Legionella pneumophila and related species are facultative intracellular parasites for which mechanisms of pathogenesis remain undefined. The legionellae are invasive and reside in the phagosomes of both professional and non-professional phagocytes where they abrogate phagolysosomal fusion by unknown mechanisms. Both chlamydiae and legionellae possess extensive disulfide cross-linking of major outer membrane proteins (MOMP) and the disulfide bonds of the chlamydiae are reduced in vivo. We propose to test the hypothesis that disulfide bonds cross-linking the MOMPs of legionella are similarly reduced in vivo and that this reduction facilitates the release of periplasic virulence factors and/or initiates de novo synthesis of proteins necessary for intracellular growth. Virulent (V), but not avirulent (AV) isogenic strains release proteins when treated with dithiothreitol suggesting that differences in MOMP structure might correlate with virulence. Differential reduction of disulfide bonds of V and AV isogenic strains will be determined by alkylating reduced sulfhydryl groups with idoacetamide. Differences in alkylation of MOMP will be visualized by SDS-PAGE and autoradiography. Released proteins following DTT challenge (V and AV) will be identified by SDS-PAGE and western blot using both polyclonal and monoclonal antibodies. A HeLa cell model has been developed that permits the study of intracellular events associated with pathogenesis. Intracellular reduction of MOMP will be examined in HeLa cells by blocking oxidation of reduced sulfhydryl groups with (14C) iodoacetamide. Protein differences will be visulized by SDs-PAGE and autoradiography. Synthesis of novel proteins within the phagosome (early and late proteins, including the extracellular protease) will be determined by inhibiting host cell protein synthesis with cycloheximide and labeling bacterial proteins with (3 5S) cysteine or methionine. Additional methods to be employed to identify proteins synthesized intracellularly include western blotting and radioimmunoprecipitation. Finally, genes encoding the MOMP, a 60 kDa DTT released protein and a protease have been identified in a cosmid library and their characterization should facilitate the proposed studies. The identification of proteins synthesized intracellularly will likely contribute significant new information to the study of Legionella pathogenesis.
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