ISOLATION AND TESTING OF P GINGIVALIS VIRULENCE GENES

此修订后的应用程序的长期目标是隔离新的 牙龈卟啉单胞菌的毒力基因及其特征 沿着先前分离的基因。 结果有望改善 我们对这种疾病的致病机制的一般理解 牙周病的病因。 该申请有三个具体目标。 第一个题目是 “构建和使用报告基因分离和表征P. gingivalis毒力基因”。 一个无启动子的报告基因， 四环素抗性将随机插入牙龈卟啉单胞菌中 使用由克隆的染色体片段提供的同源性对染色体进行测序。 的 将得到的文库富集并筛选表达 小鼠脓肿模型感染过程中报告基因的功能 但在实验室培养中不表达。 这些克隆 在重要的基因中， 牙龈卟啉单胞菌在感染过程中的存活和毒力 过程 具体目标2，题为“克隆和遗传特性鉴定”， 报告基因-标记的毒力基因”，标记的基因将 通过标记救援技术恢复。 5素数的部分测序 基因的末端将使我们能够确定那些可能是 牙龈卟啉单胞菌的致病性最重要。 的 测序数据还将为我们提供必要的信息， 合成可用于克隆整个牙龈卟啉单胞菌基因的探针 转化为大肠杆菌 选择的毒力基因的同基因突变体将 使用插入失活方法构建。 致病 这些突变体的潜力将与它们的亲本进行比较， 鼠脓肿模型，在某些情况下，还有非细菌性大鼠模型。 本申请的具体目标3的标题为“遗传和生物化学 牙龈卟啉单胞菌表面抗原和毒力因子的表征”。 特异性目标2中分离的毒力基因在遗传上和 生物化学特征 此外，我们将执行相同的 先前克隆的牙龈卟啉单胞菌编码 血凝素和另外两种未鉴定的推定表面抗原。 他们的 作为推定的毒力因子的作用将通过将它们安装在 在Specific Aim 1中开发的报告基因构建体和检测 它们在鼠脓肿模型中感染期间的表达。

The long range goal of this revised application is to isolate new virulence genes from Porphyromonas gingivaIis and to characterize them along with previously isolated genes. The results are expected to improve our general understanding of the pathogenic mechanisms employed by this organism in periodontal disease causation. The application has three specific aims. The first is entitled "Construction and Use of a Reporter Gene to Isolate and Characterize P. gingivalis Virulence Genes". A promoterless reporter gene encoding tetracycline resistance will be randomly inserted into the P. gingivalis chromosome using homology provided by cloned chromosomal fragments. The resulting library will be enriched and screened for clones which express the reporter gene function during infection in the murine abscess model but which do not express it during laboratory cultivation. Such clones win likely be labeled with the reporter gene in genes that are important to the survival and virulence of P. gingivalis during the infectious process. In Specific Aim 2, entitled "Cloning and Genetic Characterization of Reporter Gene-Labeled Virulence Genes", the labeled genes will be recovered by marker rescue techniques. Partial sequencing of the 5 prime ends of the genes will enable us to determine those likely to be of greatest importance with regard to P. gingivalis pathogenicity. The sequencing data will also provide us with the necessary information to synthesize probes which can be used to clone the entire P. gingivalis gene into Escherichia coli. Isogeneic mutants of selected virulence genes will be constructed using insertional inactivation methods. The pathogenic potential of these mutants will be compared to their parent using the murine abscess model and, in certain cases, the gnotobiotic rat model. Specific Aim 3 of the application is entitled "Genetic and Biochemical Characterization of P. gingivaIis Surface Antigens and Virulence Factors". The virulence genes isolated in Specific Aim 2 win be genetically and biochemically characterized. In addition, we will perform the same characterizations on previously cloned P. gingivalis genes encoding hemagglutinin and two other unidentified putative surface antigens. Their role as putative virulence factors will be tested by installing them in the reporter gene construct developed in Specific Aim l and examining their expression during infection in the murine abscess model.