Cloning Analysis of S. mutans Putative Collagenase

变形链球菌推定胶原酶的克隆分析

• 批准号: 6600264
• 负责人: MYLIEN DAO
• 金额: $ 14.5万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6600264
• 关键词: Streptococcus mutans; Streptococcus vaccine; active sites; bacterial proteins; collagenase; dental caries; dental caries vaccine; molecular cloning; protein structure function; vaccine development; virulence

DESCRIPTION: Dental root decay is prevalent among older individuals as their gum recesses exposing Dental root surface to attack by cariogenic bacteria. In a study involving 449 subjects of an age range of 79-101 years, 96% had coronal decay experiences, and 64% had root caries experience with 23% of the group having untreated root caries (ADA News Releases, 2000). Streptococcus mutans, an etiologic agent in the development of coronal caries, has also been implicated in Dental root decay; data in support of this implication include the finding of S. mutans in Dental root section, its ability to bind collagen, and to degrade FALGPA, a known synthetic peptide substrate for collagenase. Bacterial collagenases are considered as virulence factors as they facilitate the invasion and destruction of host tissues by the pathogens. It is not yet known whether S. mutans produces a true collagenase enzyme. Considering the increase in incidence of Dental root caries as the population lives longer, the long-term goal of the current study is to develop effective and safe methods to control this disease, and improve the nutrition and quality of life of the population at risk. In order to determine whether the collagenolytic enzyme in S. mutans is a good candidate antigen for vaccine development, the Specific Aim of the current research is to learn more about the S. mutans enzyme in order to explore this avenue. A putative S. mutans collagenase gene has been obtained previously, and sequence analysis showed a high homology with the 35-kDa collagenase of various clinical isolates of Porphyromonas gingivalis, a bacterium causing periodontitis. The plan is to clone the 1.2 kbp putative collagenase coding sequence into an expression plasmid under the control of a strong promoter in order to obtain the corresponding protein, which in turn will be isolated and characterized by biochemical methods. Antibody will be prepared against the S. mutans enzyme and tested for the ability to block collagen binding and/or collagen degrading properties. The data obtained will be compared with other known bacterial collagenases. This information is essential in determining future directions for research on the role of S. mutans in Dental root caries, and other diseases that may involve collagen binding and collagen degrading activity such as periodontitis and endocarditis.

描述：牙根腐烂在老年人中很常见，因为他们的牙龈凹陷，使牙根表面暴露于致龋细菌的攻击。在一项涉及 449 名年龄范围为 79-101 岁的受试者的研究中，96% 的人有过冠状腐烂的经历，64% 的人有过根龋的经历，其中 23% 的人有未经治疗的根龋（ADA News Releases，2000）。变形链球菌是冠状龋齿发展的病原体，也与牙根腐烂有关。支持这一结论的数据包括在牙根切片中发现变形链球菌、其结合胶原蛋白和降解 FALGPA（一种已知的胶原酶合成肽底物）的能力。细菌胶原酶被认为是毒力因子，因为它们促进病原体侵入和破坏宿主组织。目前尚不清楚变形链球菌是否产生真正的胶原酶。考虑到随着人口寿命的延长，牙根龋的发病率不断增加，当前研究的长期目标是开发有效且安全的方法来控制该疾病，并改善高危人群的营养和生活质量。为了确定变形链球菌中的胶原分解酶是否是疫苗开发的良好候选抗原，当前研究的具体目标是更多地了解变形链球菌酶，以探索这一途径。先前已经获得了假定的变​​形链球菌胶原酶基因，并且序列分析显示与引起牙周炎的细菌牙龈卟啉单胞菌的各种临床分离株的35-kDa胶原酶具有高度同源性。计划将1.2 kbp的推定胶原酶编码序列克隆到强启动子控制下的表达质粒中，以获得相应的蛋白质，然后通过生化方法对其进行分离和表征。将针对变形链球菌酶制备抗体，并测试其阻断胶原蛋白结合和/或胶原蛋白降解特性的能力。获得的数据将与其他已知的细菌胶原酶进行比较。这些信息对于确定变形链球菌在牙根龋以及可能涉及胶原蛋白结合和胶原蛋白降解活性的其他疾病（例如牙周炎和心内膜炎）中的作用的未来研究方向至关重要。