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 ΔlqsR or ΔlvbR regulatory mutants the abundance of proteins encoded by a genomic 'fitness island', metabolic enzymes, effector proteins and flagellar components (e.g. FlaA) varies. ΔlqsR or ΔflaA mutants form 'patchy' biofilms like the parental strain JR32, while ΔlvbR 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 (ΔicmT), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ΔlvbR or ΔicmT biofilms but not in ΔlqsR or ΔflaA 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, andapart from the T4SSgovern bacterial cluster formation on the amoebae.
AbbreviationsIcm/Dot intracellular multiplication/defective organelle trafficking; LAI-1 Legionella autoinducer-1; LCV Legionella-containing vacuole; Lqs Legionella quorum sensing; LvbR Legionella virulence and biofilm regulator; cdi-GMP cyclic di-guanosine monophosphate; GFP green fluorescent protein; T4SS type IV secretion system
Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa,
Acanthamoeba
are ubiquitous in the soil and aqueous environments inhabited by
Listeria monocytogenes
. Observations of predator–prey interactions between these two microorganisms revealed a predation strategy in which
Acanthamoeba castellanii
assemble
L. monocytogenes
in aggregates, termed backpacks, on their posterior. The rapid formation and specific location of backpacks led to the assumption that
A. castellanii
may recruit
L. monocytogenes
by releasing an attractant. However, this hypothesis has not been validated, and the mechanisms driving this process remained unknown. Here, we combined video microscopy, microfluidics, single-cell image analyses, and theoretical modeling to characterize predator–prey interactions of
A. castellanii
and
L. monocytogenes
and determined whether bacterial chemotaxis contributes to the backpack formation. Our results indicate that
L. monocytogenes
captures are not driven by chemotaxis. Instead, random encounters of bacteria with amoebae initialize bacterial capture and aggregation. This is supported by the strong correlation between experimentally derived capture rates and theoretical encounter models at the single-cell level. Observations of the spatial rearrangement of
L. monocytogenes
trapped by
A. castellanii
revealed that bacterial aggregation into backpacks is mainly driven by amoeboid locomotion. Overall, we show that two nonspecific, independent mechanisms, namely random encounters enhanced by bacterial motility and predator surface-bound locomotion, drive backpack formation, resulting in a bacterial aggregate on the amoeba ready for phagocytosis. Due to the prevalence of these two processes in the environment, we expect this strategy to be widespread among amoebae, contributing to their effectiveness as predators.
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