Legionella pneumophila is the etiologic agent of an increasingly recognized form of acute pneumonia, which often has a number of accompanying extrapulmonary manifestations. The ability of the bacterium to multiply within host mononuclear phagocytes appears to be an important element of the pathogenesis, but the characteristic(s) of the bacterium responsible for this aberrant phagocytosis have not been identified. L. pneumophila produces a variety of extracellular toxins and enzymes that could play a significant role in the bacterium-phagocyte interaction. The elucidation of the role of toxins in pathogenesis is the long term objective of the proposed studies. We will examine two of these exoproducts, the hemolysin and the phospholipase, that have cytotoxic and/or membrane-active potential. Purified preparations of both exoproducts will be prepared from culture supernatants by collodion bag vacuum dialysis, ion exchange chromatography, gel filtration chromatography, and chromatofocusing. These pure products will be tested primarily on human polymorphonuclear neutrophils and monocytes (although guinea pig phagocytes will also be examined). The in vitro assays will look for evidence of gross cytopathology, and effects on cell membrane integrity, cell surface hydrophobicity, and a number of phagocytic functions such as uptake and killing of bacteria, oxidative metabolism, and exocytosis. Next, the role of the toxins will be studied in guinea pig infections (both intraperitoneal and aerosol inoculation) and infection of human monocytes. Hemolysin or phospholipase-deficient mutants will be tested for alterations in pathogenicity. The presence of toxin in the tissue or fluid during lethal or sublethal infections will be determined either directly, or by measurement of an antibody response following recovery. The protective role of an antibody response to the toxins will then be evaluated by active or passive immunization of the animals. Lastly, the examination of convalescent sera from human legionellosis patients for antitoxin antibody will provide the final piece of evidence for a role of the hemolysin and phospholipase during human infections.
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