FIG 4.

The T3SS does not enhance Shigella survival in amoebae. (A) Following phagocytosis, the number of bacteria able to survive degradation by amoebae was determined after 1 h, 4 h, and 20 h of gentamicin treatment during incubation at 37°C to activate the T3SS. (B) The intracellular survival was also determined for S. flexneri M90T and S. sonnei 53G T3SS mutants and compared to those of parental strains. (C) Propidium iodide uptake was used to measure membrane integrity in amoebae exposed to the indicated strains for 3 h. All values are a percentage of the maximum PI uptake calculated for amoebae exposed to 0.25% sodium deoxycholate for 10 min. (D) The indicated bacterial strains were incubated with A. castellanii for 1 h and then washed and treated with gentamicin for 1 h to kill extracellular bacteria. Fresh medium without gentamicin was then added for a further 3 h, after which time the supernatant was harvested and the number of released bacteria determined by colony counting. For all assays, two-way ANOVA indicated no significant differences between any strains at any time point. Mean and a standard deviation of 3 repeats are shown.