NleA suppresses caspase-1 activation.

<p>(A) NleA does not interrupt IκB degradation during bacterial infection. dTHP-1 were uninfected or infected with indicated strains as described in the section of Method with modifications. Cells were centrifuged for 10 min and infected for 1.5 hrs. Then cells were washed once with PBS and directly lysed in 1x SDS sampler buffer. Proteins were separated by SDS-PAGE and probed with anti-IκB and anti-α-tubulin. (B and C) NleA does not inhibit NF-κB nuclear translocation. Differentiated THP-1 cells were uninfected (UI) or infected with wild type (WT), TOE-A5, and TOE-A5/<i>nleA</i> strains. After 1 hr of infection and 30 min of gentamicin-treatment, cells were washed, fixed and immunostained with anti-RelA antibody (green) and DAPI (blue). Images from nine random fields were captured using confocal microscopy. Cells with intense nuclear staining of RelA were counted. The percentage of such cells was calculated by dividing the total number of cells in each given field of view. Student’s t-test * <i>p</i> < 0.05 (v.s WT). (D and E) NleA inhibits secretion of IL-1β but not TNF-α. Differentiated THP-1 cells were uninfected (UI) or were infected by WT, TOE-A5, and TOE-A5/<i>nleA</i> for 1 hr, and the culture supernatants at 6 hr post-infection were analyzed by ELISA. Student’s t-test * <i>p</i> < 0.05 (D: vs WT). (F and G) NleA inhibits caspase-1 activation. After 1 hr of infection followed by (F) 6 hrs or (G) 3 hrs of incubation, culture supernatant from cells uninfected (UI) or infected with indicated strains were centrifuged and the proteins in the supernatant were TCA-precipitated. Immunoblottings were performed to assess the amount of active caspase-1 (caspase-1 p20) by anti-caspase-1 antibody. SN, supernatant; CL, cell lysate.