Date of Award:

2004

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Advisor/Chair:

Marie K. Walsh

Abstract

This study developed and characterized phospholipid vesicles, or liposomes, that mimic cell surfaces. Microemulsified liposomes contained biotinylated phosphatidylethanolamine, allowing them to be immobilized to avidin-coated glass. Laminin (LN), glycosphingolipids (GMl and GM3), and Escherichia coli's mechanosensitive channel of large conductance (EcoMscL) were embedded into liposome membranes. It was determined whether these embedded molecules exhibited their physiological roles of adhesion, cell recognition, and mechanosensation, respectively. Confocal laser scanning microscopy (CLSM) was employed to examine the interaction of fluorescently probed proteins, toxins, and bacteria with the immobilized microemulsified liposomes. Capture of individual and simultaneous multiple species of bacteria by GMl, GM3, or LN liposomes was quantified using ELISA and PCR.

Surface-bound liposomes were unilamellar and immovable, allowing removal of unincorporated probes and biomolecules. Liposomes remained intact and stable against leakage of encapsulated sulforhodamine B for several months after immobilization. Functional reconstitution of EcoMscL was examined using CLSM during modulations in the immursing solution.

Cholera toxin(Β subunit) (CTB), bovine lactoferrin (BLF), and E. coli O157:H7 were co-localized proximate to the surface of GMl liposomes. ELISAs determined E. coli O157:H7 and Salmonella enteritidis were captured on GMl liposomes containing GMI at 8.9 molar percent of total lipid. Listeria monocytogenes and Listeria innocua were not captured on the same liposomes.

PCR identified the capture of specific bacterial species from individual species and mixtures of several species on liposomes. Simultaneous assays with mixtures of multiple species showed that the receptor-associated binding of bacteria, described with PCR assays of an individual species, were independent of competitive microorganisms. L. monocytogenes and L. innocua were more frequently bound to LN liposomes than other liposomes, indicating LN promotes adhesion of both the pathogenic and a nonpathogenic strain of Listeria. E. coli O157:H7 was more frequently captured on GMI liposomes than other liposomes, indicating a specificity for this bacteria. S. enteritidis bound to all liposomes, indicating a non-specific interaction.

Known eukaryotic biomolecules implicated in cell recognition, adhesion, and mechanosensation were embedded in a system of artificial bilayers immobilized on a solid support. Liposomes constitute a biomimetic capable of specifically interacting and capturing proteins, toxins, and bacteria in solution.

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