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The objective of this research proposal is to develop a better understanding of the structural properties and mechanism of action of S. pneumoniae hyaluronate lyase enzyme. The long term objective is to better understand the mechanism of invasion of host tissue and the penetration of host defenses by S. pneumoniae and other Gram-positive bacteria.
Such understanding may lead to the development of new antibacterial therapeutic agents. S. pneumoniae is the most common cause of fatal pneumonia in the elderly, and it is one of the most common causes of middle ear infections and meningitis in children. The present vaccine consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, it is only about 60 percent effective in the elderly. In children less than 2 years of age the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the penumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk of pneumococcal disease. However, in most parts of this country, up to 35 percent of pneumococcal strains are now resistant to penicillin and the frequency of multidrug resistant strains is steadily increasing. It has been recently suggested that certain pneumococcal proteins such as hyaluronate lyase, pneumolysin, and pneumococcal surface protein A (PspA) could be used as potential vaccine or drug targets for development of new cure. Hyaluronate lyase is a major surface protein of S. pneumoniae with possibly antigenetically variable properties that might be essential for full pneumococcal virulence. Thus, hyaluronate lyase might represent one of the best alternatives for a pneumococcal vaccine or drug target especially when combined with other pneumococcal virulence factors such as PspA or pneumolysin. We have expressed in E. coli, purified, and crystallized fully active forms of hyaluronate lyase enzyme from S. pneumoniae and Group B Streptococcus. The specific aims of the research proposed in this application are directed towards: a) the determinatin of the crystal structure of the hyaluronate lyase enzyme; b) thoroughly characterizing the properties and mapping the active site of this enzyme utilizing oligonucleotide mediated site directed mutagenesis. The results will enhance our knowledge about mechanisms involved in the action of the enzyme and in the pathogenesis of S. pneumoniae bacteria.