Sandra Pepin

University of New Hampshire

Microbiology


1994

Mentor: Frank G. Rodgers - Professor of Microbiology

Isolation of Listeria monocytogenes Surface Proteins Associated with Bacterial Adherence to Host Cells

Listeria monocytogenes is a facultative intracellular pathogen, which causes listeriosis, a human and animal infectious disease. To initiate infection, bacteria adhere to and infect cells in the host. It is anticipated that the research conducted in this laboratory will lead to deterrent protocols for this potentially fatal pathogen. Little is known about the target cells in the host. However, as the organism is a foodborne pathogen, gut associated macrophages appear the most likely candidate cells.

Our research has focused on determining which surface associated proteins of L. monoctogenes were involved in bacterial adherence to murine peritoneal macrophages. A procedure to determine which proteins were involved in bacterial adherence was performed. A comparative study was done to determine which detergents, Nonidet P-40, sodium dodecyl sulfate (SDS) or urea, were most effective at removing surface proteins from the organism. It was shown that while SDS yielded a greater mass of protein, Nonidet P-40 gave additional proteins of higher molecular weight. The extracted proteins were collected and transferred to Polyvinylidine Difluoride (PVDF) sheets and subsequent in situ binding assays were undertaken to determine which of the extracted proteins were involved in adherence to biotinylated macrophages, avidin, primary and secondary antibodies and polyclonal antibodies. (Biotin/avidin were used as detecting markers).

Preliminary results indicated that a bacterial protein of approximate molecular mass 120 kilodaltons (KDa) was involved in organism binding to macrophages. Attempts are being made to isolate this 120 KDa protein using a Rotofor BioRad isoelectric focusing apparatus. This separates proteins based on pH gradient. Future research plans include purification and further characterization of the isolated protein . This purified protein will be used subsequently in competitive inhibition studies, the intent of which is to saturate macrophage receptors with the "sticky" adhesive molecule and then attempt to infect the cells with whole L. monocytogenes. It is speculated from the preliminary data that the process of binding to host cells will be blocked or inhibited by the binding proteins; thereby preventing infection of the cells by the pathogen. The raison d'etre for these studies is the development of subunit vaccines which will interrupt the infectious process.

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.

 

1993

Mentor: Frank G. Rodgers, Professor of Microbiology

Production of Monoclonal Antibodies to Components of Listeria monocytogenes Involved in Bacterial Adherence to Host Cells

This research project involved the production of monoclonal antibodies (MAbs) specific to the cell surface-associated proteins of the human pathogen Listeria monocytogenes. These MAbs will be used in competitive binding assays to define, in a blocking fashion, the process of adherence of L. monocytogenes to human host cells.

MAbs are prepared as follows: L. monocytogenes cell surface proteins were injected into mice once a week for three weeks. During this time individual B-cells of the mouse's immune system produce antibodies to each of the various components, or epitopes, of the protein. After three weeks, the B-cell enriched spleens of the mice are removed. NS-1 cells (myeloma or cancer cells) are grown at the same time and these are fused with the antibody producing B-cells. This fusion with the indefinitely reproducing cancer cells allows the hybridoma cells the ability to survive in culture. Fusion occurs by combining the collected B-cells and the cultured NS-1 cells in a 33% polyethylene glycol solution (PEG). The fused cells which result are known as hybridoma cells. After injection of individual, cloned cancerous but antibody producing hybridoma cells into mice, ascites tumors are formed in the peritoneal cavity. These tumors, derived from single cells, secrete the MAbs at very high concentrations. The MAbs will be collected from the mice for use in investigating the adherence of L. monocytogenes to host cells.

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