Legionella pneumophila, a waterborne pathogen naturally present in freshwater and capable of colonizing artificial water systems, is responsible for Legionnaires’ disease (LD), a severe form of pneumonia transmitted through inhalation of contaminated aerosols. Virulence of Legionella strains is affected by the plasticity of their genome, shaped by horizontal gene transfer and recombination events. Thus, contaminated water systems can host diverse Legionella populations with a distinct virulence potential. Here, we compare the genomic diversity of Legionella pneumophila strains isolated in water systems of academic buildings, together with their cytotoxicity and intracellular replication in THP-1-like macrophages. A six-year environmental surveillance revealed Legionella pneumophila contamination in 20 out of the 50 monitored sites, identifying five serogroups (sg) and 13 Sequence Types (STs). Phylogenetic investigations based on core genome multilocus sequence typing (cgMLST) and comparative genomics of representative isolates of each ST showed a broad diversity and a heterogeneous virulence repertoire, especially within the Dot/Icm and Lvh secretion systems. Following macrophage infection, a strain-dependent cytotoxicity and intracellular replication was observed, underlying significant pathogenic diversity within the same species and stage-dependent infection dynamics. Together, these results showed strain-specific genetic and phenotypic virulence traits to be considered during risk assessment in environmental surveillance.

Legionella pneumophila is widely recognized as the principal organism responsible for Legionnaires’ disease. Modifications of surface-exposed lipopolysaccharide in L. pneumophila are key determinants of bacterial adaptation to host cells. Using a mutant strain deficient in N-methylation of legionaminic acid and fluorescence-based biophysical analyses, it was demonstrated that N-methyl groups linked to the acetimidoylamino group of legionaminic acid modulate the physicochemical properties of the bacterial cell surface and influence the interaction of L. pneumophila with macrophages. Loss of N-methyl groups reduced the efficiency of bacterial interaction with THP-1-derived macrophages and impaired intracellular proliferation. In addition, the presence of N-methyl groups in legionaminic acid contributes to increased TNF-α production in THP-1 macrophages stimulated with L. pneumophila sg1 LPS, without affecting IL-6 induction, suggesting that N-methylation of legionaminic acid may skew the early pro-inflammatory response toward TNF-α-dominated signaling during L. pneumophila infection.