Abstract
Legionella pneumophila is a ubiquitous Gram-negative bacterium and the causative agent of Legionnaires’ disease and Pontiac fever. The facultative intracellular bacterium colonizes natural and man-made water systems. In the environment, Legionella resides in complex biofilms, that comprise several bacterial species and eukaryotic cells. Moreover, the pathogen can infect free-living amoebae such as Acanthamoeba castellanii and replicate intracellularly. Human infection occurs via inhalation of contaminated aerosols. In the lungs, the opportunistic pathogen infects alveolar macrophages and translocates more than 300 effector proteins into the host cell through an Icm/Dot type IV secretion system (Icm/Dot T4SS). This allows the pathogen to multiply within a unique compartment called the Legionella-containing vacuole (LCV). L. pneumophila employs the Legionella quorum sensing (Lqs) system to regulate various traits including the growth phase switch, virulence, and bacterial motility. The Lqs system is linked to the bacterial c-di-GMP metabolism through the pleiotropic transcription factor LvbR. The Lqs system comprises the autoinducer synthase LqsA, which produces the α-hydroxyketone LAI-1 (Legionella autoinducer-1, 3-hydroxypentadecan-4-one), the homologous sensor kinases LqsS and LqsT, and the response regulator LqsR. The interactions between L. pneumophila and its natural host cells in biofilms are poorly understood. In this study, we investigated the interactions of Legionella and the amoeba A. castellanii in mono-species L. pneumophila biofilms using confocal microscopy and flow cytometry. We found that the transcription factor LvbR, the response regulator LqsR, the bacterial flagellum (FlaA) and the Icm/Dot T4SS regulate the migration of A. castellanii through L. pneumophila biofilms. Furthermore, LvbR, LqsR and FlaA were found to govern the adherence and cluster formation of L. pneumophila on the surface of amoebae. The bacterial clusters were found to comprise motile (PflaA-positive) and virulent (PsidC-positive) bacteria. Macrophages and amoeba hosts of L. pneumophila synthesize nitric oxide (NO), a highly reactive gaseous molecule, which freely diffuses across membranes. NO has a bactericidal effect at high (micromolar) concentrations, however at low (nanomolar) concentrations, NO has been shown to act as a signalling molecule in many bacterial species. L. pneumophila possesses three NO sensors, Hnox1, Hnox2 and NosP. To investigate the role of the NO receptors in L. pneumophila signaling, marker-less L. pneumophila mutant strains lacking individual (Hnox1, Hnox2, or NosP) or all three NO receptors (triple knockout, TKO) were generated. We found that in the ΔnosP mutant, the lvbR promoter was upregulated, indicating that NosP negatively regulates LvbR. Furthermore, the NO receptor mutants were impaired for growth in A. castellanii and macrophages. In addition, we found that phenotypic heterogeneity of non-growing/growing bacteria in amoeba was regulated by the NO receptors. Moreover, the NO receptor mutant strains revealed an altered biofilm architecture and no longer responded to NO in biofilms. In summary, the NO receptors Hnox1, Hnox2 and NosP were shown to be involved in NO detection, replication in host cells, intracellular phenotypic heterogeneity, as well as biofilm formation and dispersion. Our results provide a deeper understanding of NO signaling in L. pneumophila and form the basis for further studies on the molecular components and mechanisms involved in intra-species and inter-kingdom NO signaling.
Michaelis, Sarah