Legionella pneumophila Encodes a Peptidoglycan Recycling Machinery Critical for Survival within Macrophages

Bacterial cells are surrounded by a dynamic cell wall which is made up of a mesh-like peptidoglycan (PG) layer that provides the cell with structural integrity and resilience. In Gram-positive bacteria, this layer is thick and robust, whereas in Gram-negative bacteria, it is thinner and flexible as the cell is supported by an additional outer membrane. PG undergoes continuous turnover, with degradation products being recycled to maintain cell wall homeostasis. Some Gram-negative species can bypass de novo PG biosynthesis, relying instead on PG recycling to sustain growth and division. Legionella pneumophila (hereafter Legionella), the causative agent of Legionnaires' disease, encodes such recycling machinery within its genome. This study investigates the biochemical, genetic, and pathogenic roles of PG recycling in Legionella. Here, two PG recycling gene homologues in the Legionella genome lpg0296 (amgK) and lpg0295 (murU) were identified; chemical biology strategies were used to illuminate incorporation of "click"-PG-probes into whole PG. Copper-free click chemistry with ultrafast tetrazine-NAM probes enabled live-cell PG labeling further supported the use of recycling programs in Legionella. Deletion of amgK abolished PG labeling, while genetic complementation restored labeling. The data suggest that under conditions where de novo peptidoglycan synthesis is blocked, amgK plays a critical role in maintaining cell wall integrity, as its deletion led to increased antibiotic susceptibility and impaired survival in host alveolar macrophages. An intracellular survival assay demonstrated that while PG recycling is not essential for internalization, survival of Legionella within MH-S murine alveolar macrophages requires functional amgK. These findings underscore the essential role of AmgK in Legionella's intracellular survival, emphasizing the importance of PG recycling in pathogenicity, and establishing a foundation for developing novel Legionella-specific antibiotic strategies.