Fig. 5. Simultaneous mutation of multiple recognition motifs is required to evade NAIP5 or TLR5 recognition but disrupts flagellar motility.

(A to C) The indicated mutations were introduced at the endogenous FlaA locus of L. pneumophila strain LP02. (A) BMMs were infected with L. pneumophila strains at multiplicity of infection (MOI) = 3, and cell death was measured by lactate dehydrogenase (LDH) release at 4 hours. The dashed line indicates Nlrc4-independent LDH release in wild-type LP02 infection. (B) NAIP5- and FlaA-dependent restriction of L. pneumophila replication in BMMs. BMMs were infected at MOI = 0.01, and colony-forming units (CFU) were measured at the indicated time points. hpi, hours post-infection. (C) L. pneumophila were classified as motile (Y) or nonmotile (N) on the basis of observation of swimming runs. Bacteria were vortexed to dissociate cell-surface flagella, and supernatants were analyzed by Coomassie stain. (D to F) S. typhimurium strain LT2ΔfliCΔfljAB was transformed with an expression vector encoding wild-type FliC or the indicated variants. (D) Overnight culture supernatants were incubated 6 hours with CHO cells expressing HsTLR5 and a nuclear factor κB (NFκB) luciferase reporter. Reporter cells were analyzed for luciferase expression. (E) Diameter of colonies incubated on 0.4% agarose plates for 8 hours. (F) Culture supernatants and the supernatants of vortexed bacteria were analyzed for the presence of secreted or cell-dissociated flagellin, respectively. Results are representative of at least three independent experiments (error bars, SD; n = 3 biological replicates). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ANOVA (analysis of variance) comparing across BMM genotype [(A) and (B)] or against wild-type FliC [(D) and (E)]).