B streptococci (OBS) are presently the most frequent cause of serious, often fatal, bacterial infections of neonates in the United States and are also a common cause of peripartum maternal sepsis. There is evidence that a hyaluronate lyase secreted by the bacteria is important for systemic invasion and also may interfere with some normal host defense mechanisms. Similar enzymes are produced by the human pathogens Streptococcus pneumoniae and Staphylococcus aureus. Information on the properties and specificity of the GBS enzyme, therefore, may result in an improved understanding of the invasive capacities of all three pathogens, and possibly lead to effective means for prevention and control of infections caused by the bacteria. The first specific aim is to biochemically characterize GBS hyaluronate lyase. This will involve identifying amino acids important in the active site, identifying hyaluronan- and calcium-binding regions, and studying the molecular basis for the observed processive mode of action of the enzyme. Certain domains in the GBS enzyme are very similar to hyaluronan- and calcium binding domains identified in other proteins. The effects of replacing selected amino acid residues in these domains using site-directed mutagenesis will be determined. 0ther candidate amino acids will be picked for replacement based upon a variety of assays and the extent to which the residues have been conserved in related enzymes. The second specific aim is to determine the specificity of GBS hyaluronate lyase for chondroitin sulfates. Preliminary experiments revealed that GBS hyaluronate lyase cleavage of chondroitin sulfate occurs only at (31-4 galactosamidic bonds involving an unsulfated disaccharide repeat. Such specificity makes it possible to use the enzyme in studies of chondroitin sulfate chain sequence. This is important since it is clear that several chondroitin sulfates have precise biological functions that must be related to their structures. In addition, detailed knowledge of the cleavage specificity of the enzyme will help clarify its effects on the extracellular matrix and basement membranes of tissues exposed to it during infection. The third specific aim is to assess the contribution of GBS hyaluronate lyase to the invasive potential of the bacteria. The invasive capacity of a new GBS hyaluronate lyase-negative mutant will be compared to that of the parental strain in a neonatal rat model of GBS lung invasion. In addition, the ability of passively administered antibody to the enzyme to abolish its invasion-enhancing effects will be assessed.
|Baker, J R; Pritchard, D G (2000) Action pattern and substrate specificity of the hyaluronan lyase from group B streptococci. Biochem J 348 Pt 2:465-71|
|Pritchard, D G; Trent, J O; Li, X et al. (2000) Characterization of the active site of group B streptococcal hyaluronan lyase. Proteins 40:126-34|