Figure 2.

α-, β- and γ-hemolysins secreted during S. aureus growth activate caspase-1. A,, S. aureus secretes α-hemolysin during in vitro growth. S. aureus (S a.) was subcultured for the indicated amount of time and supernatants were immunobloted with an anti-α-hemolysin antiserum. An isogenic strain deficient in α-hemolysin was used as a specificity control. The arrow indicates the α-hemolysin 33 kDa monomer. B, Production of α-hemolysin during infection of macrophages with S. aureus under conditions similar to those used for Fig. 1A. Cell extracts were immunoblotted with anti-α-hemolysin antiserum. Culture supernatant from growing S. aureus (wt) was used as a positive control. C and D, α-, β- and γ-hemolysins mediate caspase-1 activation induced by S. aureus supernatant. Macrophages were stimulated for 3 h with supernatant from wt S. aureus or its isogenic hemolysin mutants. Cell extracts were immunobloted with anti-caspase-1 antibody and IL-1β measured in the cell supernatants. Values represent mean ± SEM (n = 3). ND, not detectable. *, indicates a no-specific band. Results are representative of at least three separate experiments. C, * p < 0.05, ** p < 0.01 (Student’s t test).