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. Author manuscript; available in PMC: 2009 Apr 1.
Published in final edited form as: Nat Immunol. 2008 Aug 24;9(10):1171–1178. doi: 10.1038/ni.1646

Figure 3. Leucines in the C-terminus of L. pneumophila flagellin are critical for activation of the inflammasome.

Figure 3

(a) Flow cytometry of wild-type and IPAF-deficient macrophages transduced with GFP-C35 harboring the mutations L470A (GFP-C35A), L472A and L473A (GFP-C35AA), or L470A, L472A and L473A (GFP-C35AAA). (b) Immunoblot of IPAF-deficient macrophages expressing the constructs in a with anti-GFP. (c) Cell death indicated by release of lactate dehydrogenase (LDH) ±s.d. from wild-type macrophages infected with wild-type (WT), flagellin-deficient (ΔflaA) or ΔflaA L. pneumophila complemented with wild-type flagellin (ΔflaA ::flaA) or the leucine to alanine mutants L470A (ΔflaA::flaA-A), L472A and L473A (ΔflaA::flaA-AA), all three mutations (ΔflaA::flaA-AAA), salmonella flagellin ((ΔflaA ::fliC), or the fliI mutant (fliI::Cm), which is non-flagellated but still expresses flaA21, 53. Macrophages were assayed for release of LDH after 4 hours of infection. (d) ELISA assay for release of IL-1β from Pam3CSK4-primed bone-marrow-derived macrophages infected at an MOI of 1 with the indicated strains. (e) Replication of the indicated L. pneumophila strains, in wild-type and IPAF-deficient macrophages, measured by colony forming units. Results are representative of 2–3 independent experiments.