BIOCHEMICAL CHARACTERIZATION OF GBS HYALURONATE LYASE

乙型链球菌(OBS)是目前最常见的 美国新生儿严重的、通常是致命的细菌感染 国家和地区也是围产期母体败血症的常见原因。 有证据表明，这种细菌分泌的一种透明质酸裂解酶 对全身性入侵很重要，也可能干扰 一些正常的宿主防御机制。类似的酶有 由人类病原体肺炎链球菌和 金黄色葡萄球菌。有关属性的信息和 因此，GBS酶的特异性可能导致 提高了对这三种病毒侵袭能力的理解 病原体，并可能导致有效的预防和 控制由细菌引起的感染。第一个具体目标 是为了对GBS透明质酸裂解酶进行生化表征。这将是 包括识别活性部位中重要的氨基酸， 鉴定透明质酸和钙结合区，并进行研究 观察到的进行性作用方式的分子基础 这种酶。GBS酶中的某些结构域非常相似 到透明质酸结合区域和钙结合区域 蛋白质。替换部分氨基酸残基的效果 使用定点突变的这些结构域将被确定。 其他候选氨基酸将被挑选出来进行替代 根据各种化验和残留物的含量 在相关酶中是保守的。第二个具体目标是 GBS透明质酸裂解酶对软骨素的特异性测定 硫酸盐。初步实验表明，GBS透明质酸盐 硫酸软骨素裂解酶仅在(31-4)位发生裂解 含有未硫化二糖重复的半乳糖胺键。 这种特异性使这种酶有可能用于研究 硫酸软骨素链序列。这很重要，因为它是 显然，几种硫酸软骨素具有精确的生物学特性 必须与其结构相关的功能。此外, 对酶的切割特异性的详细了解将 有助于阐明其对细胞外基质和基底物的影响 在感染期间暴露于它的组织的膜。第三 具体目的是评估GBS透明质酸裂解酶的贡献 这种细菌的入侵潜力。一种细菌的侵袭能力 新的GBS透明质酸裂解酶阴性突变体将与 新生鼠GBS肺模型亲本株的致病作用 入侵。此外，被动给药的能力 该酶的抗体可消除其促进侵袭的作用 将会被评估。

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.