indicated Shigella M90T strains in sub-confluent Caco-2 cell layers. Cells were infected at MOI 100 for 1h, and analysed by selective plating of intracellular bacteria. Shown are CFU data expressed as the percentage of the inoculum retrieved in the intracellular population. Shown are CFU data for 7 (M90T pControl) and 9 (M90T ΔmxiD, M90T pVirF₂₁) biological replicates from 3 independent experiments. Bars represent mean and standard deviation. Statistical significance was determined by Mann Whitney U test, **P < 0.01.