Temporal resolution of two-tracked NF-κB activation by Legionella pneumophila

P>The intracellular pathogen Legionella pneumophila activates the transcription factor NF-kappa B in macrophages and human epithelial cells, contributing to cytokine production and anti-apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF-kappa B by L. pneumophila in human epithelial cells, using a p65-GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellin-deficient (Delta fliA and Delta flaA) mutants could not induce this first, TLR5 and MyD88-dependent activation. The second p65 translocation event, however, is a long-term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65-GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of I kappa B alpha and upregulation of anti-apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (Delta dotA; Delta icmB/dotO), Dot/Icm effectors (Delta sdbA; Delta lubX) and two bacterial effector mutants (Delta enhC; Delta ptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF-kappa B activation and intracellular replication and hints at an active interference of NF-kappa B signalling by L. pneumophila.