Genetic dissection of the regulatory pathway that controls the <italic>Legionella pneumophila</italic> transmission phenotype.

Abstract

The gram negative bacterium <italic>Legionella pneumophila </italic> is an intracellular pathogen of alveolar macrophages and freshwater amoebae. When inhaled in contaminated water, these bacteria can cause the severe pneumonia, Legionnaires' Disease. To succeed as a pathogen, <italic> L. pneumophila</italic> must not only colonize phagocytes, but also be transmitted from host to host. To determine the molecular mechanisms that allow <italic> L. pneumophila</italic> to invade and to replicate intracellularly, the regulatory circuit that controls virulence traits must be identified. Genetic analysis of <italic>L. pneumophila</italic> cultured in broth and macrophages support a model in which virulence traits promote transmission to a new host phagocyte. These traits are induced coordinately by the sigma factor RpoS, the sensor kinase-response regulator LetA/S, the novel regulator LetE, and the flagellar sigma factor FliA in response to the alarmone guanosine 3<super>'</super>,5<super> '</super>-bispyrophosphate (ppGpp) which accumulates when amino acids are limiting. RpoS is required for evasion of endosomes, expression of the infectivity gene <italic>mip</italic>, efficient replication within macrophages, sodium sensitivity and full flagellar motility. Independently of RpoS, the <italic> letE</italic> gene product enhances LetA/S dependent traits and is required for maximal contact-dependent cytotoxicity, infection of macrophages, pigment production, reduced cell size, and thermotolerance. Similarly to the <italic> E. coli</italic> regulatory RNA CsrB, <italic>letE</italic> counteracts repression of transmission phenotypes by the mRNA-binding protein CsrA; however, site-directed mutagenesis indicates LetE functions as a protein. RpoS, LetA/S and LetE activate transmission traits by acting at distinct levels within the FliA regulon, as revealed by Northern analysis. When conditions are favorable for growth, intracellular bacteria down regulate motility, cytotoxicity, and the capacity to block phagosome maturation by a mechanism that may employ RpoS. Multiple copies of <italic>rpoS</italic> repress LetA/S dependent motility, cytotoxicity, and infectivity, and decrease transcript levels of <italic>fliA, flaA</italic>, and <italic>letE</italic>. Consistent with a role during growth, <italic> rpoS</italic> transcripts peak in the exponential phase and ectopic LetA expression in replicating cells induces cytotoxicity only in the absence of RpoS. This thesis supports a model in which <italic>L. pneumophila</italic> transmission traits are repressed during the replicative phase and induced during the stationary phase of growth by the combined activities of RpoS, LetA/S, LetE, FliA, and CsrA.