Melissa M. Bourque

1998 PhD - Microbiology and Immunology - Alleghany University


University of New Hampshire

Microbiology

1992

Mentor: Dr. Frank G. Rodgers - Professor of Microbiology

Isolation and Characterization of an Opsonin-Independent U-937 cell Receptor Responsible for Attachment of Legionella pneumophila

Isolation of host-cell outer membranes from the macrophage-like U-937 cell is a necessary prelude to elucidating host-cell surface structures or receptors responsible for opsonin-independent binding of the human pneumonic pathogen Legionella pneumophila. A method of isolating U-937 cell outer membranes has been developed. This involves sucrose density gradient separation of biotinylated cell outer membranes, followed by an avidin detection system for localizing the sucrose gradient fraction rich in macrophage outer membranes. The biotin-avidin detection system has a high sensitivity and a specificity similar to antibody/antigen interactions. Outer membranes were separated from other cell components by sucrose gradient centrifugation and the fractions containing 37.6%/42.9% and 26.6%/37.6% interfaces were expected to contain the biotinylated outer membrane proteins. Avidin-horse radish peroxidase (HRP) western blot analysis was used to detect biotinylated U-937 cell outer membranes. Similar outer membranes were prepared without biotin. These non-biotinylated macrophage membranes were generated to determine the presence of the biotin label interfered with L. pneumophila attachment. Following the U-937 cell outer membrane isolation L. pneumophila was used to probe potential receptors specific for Legionella binding to the nonbiotinylated macrophage outer membranes in an opsonin independent manner. Such binding will be determined using an anti-Legionella antibody and HRP assay system.

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