Fig. 4.

LasB-activated IL-1β is active. (a) IL-1 signalling activity by 100 ng human pro-IL-1β after 2 h incubation with titrations of recombinant LasB. (b) SDS-PAGE analysis of the kinetics of cleavage and maturation of recombinant human pro-IL-1β (1 μg) by recombinant LasB (50 ng). (c) Signalling activity of recombinant IL-1β N-terminal truncations generated using in vitro transcription/translation from the human il1b gene with coding beginning at the indicated codon, 1 is full-length pro-IL-1β, 117 corresponds to the fragment generated by caspase-1 cleavage. (d) Cleavage of internally-quenched fluorescent IL-1β peptide fragments (amino acids 103–123 of human IL-1β) by recombinant LasB or caspase-1. (e) IceLogo frequency plot showing amino acids significantly enriched (above X-axis) and de-enriched (below X-axis) in the P2 to P2ʹ positions following incubation of LasB with a mixture of 228 tetradecapeptides. Cleavage occurs between P1 and P1ʹ, lowercase “n” is norleucine. (f) Cleavage of internally-quenched fluorescent IL-1β peptide fragments by proteases within BAL collected from C57BL/6 or casp-1/11^−/− mice 24 h post-intratracheal infection with 10⁷ CFU of PAO1 or ΔlasB. Error bars show mean ± s.d, n = 4 (a-e), n = 5 (f), and represent at least 3 independent experiments. Statistical analysis by ANOVA with Tukey post-test, *P < 0.05, **P < 0.005, ***P < 0.0005.