Quorum sensing mediates the emergence of peripheral transmissive Legionella within the pathogen vacuole, selective escape and efficient spread

Legionella pneumophila, the causative agent of the severe pneumonia Legionnaires’ disease, replicates in free-living protozoa as well as in macrophages within a distinct compartment, the Legionella-containing vacuole (LCV). switches between a replicative, non-virulent and a motile, virulent phase. The timing, spatial organization, cues and functional consequences of the biphasic life cycle are not well understood. In this study, we show on a single cell level that bacterial clusters within a distinct pathogen vacuole develop spatially organized and functionally distinct subpopulations over time. At late stages of infection (>42 h), individual motile (PflaA-GFP-positive) and virulent (PralF-GFP-positive) L. pneumophila emerged at the periphery of clusters comprising non-growing bacteria. Comparative proteomics of PflaA-GFP-positive and PflaA-GFP-negative L. pneumophila subpopulations revealed distinct proteomes with flagellar proteins or cell division proteins being exclusively produced by the former or the latter, respectively. Towards the end of an infection cycle (~48 h), PflaA-GFP-positive L. pneumophila preferentially escaped the LCV and the bursting host cell, and the motile subpopulation promoted spreading of L. pneumophila. The emergence of peripheral motile L. pneumophila, LCV escape, host cell lysis and spreading of the bacteria to the environment is controlled by the Lqs quorum sensing system. Thus, quorum sensing controls the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intracellular bi-phasic life cycle of L. pneumophila.