Molecular Assembly on the Cell Surface of Actinomyces

描述（由申请方提供）：细菌病原体的成功感染需要附着于宿主组织并定殖。表面决定簇（菌毛和非菌毛表面蛋白）是所有细菌病原体中这些过程所必需的，因为它们提供与组织因子的特异性受体-配体相互作用，所述组织因子决定细菌宿主范围和感染部位。在第一批定植于人类牙齿的细菌中，放线菌与口腔链球菌一起可能作为与携带和牙周疾病的病因学相关的其他物种定植的基础。由于放线菌的菌毛和非菌毛表面成分能够与宿主细胞相互作用并激活宿主细胞，因此它们可能在发病机制中发挥重要作用。这些相互作用的潜在机制仍然模糊不清，由于缺乏详细的分子信息的组成的放线菌细胞表面和缺乏一个简单的遗传系统来调查的作用，不同的组件。利用生物信息学的方法，我们已经确定了两个菌毛基因簇的未完成的基因组中的内氏放线菌MG-1。通过生物化学和电子显微镜分析，我们已经证明，每个基因簇编码一个独特的菌毛结构组成的菌毛轴蛋白和一个小亚基主要位于尖端区域。它们组装成菌毛结构需要一种在基因簇中发现的特异性转肽酶，分选酶。此外，我们还确定了许多推定的表面蛋白，其中大部分被预测参与细胞间相互作用的基础上的序列特征。我们推测放线菌与宿主细胞受体和感染伙伴的相互作用可能涉及各种菌毛和非菌毛因子。因此，本研究的长期目标是阐明放线菌表面分子的组装机制，并研究它们在细菌感染中的作用。使用电子显微镜，质谱和分子生物学的组合，我们的目标是确定放线菌菌毛的分子结构和它们的蛋白质交联的化学性质。通过遗传学和生物化学实验来确定菌毛组装的酶、底物和产物。重要的是，我们将确定菌毛和非菌毛因子是否有助于放线菌与宿主细胞和其他病原组分的相互作用。所产生的结果应提供足够的，强大的实验系统，为未来的研究这些生物体在其生态位的生物过程。我们的建议旨在表征口腔细菌放线菌的表面结构，可能在牙菌斑形成中发挥重要作用。所产生的结果将允许开发抑制剂，其阻断表面蛋白或菌毛的组装，这可能被证明是预防携带和牙周疾病的有用策略。

DESCRIPTION (provided by applicant): Successful infections by bacterial pathogens require attachment to and colonization of host tissues. Surface determinants (fimbrial and non-fimbrial surface proteins) are essential for these processes in all bacterial pathogens as they provide specific receptor-ligand interactions with tissue factors that determine both bacterial host range and sites of infection. Among the first bacteria to colonize human teeth, actinomyces together with oral streptococci may serve as a foundation for the colonization of other species that are associated with the etiology of carries and periodontal diseases. Fimbriae and non-fimbrial surface components of Actinomyces may have important roles in pathogenesis owing to their ability to interact with and activate host cells. The underlying mechanisms of these interactions remain obscure due to the absence of detailed molecular information on the composition of the Actinomyces cell surface and the lack of a facile genetic system to investigate the role of different components. Using a bioinformatics approach, we have identified the two fimbrial gene clusters in the unfinished genome of Actinomyces naeslundii MG-1. By biochemical and electron microscopy analysis, we have demonstrated that each gene cluster encodes a distinct fimbrial structure comprised of a fimbrial shaft protein and a minor subunit located largely at the tip region. Their assembly into fimbrial structures requires a specific transpeptidase, sortase, found in the gene cluster. Furthermore, we also have identified many putative surface proteins, a majority of which are predicted to participate in cell-cell interactions based on sequence features. We hypothesize that interactions of Actinomyces with host cell receptors and infectious partners may involve various fimbrial and non- fimbrial factors. Thus, the long term goal of this proposal is to delineate the mechanisms of assembly of the surface molecules of Actinomyces and to examine their role in bacterial infection. Using a combination of electron microscopy, mass spectrometry and molecular biology, we aim to determine the molecular architecture of Actinomyces fimbriae and the chemical nature of their protein cross-linkages. Genetic and biochemical experiments will be employed to define the enzymes, substrates and products of fimbrial assembly. Importantly, we will determine whether fimbrial and non-fimbrial factors contribute to the interactions of Actinomyces with host cells and other etiological components. The results generated should provide ample, powerful experimental systems for future studies on the biological processes of these organisms in their ecological niche. Our proposal aims to characterize surface structures of oral bacteria Actinomyces that may play an important role in plaque formation. The generated results will permit the development of inhibitors which block the assembly of surface proteins or fimbriae that may prove a useful strategy for preventing carries and periodontal diseases.