University of New Hampshire
Mentor: Frank G. Rodgers - Professor of Microbiology
Isolation of Lipopolysaccharide Produced by Legionella pneumophila and its role in adherence
Legionella pneumophila is a pleomorphic Gram negative rod-shaped bacterium which causes two distinct diseases: Pontiac fever, a self limiting non-fatal disease and Legionnaires disease, an acute lobar pneumonia. In order to cause respiratory disease, the organism must first adhere to and penetrate alveolar macrophages, the target cells in the lungs. I am interested in the adherence of this organism to host cells. Normal human alveolar macrophages are not available. !As a consequence I use U-937 cells which are derived from a human histiocytic lymphoma and mimic many of the properties of alveolar macrophages. Previous research in our laboratory showed that the adhesion may be a glycoprotein, which binds to a glycolipid receptor on host cells.
Growth curves were conducted (a total of four) to determine the concentration of bacteria in a known volume of media at a specific time in order to accurately infect the host cells with a multiplicity of infection (MOI) of 100 bacteria per host cell. L.ipopolysaccharide (LPS) extractions were performed to separate the bacterial component from Legionella. The lipid A portion was cleaved from the polysaccharide and O-antigenic side chain via acid hydrolysis. The polysaccharide portion was electrophoresed on a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and stained with a periodic acid to determine the presence of polysaccharides. The lipid depleted polysaccharide was used to treat U-937 cells to determine if the polysaccharide component from the LPS is involved in Legionella adherence. Several distinct sugars which comprise the LPS were also assayed for their specific contributions to pathogen binding.
Thus far, preliminary results show that D-glucosamine which comprises approximately 20% of the bacterial polysaccharide of the LPS, showed a significant (greater than 50%) reduction in binding. Ultimately, the receptors on the host cell and the adhesion on the bacterium will be isolated and characterized with a view to potential vaccine development.