All intracellular pathogens possess specialized strategies for invading their host cell, evading its defenses, and multiplying at the expense of its metabolism. The long term goal of the research described in this proposal is to understand the molecular basis of these events in a specific host-parasite system. To do this an extensive molecular genetic analysis has been undertaken of a facultative bacterial pathogen, Legionella pneumophila, the causative agent of legionnaires' disease and related respiratory ailments. This organism invades human macrophages and monocytes, evades their microbicidal systems, and grows exponentially within a specialized vacuole which does not acidify or fuse with lysosomes, but which recruits organelles from the cytoplasm. These events are also observed with other intracellular pathogens. This system is ideally suited to molecular genetic analysis because L. pneumophila can be grown on bacterial culture media but grows exclusively intracellularly under tissue culture conditions.
The specific aims are to: (i) Genetically characterize loci that are required for intracellular growth and host cell killing (icm, intracellular multiplication) and identify their products. This will be done by complementation analysis, physical mapping and DNA sequence analysis; (ii) Define the phenotypes of the different icm mutants with respect to prevention of vacuole acidification, inhibition of phagosome- lysosome fusion and organelle recruitment. This will be done using a combination of electron microscopy, fluorescence imaging and biochemical approaches; (iii) Measure the expression of """"""""indicator genes"""""""" during macrophage infection to learn more about the intracellular environment and the ability of Legionella to modify the properties of the phagosome. To do this the beta-galactosidase activity of lacZ fusions to icm and other reporter genes which are regulated by a variety of factors including: amino acid availability, oxidative damage, low pH and iron limitation will be measured; (iv) Identify genes that regulate icm gene expression. To do this libraries of wild-type L. pneumophila DNA cloned in a vector with a regulatable promoter (Ptac) will be introduced to various icm::lacZ strains and examined for clones that increase or decrease beta- galactosidase activity upon induction with IPTG, (a specific inducer of the Ptac promoter.)
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