The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote inter-bacterial and inter-kingdom signaling

Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires disease. The opportunistic pathogen employs the -hydroxyketone compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. LAI-1 is produced by the autoinducer synthase LqsA, but it is not known, how LAI-1 is released by the pathogen. Here, we use a V. cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in E. coli generated LAI-1, which partitions to outer membrane vesicles (OMVs), and slightly reduces OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L. pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favour of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes inter-bacterial communication as well as interactions with eukaryotic host cells. Originality - Significance StatementInter-kingdom signaling involving low molecular weight bacterial compounds that are detected by eukaryotic cells represents an important, yet incompletely understood aspect of pathogen-host interactions. In many cases, the small signaling molecules are produced in only little amounts, their secretion mechanism is not known, and their effects on eukaryotic host cells are barely studied. Here, we reveal that the -hydroxyketone compound LAI-1 of L. pneumophila is released from the bacteria by outer membrane vesicles, which promote inter-bacterial communication as well as inter-kingdom signaling.