Rapid Icm/Dot T4SS Inactivation Prevents Resuscitation of Heat‐Induced VBNC Legionella pneumophila by Amoebae

ABSTRACT Legionella pneumophila , the causative agent of Legionnaires' disease, employs the Icm/Dot Type IV secretion system (T4SS) to replicate in amoebae and macrophages. The opportunistic pathogen responds to stress by forming ‘viable but non‐culturable’ (VBNC) cells, which cannot be detected by standard cultivation‐based techniques. In this study, we document that L. pneumophila enters the VBNC state after exposure to heat stress at 50°C for 30 h, at 55°C for 5 h or at 60°C for 30 min, while still retaining metabolic activity and intact cell membranes. Resuscitation of heat‐induced VBNC L. pneumophila neither occurred in amoebae nor in macrophages. VBNC L. pneumophila showed impaired uptake by phagocytes, formation of Legionella ‐containing vacuoles (LCVs), and Icm/Dot‐dependent secretion of effector proteins. The T4SS was rapidly inactivated already upon exposure to 50°C for 3–5 h, while the bacteria were still culturable. The Legionella quorum sensing (Lqs)‐LvbR network is implicated in VBNC induction, since the ∆ lvbR and ∆ lqsR mutant strains showed a more pronounced heat sensitivity than the parental strain, and the ∆ lqsA mutant was less heat sensitive. Taken together, our results reveal that heat exposure of L. pneumophila rapidly inactivates the Icm/Dot T4SS before the VBNC state is induced, thus impairing resuscitation by amoebae.