Studies of the Flp operon in A. actinomycetemcomitans

性状：（由申请人提供）伴放线放线杆菌（Aa） 是一种革兰氏阴性细菌，其在人体内的主要栖息地是龈下 沟Aa与青少年侵袭性牙周炎的关系 (e.g.局限性青少年牙周炎（LJP）提供了最引人注目的 牙周炎细菌特异性的证据。新鲜分离株， “粗糙的”粘附集落表型自发地且不可逆地转变为 在肉汤中生长时为非粘附的光滑菌落表型。近期初步 对大鼠的研究发现，只有粗糙表型细胞能够在 口腔粗糙表型主要与许多 在细胞表面形成的菌毛。这些菌毛由一个 菌毛操纵子（flp操纵子），最近已确定在Aa。 转座子分析表明，该操纵子在菌毛中是重要的， 表情它可能会影响相变化，生物膜形成， 可能是白细胞毒素与细菌细胞的结合。虽然Flp 该操纵子的第一个基因编码的菌毛蛋白亚基具有同源性 与V型一样，它在几个方面是独特的，特别是它的 小尺寸（6.5 kb）。我们对监管的研究重点将是5'结束 操纵子，flp到tadA序列，基于先前的互补研究 转座子突变体。本提案的具体目标之一是1。）到 通过研究确定菌毛相位变化的分子基础， Flp菌毛蛋白亚基的转录、翻译和细胞定位。通过 比较flp的DNA序列、转录和细胞定位， 亚基的粗糙和自发的同基因光滑的变体，我们将确定在 在光滑变体中中断了什么样的分子水平的菌毛表达， 并鉴定潜在的顺式调节序列。我们的初步 对这些基因的转录分析表明， 多顺反子信息和操纵子内的多于一个启动子。我们的目标是 2.）的情况。进行潜在启动子区域的功能分析， 通过研究确定flp操纵子的转录组织 选择的缺失突变体。本申请中提出的实验， 成功完成后，将提供必要的初步结果， 随后的实验来确定Aa中菌毛调节的基础。的 通过这些研究获得的信息最终将有助于发展 预防Aa定植和随后的牙周炎的新策略 疾病

DESCRIPTION: (provided by applicant) Actinobacillus actinomycetemcomitans (Aa) is a gram-negative bacterium whose primary habitat in humans is the subgingival sulcus. The association between Aa and aggressive periodontitis in adolescents (e.g. localized juvenile penodontitis, LJP) provides the most compelling evidence for bacterial specificity in periodontitis. Fresh isolates with a "rough," adherent colony phenotype spontaneously and irreversibly switch to a non-adherent, smooth colony phenotype when grown in broth. Recent preliminary studies in rats found only the rough phenotype cells capable of colonizing the oral cavity. The rough phenotype is primarily associated with numerous bundle-forming fimbriae on the cell surface. These fimbriae are encoded by a fimbrial operon (the flp operon) that has recently been identified in Aa. Transposon analysis has demonstrated that this operon is important in fimbrial expression. It likely influences phase variation, biofilm formation, and possibly leukotoxin association with the bacterial cell. Although the Flp fimbrillin subunit encoded by the first gene of this operon shares homology with type V-like fimbrillin, it is unique in several respects, especially its small size (6.5 kb). The focus of our study on regulation will be the 5' end of the operon, the flp to tadA sequence, based on previous complementation studies in transposon mutants. One of the SPECIFIC AIMS of this proposal is 1.) to determine the molecular basis of fimbrial phase variation through the study of transcription, translation, and cell localization of Flp fimbrillin subunit. By comparing the DNA sequence, transcription, and cell localization of the flp subunit in rough and spontaneous isogenic smooth variants, we will identify at what molecular level fimbrial expression is interrupted in the smooth variant, and identify potential cis regulatory sequences. Our preliminary transcriptional analysis of these genes suggests there may be more than one polycistronic message and more than one promoter within the operon. Our goal is 2.) to perform functional analysis of potential promoter regions and to determine the transcriptional organization of the flp operon through the study of selected deletion mutants. The experiments proposed in this application, when successfully completed, will provide the essential preliminary results for subsequent experiments to define the basis of fimbrial regulation in Aa. The information gained by these studies will ultimately aid in the development of novel strategies for preventing colonization of Aa and subsequent periodontal disease.