FIGURE 2.

T3SS activity in Pseudomonas PscF mutants. (A) The cytotoxicity of P. aeruginosa strains toward macrophage cells was measured by monitoring LDH release after 2 and 3 h of infection using a multiplicity of infection (MOI) of 5. The PscF deletion mutant (ΔF), D76A, and P47A/Q54A strains show a higher statistical difference at both 2 and 3 h of infection compared to wild-type complemented strain (ΔF/Fwt). LDH measurements were corrected with values corresponding to cells that were not infected; we considered as 100% of cytotoxicity a 1% Triton X-100 treated well. Pairwise differences relative to ΔF/Fwt based on the Tukey test are indicated: ns, non-significant, ^∗∗∗P < 0.001, ^∗P < 0.05. Cytotoxicity of original CHA strain is also reported. (B) Western blot of the supernatant (secreted proteins) and total bacteria (expression) fractions after centrifugation were developed with anti-PopB and anti-PcrV antibodies. The expression of PscF variants was confirmed by loading total bacterial extracts on SDS- 15% PAGE. The Western blot was developed using anti-PscF antibodies obtained using a monomeric 6His-PscF (see section “Materials and Methods”). The D76A strain secretes almost no PopB or PcrV while P47A/Q54A secretes less of both translocators as compared to other mutants and the wild-type strain. LasB was used as loading control for the secreted protein, and DsbA as loading control for bacterial expression and as a lysis control for the supernatant (data not shown).