Figure 4. Analyses of class II mutations contributing to the noncytotoxic/constitutive-secretion phenotype.

(A) Translocation profiles of ExoU by the EZ-linker mutants during HeLa cell infection. After infection, bacterial cells (b) harvested from the cell culture medium and soluble fractions of HeLa cells (cs) were subjected to Western blot analysis. An anti-SOD antibody was used to detect SOD1 present in HeLa cell soluble fractions. (B) Western blot analysis of ExoU or ExoU-S142A release into cell culture medium during infection. Expression of PcrV::EZ derivatives or parental PcrV in bacterial fractions was detected with anti-PcrV IgG. (C) Cytotoxicity profiles of the strains that co-express the nontoxic derivatives with PcrV. Kinetics of cytotoxicity was measured by LDH release from infected HeLa cells. (D) Immunoblot analysis of secreted PcrV and PcrV derivatives (+NTA) or ExoU (±NTA). Regulation of ExoU secretion was examined when co-expressed with a chromosomal parental copy of pcrV. EZ138 contains two cysteine residues in the inserted linker, leading to the extra conformational species observed in the immunoblot (lane 7). (E) Bacterial surface localization of PcrV and class II derivatives. Surface-localized V proteins were quantified by flow cytometry.