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.