Gene Regulation of Fimbriae in Actinomyces naeslundii

奈氏放线菌是正常口腔菌群的居民，但它也有可能引起疾病，即根面龋病和牙周炎(3，4)。奈氏假丝酵母菌的定植可以在婴儿期开始；它可以通过1型菌毛附着在牙齿表面的唾液富含脯氨酸的蛋白质上，和/或通过2型菌毛附着在周围的链球菌物种如口腔链球菌上。此外，2型菌毛的黏附与宿主中的乳糖敏感受体有关，如粘膜上皮细胞、红细胞和多形核白细胞(55，57)上的受体。由于对革兰氏阳性细菌中菌毛相关的黏附机制知之甚少，因此研究OFA。内氏杆菌1型和2型菌毛使这种生物成为研究细菌对宿主细胞的黏附以及对周围细菌细胞聚集的理想模型。 生物膜。这项研究将检测环境信号对1型和2型菌毛合成的影响，以检验环境因素可以调节菌毛基因表达的假说。这项提案的重点将包括： 目的1.测定调节菌毛生物合成的环境信号 实时逆转录聚合酶链式反应将被用来监测1型和2型菌毛基因OFA的表达。在不同条件下，在连续培养体系中生长。 目的2.2型菌毛启动子的特征通过引物延伸和S 1核酸酶实验定位2型菌毛基因的转录起点。然后，2型菌毛启动子区域将被克隆到报告基因载体中，用于未来的启动子突变研究。

Actinomyces naeslundii is a resident of the normal oral flora, however, it also has the potential to cause disease, namely root caries and periodontitis (3, 4). Colonization by A. naeslundii can begin during infancy; it can adhere to salivary proline rich proteins on a tooth surface via type 1 fimbriae and/or to surrounding Streptococcus species such as S. oralis via the type 2 fimbriae. In addition, adherence by type 2 fimbriae is associated with lactose sensitive receptors in the host, such as those on mucosal epithelial cells, erythrocytes, and polymorphonuclear leukocytes (55, 57). Since little is known about the fimbriae associated adherence mechanisms in gram-positive bacteria, studies ofA. naeslundii type 1 and type 2 fimbriae make this organism an ideal model to investigate adherence to host cells as well as aggregation to surrounding bacterial cells in a biofilm. This investigation will examine the effects of environmental signals on the synthesis of type 1 and type 2 fimbriae to test the hypothesis that environmental factors can regulate gene expression of fimbriae. The focus of this proposal will include: Aim 1. Determination of environmental signals that regulate fimbrial biosynthesis Real-time reverse transcriptase PCR will be used to monitor the expression of the type 1 and type 2 fimbrial genes ofA. naeslundii while growing in different conditions in a continuous culture system. Aim 2. Characterization of the type 2 fimbrial promoter The transcriptional start site for the type 2 fimbrial locus will be mapped by primer extension and S 1 nuclease experiments. The type 2 fimbrial promoter regions will then be cloned into a reporter gene vector for use, in future grant periods, in promoter mutation studies.