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. <P> 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. <P> 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. <P> 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. <P> 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. <P> PUBLIC HEALTH RELEVANCE: We will examine how phage resistance confers attenuation while preserving immunogenicity in a mouse oral-virulent Listeria monocytogenes strain. We feel that the results of our studies will constitute a substantive improvement in our understanding of the steps in the pathogenesis of the natural form of listeriosis (i.e., via an oral inoculation route). Further, the characterizations may support the 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, leading to improvements in vaccines to prevent infectious disease and in immunotherapeutics to combat tumor growth. 2
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