Infections were carried out over a period of 12 h and fixed for Giemsa staining (a), (c), (d), or lysed to release internalised bacteria which were then plated for enumeration of cfu per well (b). (a) Formation of MNCs is increased in the triple ΔbpsI123 mutant as compared with wt (MSHR520); comparison of mutant to wt is statistically significant for the 51+ group. >1000 nuclei were counted for each well and are expressed as the number of nuclei per group. Columns show the mean values with SEM. (b) No significant variation in intracellular bacteria is seen between wt and ΔbpsI1, ΔbpsI2, ΔbpsI3, ΔbpsI123. (c) MNC formation in single AHL mutants: In contrast to the triple mutant strain (comparison of mutant to wt is statistically significant for the 51+ group), formation of MNCs does not significantly differ between the wt and any of the single AHL mutants. (d) MNC formation in double AHL mutants: In contrast to the triple mutant strain (comparison of mutant to wt is statistically significant for the 11–25 and 26–50 groups), formation of MNCs does not differ between the wt and any of the double AHL mutants. Data in each figure are derived from 3 separate experiments, each including duplicate or triplicate wells.