Faculty

Legionella pneumophila growth in macrophages 

    Innate and adaptive immune responses are initiated when macrophages ingest microbes. To investigate the mechanisms that govern the outcome of this encounter, we exploit a bacterial pathogen as a genetic probe of macrophage function. Legionella pneumophila is an opportunistic human pathogen whose natural reservoir is fresh water amoebae. When inhaled, the gram-negative bacteria can colonize alveolar macrophages and cause the severe pneumonia, Legionnaires' disease. Our genetic, microbiological, and cell biological studies support the following working model for the encounter between macrophages and L. pneumophila:

    To persist in fresh water reservoirs, L. pneumophila alternates between an intracellular replicative form and an extracellular transmissive form, a cellular differentation governed by the nutrient supply. When a transmissive bacterium is ingested by an amoebae or macrophage, L. pneumophila isolates its vacuole from the endosomal network. To do so, L. pneumophila not only exploits Type IV secretion, but also sheds LPS-rich vesicles that can block phagosome-lysosome fusion. Before replicating within its stalled phagosome, L. pneumophila must first acquire essential amino acids via Pht transporters, a subclass of the Major Facilitator Superfamily that is also conserved in Coxiella burnetii and Francisella tularensis. If nutrients are adequate, the microbe exits lag phase, down-regulates transmission factors, and converts to a replicative form, a cellular differentiation mediated by the CsrA repressor.

   Meanwhile, macrophages recognize the paused pathogen phagosome as cargo for autophagy, a mechanism to engulf defective organelles within membrane derived from the secretory pathway for delivery the lysosomes. A variety of microbes stimulate robust autophagy, suggesting this broadly conserved membrane traffic pathway is a component of the innate immune system. Consequently, L. pneumophila and other intracellular pathogens have acquired mechanisms to perturb autophagosome maturation to avoid or delay their delivery to toxic lysosomes. Indeed, autophagosome maturation is sluggish in the macrophages of A/J naip5 mutant mice, one of the few strains known to be susceptible to infection by L. pneumophila.

    As the intracellular pathogens replicate within autophagosome-like vacuoles, they deplete the local nutrient supply. As a result, a stringent response mechanism coordinates L. pneumophila differentiation to the transmissive form. The sensor RelA synthesizes (p)ppGpp, a second messenger that positively activates the sigma factors RpoS and FliA, the two component regulator LetA/S, and the co-activator LetE. In particular, once LetA/S relieves CsrA repression, the flagellar sigma factor FliA coordinately activates expression of multiple virulence traits: Motility promotes contact with host cells, FliA induces expression of factors required for lysosome avoidance, pigmentation, and surface modifications, and flagellin in combination with Type IV secretion promotes an acute proinflammatory response from murine macrophages.

    To test key aspects of our working model of the encounter between macrophages and L. pneumophila, the laboratory exploits our knowledge of microbial differentiation and the numerous molecular genetic and cell biology tools now available. Understanding the mechanisms that govern the response of macrophages to microbes can inform the design of new strategies to prevent and treat a wide variety of infectious diseases.

Selected Publications:

   Molofsky, A. B. and M. S. Swanson: MicroReview: Differentiate to thrive: Lessons from the Legionella pneumophila life cycle. Mol. Microbiol., 53: 29-40, 2004.

   Amer, A. O. and M. S. Swanson: Autophagy is an immediate macrophage response to Legionella pneumophila. Cell. Microbiol., 7:765-778, 2005.

   Sauer, J.-D., M. A. Bachman, and M. S. Swanson: The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages. Proc. Natl. Acad. Sci., 102: 9924-9929, 2005.

   Fernandez-Moreira, E., J. H. Helbig, M. S. Swanson: Membrane vesicles shed by Legionella pneumophila inhibit fusion of phagosomes with lysosomes. Infect. Immun., 74: 3285-3295, 2006.

   Dubuisson, J.-F., and M. S. Swanson. Mouse infection by Legionella, a model to analyze autophagy. Autophagy 2:179-82, 2006.
  
   Molofsky, A. B., B. G. Byrne, N. N. Whitfield, C. A. Madigan, E. T. Fuse, K. Tateda, and M. S. Swanson: Cytosolic recognition of flagellin by murine macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203:1093-104, 2006.

   Swanson, M. S. Autophagy: Eating for good health, J. Immunol., 177(8), 4945-4951, 2006.