Project description:Legionella pneumophila cells were harvested during exponential growth (RP) and stationary growth (TP). VBNC cells were also anylzed. Protein subfractions were studied.
Project description:Legionella pneumophila is a water-borne pathogen, and thus survival in the aquatic environment is central to its transmission to humans. Hence, identifying genes required for its survival in water could help prevent Legionnaires’ disease outbreaks. In the present study, we investigate for the first time the role of the sigma factor RpoS in promoting the survival in water, where L. pneumophila experiences total nutrient deprivation. The rpoS mutant showed a significant survival defect compared to the wild-type strain in defined water medium (DFM). Then, we analyzed the transcriptome of the rpoS mutant during exposure to water using whole genome microarray analysis. We found that RpoS negatively affects the expression of several genes, including genes required for replication, cell division, translation and transcription, suggesting that the mutant fails to shutdown major metabolic programs.
Project description:Legionella pneumophila are important opportunistic pathogens for which environmental reservoirs such as protists are crucial for the infection of humans. Free-living amoebae are considered key hosts providing nutrients and shelter for highly efficient intracellular proliferation of L. pneumophila, which eventually leads to lysis of the amoeba host cell. Yet, the significance of other bacterial players for L. pneumophila ecology is poorly understood. In this study we used a ubiquitous amoeba and their bacterial endosymbiont to investigate the impact of this common association on L. pneumophila infection. We demonstrate that Acanthamoeba castellanii harboring the chlamydial symbiont Protochlamydia amoebophila were able to erase L. pneumophila and, in contrast to symbiont-free amoebae, survived the infection and were able to resume growth. Environmental amoeba isolates harboring P. amoebophila were equally well-protected, and fresh environmental isolates of L. pneumophila were equally well-erased, suggesting ecological relevance of this symbiont-mediated protection. We further show that protection was not mediated by impaired L. pneumophila uptake. Instead, we observed reduced virulence of L. pneumophila released from symbiont-containing amoebae that is strongly supported by transcriptome data. Interference with transition to the transmissive phase is thus likely the basis for this protection. Finally, our data indicate that the defensive response of amoebae harboring P. amoebophila leaves the amoebae with superior fitness reminiscent of immunological memory. Given that mutualistic associations between bacteria and amoebae are widely distributed, P. amoebophila and potentially other amoeba endosymbionts could be key elements in shaping environmental survival, abundance and virulence of this important pathogen thereby affecting frequency of human infection.
Project description:Legionella pneumophila Philadelphia-1 strain was grown to stationary phase in AYE broth and starved in freshwater for 2 hours and RNA was harvested with or without sublethal heat shock via immersion in a 55 degree C hot water bath for 5 minutes