Regulation of ribosome hibernation controls <i>Legionella</i> survival, infection, antibiotic tolerance, and phenotypic heterogeneity

Bacterial virulence is regulated by the growth phase and ribosome activity, implicating the formation of translationally silent ("hibernating") ribosomes. <i>Legionella pneumophila</i>, the causative agent of Legionnaires' disease, is a facultative intracellular bacterium that grows in both environmental amoebae and mammalian macrophages. Thus far, ribosome hibernation factors of <i>L. pneumophila</i> have not been characterized. Here, we show that <i>L. pneumophila</i> encodes homologs of the ribosome hibernating factors LhpF (Lpg1206), RaiA (Lpg0467), RsfS (Lpg1377), and the GTPase HflX (Lpg0010), which define the ribosome populations by mediating 100S ribosome dimerization, 70S inactivation, ribosome assembly inhibition, and ribosome splitting, respectively. Exceptional among γ-proteobacteria, <i>L. pneumophila</i> forms 100S ribosome dimers during exponential growth. Functional studies show that the hibernation factors support survival upon starvation, regrowth, efficient host infection, and virulence factor production of <i>L. pneumophila</i>. Furthermore, they enhance antibiotic tolerance and shape intracellular heterogeneity of bacterial growth and motility. Our findings identify ribosome hibernation as a central mechanism by which <i>L. pneumophila</i> orchestrates survival, persistence, and infection, highlighting its critical role in bacterial physiology and pathogenesis.IMPORTANCEDue to nutrient limitation and adverse conditions in the environment, bacteria mostly do not grow exponentially but adopt a resting ("dormant") state. Bacterial dormancy usually coincides with the formation of translationally silent ("hibernating") ribosomes; however, the role of ribosome hibernation in intracellular pathogens is poorly understood. The facultative intracellular bacterium <i>Legionella pneumophila</i> is virulent in the stationary but not in the exponential growth phase, and therefore, an in-depth characterization of the pathogen's physiological states and ribosome profiles is crucial for understanding its virulence. Using bioinformatics, bacterial genetics, biochemical, and cell biological approaches, in this study, we reveal that the <i>L. pneumophila</i> ribosome hibernation factors LhpF, RaiA, RsfS, and HflX determine distinct ribosome subpopulations and are implicated in starvation survival and regrowth, as well as in host cell infection, intracellular replication, and phenotypic heterogeneity. Collectively, our data highlight the critical importance of ribosome hibernation for the physiology and virulence of <i>L. pneumophila</i>.