BIOCHEMICAL CHARACTERIZATION OF GBS HYALURONATE LYASE

GBS 透明质酸裂解酶的生化特征

• 批准号: 2382616
• 负责人: DAVID G PRITCHARD
• 金额: $ 24.07万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2382616
• 关键词: SDS polyacrylamide gel electrophoresis; Streptococcus agalactiae; active sites; bacterial proteins; calcium binding protein; chemical binding; chemical cleavage; chondroitin sulfates; enzyme activity; enzyme linked immunosorbent assay; enzyme structure; host organism interaction; laboratory rat; molecular pathology; monoclonal antibody; polysaccharide carbon oxygen lyase; protein sequence; site directed mutagenesis; solvolysis; spectrometry; virulence

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.

B链球菌（OBS）是目前最常见的原因， 严重的，往往是致命的，细菌感染的新生儿在美国 也是围产期产妇败血症的常见原因。 有证据表明细菌分泌的透明质酸裂解酶 对系统性侵袭很重要，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产生， 金黄色葡萄球菌。关于财产的信息和 因此，GBS酶的特异性可能导致 更好地了解所有这三种病毒的入侵能力 病原体，并可能导致有效的预防手段， 控制由细菌引起的感染。第一个具体目标 是对GBS透明质酸裂解酶进行生物化学表征。这将 包括鉴定在活性位点中重要的氨基酸， 识别透明质酸和钙结合区域，并研究 观察到的进行性作用模式的分子基础 酶GBS酶中的某些结构域非常相似 在其他研究中鉴定的透明质酸和钙结合结构域 proteins.替换选择的氨基酸残基的影响， 将使用定点诱变确定这些结构域。 其他候选氨基酸将被挑选用于基于 在各种测定和残留物具有的程度上， 在相关酶中是保守的。第二个具体目标是 测定GBS透明质酸裂解酶对软骨素的特异性 硫酸盐初步实验表明，GBS透明质酸 硫酸软骨素的裂解酶裂解仅发生在（31-4 包含未硫酸化的二糖重复的半乳糖糖键。 这种特异性使得有可能使用该酶研究 硫酸软骨素链序列。这一点很重要，因为 很明显，几种硫酸软骨素具有精确的生物活性， 必须与其结构相关的功能。此外，本发明还提供了一种方法， 酶的切割特异性的详细知识将 有助于阐明其对细胞外基质和基底的影响 在感染时暴露于其下的组织膜。 第三 具体目的是评估GBS透明质酸裂解酶的贡献 细菌的入侵潜力。入侵能力 新的GBS透明质酸裂解酶阴性突变体将与 在新生大鼠GBS肺模型中的亲本菌株 入侵此外，被动管理的能力 酶的抗体，以消除其入侵增强作用 将被评估。