FIG. 1.

Genes required for T3SS expression in response to low-Ca²⁺ and CHO cell signals. (A) The indicated strains carrying the P_exsD-lacZ reporter were grown under noninducing (lacking EGTA; white bars) or inducing (with EGTA; hatched bars) conditions for the expression of the T3SS and assayed for β-galactosidase (β-gal) activity (reported in Miller units). (B) P. aeruginosa strains were cultured in Ham's F12 medium alone (white bars) or in the presence (hatched bars) of CHO cells (10:1 MOI) for 4 h and assayed for the expression of the P_exsD-lacZ reporter and for T3SS-dependent cytotoxicity, respectively. Percent cytotoxicity (based on the release of LDH) was calculated relative to an uninfected control (0% cytotoxicity) and the amount of LDH released by coculture with the wild-type (wt) strain (100% cytotoxicity). Under these conditions, the wild-type strain released 72% of the LDH compared to cells completely lysed with Triton X-100. (C) Time course analysis of T3SS-dependent cytotoxicity towards CHO cells. P. aeruginosa strains were cocultured with CHO cells (10:1 MOI) for the indicated times and then assayed for LDH release. The reported values represent averages from at least three independent experiments, and error bars indicate the standard errors of the means.