Migration of Acanthamoeba through Legionella biofilms is regulated by the bacterial Lqs‐LvbR network, effector proteins and the flagellum

Summary The environmental bacterium Legionella pneumophila causes the pneumonia Legionnaires' disease. The opportunistic pathogen forms biofilms and employs the Icm/Dot type IV secretion system (T4SS) to replicate in amoebae and macrophages. A regulatory network comprising the Legionella quorum sensing (Lqs) system and the transcription factor LvbR controls bacterial motility, virulence and biofilm architecture. Here we show by comparative proteomics that in biofilms formed by the L . pneumophila Δ lqsR or Δ lvbR regulatory mutants the abundance of proteins encoded by a genomic ‘fitness island’, metabolic enzymes, effector proteins and flagellar components (e.g. FlaA) varies. ∆ lqsR or ∆ flaA mutants form ‘patchy’ biofilms like the parental strain JR32, while ∆ lvbR forms a ‘mat‐like’ biofilm. Acanthamoeba castellanii amoebae migrated more slowly through biofilms of L . pneumophila lacking lqsR , lvbR , flaA , a functional Icm/Dot T4SS (∆ icmT ), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ∆ lvbR or ∆ icmT biofilms but not in ∆ lqsR or ∆ flaA biofilms. The amoeba‐adherent bacteria induced promoters implicated in motility (P flaA ) or virulence (P sidC , P ralF ). Taken together, the Lqs‐LvbR network (quorum sensing), FlaA (motility) and the Icm/Dot T4SS (virulence) regulate migration of A . castellanii through L . pneumophila biofilms, and – apart from the T4SS – govern bacterial cluster formation on the amoebae.