EXPRESSION OF FIMBRIAE IN A ACTINOMYCETEMCOMITANS

放线菌共生菌中菌毛的表达

• 批准号: 2679646
• 负责人: John K Spitznagel
• 金额: $ 11.04万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2679646
• 关键词: Actinobacillus actinomycetemcomitans; DNA; bacterial genetics; gene complementation; gene expression; genetic library; genetic regulation; genetic transcription; growth media; molecular cloning; molecular genetics; northern blottings; nucleic acid sequence; oral bacteria; phenotype; pilus; polymerase chain reaction; restriction mapping; virulence

DESCRIPTION (Adapted from investigator's Abstract): This proposal addresses the molecular basis for fimbrial expression by Actinobacillus actinomycetemcomitans (Aa). It is stated that the periodontal pathogen Aa expresses fimbriae when freshly isolated from the oral cavity. Fimbriate Aa are adherent in broth culture, while non-fimbriated Aa are much less adherent. This behavior led many investigators to reason that fimbriae are an important virulence factor for Aa. Fimbrial expression by Aa may be important to that organism during colonization of the periodontal tissues. The long term objectives of this proposal are to determine the genetic organization of the fimbrial genes and to ascertain the molecular basis for regulation of this potential virulence factor. Specific Aim 1 is to isolate and clone the Aa fimbrial gene(s). Aa fimbrial clones will be selected from genomic libraries of DNA from fimbriated strain UTP001 using PCR and immunologic probes. A genomic library, constructed in a broad range mobilization cosmid vector, will be used in complementation studies with a non-fimbriated, rifampicin resistant, Aa strain generated from UTP001. Specific Aim 2 is designed to determine the DNA sequence and transcriptional organization of the Aa fimbrial gene(s). Fimbrial gene mapping and sequencing will be accomplished by DNA restriction analysis, Sanger sequencing, and by analysis of fimbrial RNA transcripts. Fimbrial RNA will be studied by Northern analysis, S1 nuclease, and primer extension mapping to determine the transcriptional organization, locate the transcriptional start site, and the promoter region of the gene. Specific Aim 3 will define the environmental growth conditions that direct regulation of fimbrial expression in Aa strain UTP001, and to determine the molecular basis for the regulation. The regulatory effects of environmental growth conditions on fimbrial expression will be studied first at the phenotypic level, then at the molecular level. Molecular characterization studies will begin as soon as initial DNA sequence and transcriptional organization are ascertained. It is felt that understanding the molecular basis for fimbrial expression by Aa will enable researchers to develop better therapies to combat this pathogen in the most susceptible patient populations.

描述（改编自研究者摘要）：本提案涉及 放线杆菌菌毛表达分子基础 伴放线菌（Aa）。 据指出，牙周病原体Aa 当从口腔新鲜分离时表达菌毛。 流苏Aa 在肉汤培养基中，Aa是粘附的，而非菌毛Aa则少得多 粘附性。 这种行为导致许多研究人员推断菌毛是 Aa的一个重要毒力因子。Aa的菌毛表达可能是 在牙周组织定植期间对生物体是重要。 这项提案的长期目标是确定遗传 组织的菌毛基因，并确定分子基础， 这种潜在的毒性因子的调节。 具体目标1是分离 并克隆Aa菌毛基因。 Aa菌毛克隆将选自 使用PCR的来自有菌毛的菌株UTP001的DNA的基因组文库和 免疫探针 一个基因组文库，在广泛的范围内构建， 动员粘粒载体，将用于互补研究， 从UTP001产生的无菌毛、利福平抗性Aa菌株。 特异性目的2旨在确定DNA序列和转录 Aa菌毛基因的组织。 菌毛基因定位和 测序将通过DNA限制性分析完成，桑格 测序和分析菌毛RNA转录物。 菌毛RNA将 通过北方分析、S1核酸酶和引物延伸作图进行研究 为了确定转录组织， 起始位点和基因的启动子区。 具体目标3将定义 环境生长条件直接调控菌毛生长 在Aa菌株UTP001中表达，并确定表达的分子基础。 调控 环境生长条件的调节作用 菌毛的表达将首先在表型水平上进行研究，然后在 分子水平。 分子表征研究将尽快开始 随着初始DNA序列和转录组织的确定。 我们认为，理解菌毛表达的分子基础， aa将使研究人员能够开发更好的疗法来对抗这种情况 最易感染的患者群体中的病原体。