Legionella pneumophila is a Gram negative bacterial pathogen that replicates within fresh water amoeba species and within alveolar macrophages. Its growth inside of a membrane-bound vacuole depends on a chromosomally encoded type IV secretion system (T4SS) that delivers bacterial proteins into the host cell. Here, host cell signaling pathways, which control L. pneumophila intracellular growth in mammalian cells were identified. A microarray-based approach showed that L. pneumophila activates an anti-apoptotic pathway controlled by NF-κB in human macrophages. NF-κB is a transcription factor known to play an important role in the host innate immune response to bacterial infection. NF-κB activation is dependent on L. pneumophila’s T4SS and required for macrophage survival and intracellular growth. The L. pneumophila T4SS is predicted to deliver more than 150 proteins into the host cell. Thus, a mammalian expression library was generated and screened for the ability of candidate L. pneumophila type IV translocated substrates (T4Ts) to activate the NF-κB signaling pathway. Two genes, lnaB and legK1, were identified that strongly activated NF-κB. A Legionella strain carrying a null mutation of lnaB (L&barbelow;egionella N&barbelow;F-κB a&barbelow;ctivator B&barbelow;) reduced NF-κB activation by 2-fold, suggesting that proteins delivered by the T4SS can elicit an innate immune response. Using this library, I also investigated the subcellular localization of L. pneumophila proteins when expressed in mammalian cells. One third of the gene products in the library localized to discrete organelles suggesting that these bacterial proteins may target these sites when delivered by the T4SS.

Besides NF-κB signaling, the role of other genetic determinants in Legionella intracellular growth in murein macrophages was examined. Macrophages derived from inbred mouse strains are known to either restrict or permit L. pneumophila intracellular growth depending on their Naip5 allele. Six amino acid polymorphisms in Naip5 were predicted to control L. pneumophila restriction. However, we found that the only amino acids that could be correlated to the L. pneumophila growth restriction phenotype map to the predicted NACHT domain. Therefore, the NACHT domain is likely critical for Naip5’s function, as has been observed with other NACHT domain-containing proteins.