The goal of this research proposal is to identify and characterize the molecular mechanisms of oral streptococci that contribute to the pathogenesis of streptococcus-associated nephritides and to develop specific prophylactic and therapeutic measures for these diseases. We have shown that S. mutans, S. pyogenes and S. sanguis produce a heparin-- binding protein (HBP) that also binds to heparan proteoglycans in basal laminae of animal tissues, particularly in kidney, heart and liver. This cationic protein is loosely associated with the bacterial cell wall and is released along with other components, such as lipoteichoic acid (LTA), into the cell environment during growth.
One specific aim i s to define the pathogenic properties of purified HBP and LTA in rabbits particularly their abilities to mediate deposition in kidneys of circulating immune complexes and/or formation of immune complexes in situ. Kidney tissue will be analyzed for pathology using histologic and immunohistologic stains.
A second aim i s to evaluate antibody production to HBP and LTA by rabbits during immunization with nephritis-associated strains of streptococci or with other streptococcal strains. The relative quantities and properties of HBP on bacterial surfaces and in culture medium will be determined using monoclonal antibodies. Ultrastructural location of the antigens on streptococci will be determined using immunoelectron microscopy.
The third aim i s to isolate the structural genes of HBP from these bacteria and determine the nucleotide sequences to further characterize this virulence factor.
The fourth aim i s to generate HBP-deficient strains of streptococci, using insertion mutagenesis, and to compare them with isogeneic parental strains to prove the pathogenic properties of this protein. The information derived from the experiments proposed in this application will further our understanding of all infection-associated nephritides in humans and may ultimately permit the development of safe streptococcal vaccines to protect against infection without risk of tissue injury.
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