The water-borne bacterium Legionella pneumophila is the causative agent of Legionnaires’ disease. In the environment, the opportunistic pathogen colonizes different niches, including free-living protozoa and biofilms. The physiological state(s) of sessile Legionella in biofilms and their functional consequences are not well understood. Using single-cell techniques and fluorescent growth rate probes as well as promoter reporters, we show here that sessile L. pneumophila exhibits phenotypic heterogeneity and adopts growing and nongrowing (“dormant”) states in biofilms and microcolonies. Phenotypic heterogeneity is controlled by the Legionella quorum sensing (Lqs) system, the transcription factor LvbR, and the temperature. The Lqs system and LvbR determine the ratio between growing and nongrowing sessile subpopulations, as well as the frequency of growth resumption (“resuscitation”) and microcolony formation of individual bacteria. Nongrowing L. pneumophila cells are metabolically active, express virulence genes and show tolerance toward antibiotics. Therefore, these sessile nongrowers are persisters. Taken together, the Lqs system, LvbR and the temperature control the phenotypic heterogeneity of sessile L. pneumophila, and these factors regulate the formation of a distinct subpopulation of nongrowing, antibiotic tolerant, virulent persisters. Hence, the biofilm niche of L. pneumophila has a profound impact on the ecology and virulence of this opportunistic pathogen.
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We thank Selina Niggli for initial cloning and Ramon Hochstrasser for comments and discussions. We would also like to thank Sébastien Faucher (McGill University, Canada) for providing the Δ6S RNA mutant and parental strains. Research of NP in the laboratory of HH was supported by the Swiss National Science Foundation (SNF) Ambizione program (PZ00P3_161492 & PZ00P3_185529) awarded to NP. Research in the laboratory of HH was supported by the SNF (31003A_153200, 31003A_175557), the Novartis Foundation for Medical-Biological Research, the OPO Foundation, and the German Research Foundation (DFG; SPP 1617). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Personnic, N., Striednig, B. & Hilbi, H. Quorum sensing controls persistence, resuscitation, and virulence of Legionella subpopulations in biofilms. ISME J (2020). https://doi.org/10.1038/s41396-020-00774-0