Grant: $186,024 - National Institutes of Health - Jul. 20, 2009
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Award Description: The purpose of this R21 application is to examine how bacteriophage resistance confers attenuation, while preserving immunogenicity of a Listeria monocytogenes strain that is oral-virulent for mice. Mutants of listerial strain F6214-1 that are resistant to phage P35h4 are attenuated when inoculated orally into female A/J mice and show impaired replication in cultured mouse enterocytes. One of these mutants, containing a Tn917 insertion in the glcV gene, has been extensively characterized. Phage binding studies indicate that the mutant has a cell surface alteration that precludes phage attachment. All phenotypes associated with the mutation are complemented in trans by a parental copy of the glcV gene. The glcV gene is predicted to encode a group 2 glycosyl transferase. The loss of this product results in a defective phage receptor and alters a normal host-pathogen interaction required for virulence. Interestingly, the glcV lesion, while preventing phage attachment, does not affect the mutant?s ability to bind to cultured mouse enterocyte monolayers. Rather, the mutation appears to alter a subsequent step in intracellular replication measured as a reduction in plaque forming efficiency and plaque size. In vivo, the mutant is, in contrast to the highly invasive parent strain, undetectable in the liver and spleen 48 h post oral inoculation. The mutant is important because its documented properties (and our preliminary studies) indicate its outstanding potential as a live oral vaccine platform. Impediments to exploiting the mutant in this fashion include an incomplete understanding of the nature of the cell surface alteration that precludes phage binding, the mutant?s intracellular growth properties, and the stage in the infectious process when the host eradicates the mutant. Accordingly, our specific aims are directed towards a characterization of (1) the nature of the bacterial cell surface defect that eliminates phage binding, (2) the defect in growth and cell-to-cell spread of the phage resistant mutant in vitro, and (3) the extent of organ involvement and development of immunity following the translocation of the mutant from the intestinal lumen. We think that the results of our studies will constitute a substantive improvement in our understanding of the steps in the pathogenesis of naturally acquired listeriosis (i.e., acquired via an oral inoculation route). Furthermore, the characterizations may support the further development of the phage resistant mutant as a vaccine platform. Such a platform has the potential to provide a practical, safe and efficient means by which antigens are presented to the immune system. This could lead to improvements in vaccines to prevent infectious disease and in immunotherapeutics to combat tumor growth.
Project Description: Regarding determining the biochemical nature of the phage receptor, fractionation of the cell wall of the parent and phage resistant mutant strain were carried out, to Fraction D, which is after protease treatments and SDS precipitation. However, the binding ability of the CHO component was lost upon treatment with protease. This leads us to believe the binding may have carbohydrate (periodate sensitive) and protein (protease sensitive) components involved. We are evaluating a polyclonal antibody in our laboratory and have preliminary western analysis and recently adsorbed the antibody with the phage resistant mutant to further our understanding of the phage receptor. We have tested several saccharides for phage binding inhibition. We found that glucosamine inhibits phage binding to parental listeria in a dose dependent manner with a > 85% inhibition in 500mM of glucosamine. This result confirms our hypothesis that there is a carbohydrate component necessary for phage binding and we plan to investigate this further. We have initiated experiments to evaluate the cell to cell spread defect in the phage resistant mutant. We have used FITC-phalloidin to stain actin within MODE K cells and our polyclonal antibody to visualize listeria (Texas Red). Visualization of plaques formed by the parent and the mutant after 48 hours of growth is dramatically different. The parental plaque has many listeria in adjacent cells to the cleared plaque, whereas the mutant has very few listeria in the adjacent MODE K cells, and many listeria and CW debris in the middle of the plaque. Work continues with different infection times and actin tail evaluations. We have also measured IFN in the culture supernatants of parent and mutant 48 hours PI. IFN is detected in the mutant, but at much lower than as is produced in the parent. Work continues with evaluation of TNF and IFN in MODE K enterocytes as well as J774 macrophage cell lines.
Jobs Summary: This project includes employees classified as SPA Employee, , and EPA Employee. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on Jul. 20, 2009. Help expand these official descriptions using the wiki below.