Molecular Assembly on the Cell Surface of Actinomyces

描述(申请人提供)：成功感染细菌病原体需要附着和定植宿主组织。表面决定因素(菌毛和非菌毛表面蛋白)在所有细菌病原体的这些过程中是必不可少的，因为它们提供了与组织因子的特定受体-配体相互作用，这些组织因子决定了细菌的宿主范围和感染部位。在最早在人类牙齿中定植的细菌中，放线菌和口腔链球菌可能是与携带者和牙周病的病因学相关的其他物种定植的基础。放线菌的菌毛和非菌毛表面成分可能由于其与宿主细胞相互作用并激活宿主细胞的能力而在致病过程中发挥重要作用。由于缺乏关于放线菌细胞表面组成的详细分子信息，以及缺乏简单的遗传系统来研究不同成分的作用，这些相互作用的潜在机制仍然不清楚。利用生物信息学方法，我们鉴定了奈氏放线菌MG-1未完成基因组中的两个菌毛基因簇。通过生化和电子显微镜分析，我们已经证明了每个基因簇都编码了一个不同的菌毛结构，包括一个菌毛轴蛋白和一个主要位于顶端的次要亚基。它们组装成菌毛结构需要在基因簇中发现一种特殊的转肽酶，即索尔特酶。此外，我们还鉴定了许多可能的表面蛋白，根据序列特征预测其中大部分参与细胞与细胞之间的相互作用。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，这项提议的长期目标是描绘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。利用电子显微镜、质谱学和分子生物学相结合的方法，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键的化学性质。基因和生化实验将用于确定菌毛组装的酶、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学成分的相互作用。所产生的结果应该为未来研究这些生物在其生态位中的生物过程提供充足、强大的实验系统。我们的建议旨在描述口腔细菌放线菌的表面结构，这些结构可能在菌斑形成中发挥重要作用。产生的结果将允许开发阻止表面蛋白或菌毛组装的抑制剂，这可能被证明是预防携带者和牙周病的有用策略。

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.