Molecular Assembly on the Cell Surface of Actinomyces

描述(申请人提供)：牙菌斑代表已知的困扰人类的最复杂的微生物群落或生物膜之一。与口腔生物膜有关的疾病--携带牙齿、牙周炎和牙周炎--摧毁了大量人口，并由于缺乏有效的治疗方法而继续构成巨大的经济负担。该项目的长期目标是阐明口腔生物膜发育的基本机制，并确定可能成为开发药物和疫苗的有吸引力的目标的关键角色。牙菌斑的形成始于早期的细菌定殖者附着到牙釉质上，产生一种粘附性基质，然后吸引中晚期定殖者。放线菌是一种关键的早期定植生物，它不仅可以直接与牙齿表面相互作用，而且可以与许多早期和中期定植生物相互作用，在生物膜的发育过程中发挥着重要的作用。因此，我们的研究集中在对菌毛和其他表面蛋白的黏附原理进行剖析，特别是涉及到这些相互作用以及它们在细菌表面组装的机制。在过去的资助期间，我们成功地为口腔放线菌开发了一种简便的新基因破坏技术，并通过它确定了在上述细胞-细胞相互作用中起关键作用的两个不同菌毛的关键成分。我们证明了尖端fimbrins FimQ具有双重功能，促进1型菌毛的组装，并直接介导细菌对唾液中富含脯氨酸的蛋白质的黏附，这些蛋白质被认为是覆盖在牙齿表面的。相反，2型菌毛的菌丝介导了受体多糖依赖的与口腔链球菌的共聚集、与红细胞的黏附和生物膜的发育。结构研究揭示了FIMA多价功能所必需的两个粘附性Ig G样模块。值得注意的是，我们发现这些菌毛蛋白聚合成菌毛聚合物需要它们的同源菌毛特异性分类酶，这是革兰氏阳性细菌中一种保守的转肽酶。产生的聚合物通过看家分类酶SrtA锚定在细胞壁上，这种酶对许多表面蛋白的细胞壁锚定也是必不可少的，其中一个细胞壁分离信号AcaF被发现在细菌共聚集中发挥重要作用。最重要的是，我们发现srtA的失活极大地扰乱了细菌的形态，并伴随着细胞壁和隔膜的异常。因此，分解酶机制是细菌致病和适应能力的关键因素。在这些重大进展和几个新假说的推动下，继续的提议有三个主要目的：(1)揭示细胞表面稳态中索酸酶srtA的生理功能和调节；(2)剖析菌毛蛋白和非菌毛表面蛋白与各种细胞受体的分子相互作用；(3)进一步阐明放线菌菌毛组装的基本机制，描述顶端菌毛蛋白FimQ介导的菌毛组装的不同机制，并鉴定索酸酶介导的菌毛组装所需的反式作用因子。

DESCRIPTION (provided by applicant): Dental plaque represents one of the most complex microbial communities or biofilms known to afflict man. Oral biofilm-related diseases - dental carries, gingivitis and periodontitis - devastate a large human population and continue to pose a huge economic burden due to the lack of effective therapies. The long-term goal of this project is to elucidate the basic mechanisms of oral biofilm development and identify key players that may be attractive targets for developing drugs and vaccines. The development of dental plaque begins with the attachment of early bacterial colonizers to the tooth enamel, generating an adhesive matrix that then attracts the intermediate and late colonizers. Actinomyces is a key early colonizer that plays a prominent role in biofilm development by virtue of its ability to directly interact not only with the tooth surface but also with a number of both early and intermediate colonizers. Therefore, our studies have focused on dissecting the adhesive principles, i.e. fimbriae and other surface proteins, specifically involved in these interactions ad the mechanism of their assembly on the bacterial surface. During the past grant period, we succeeded in developing a facile new gene disruption technology for Actinomyces oris, and through it, identified the key components of two distinct fimbriae that are pivotal in the aforementioned cell-cell interactions. We showed that the tip fimbrillin FimQ serves dual functions, facilitating the assembly of type 1 fimbriae and directly mediating bacterial adherence to salivary proline-rich proteins known to coat the tooth surface. In contrast, the shaft fimbrilli FimA of the type 2 fimbriae mediates the receptor polysaccharide-dependent coaggregation with oral streptococci, adherence to erythrocytes and biofilm development. Structural studies revealed two adhesive IgG-like modules of FimA essential for its multivalent functions. Significantly, we showed that polymerization of these fimbrillins into fimbrial polymers requires their cognate fimbriae-specific sortase, a conserved transpeptidase in Gram-positive bacteria. The resulting polymers are anchored to the cell wall by the housekeeping sortase SrtA, which is also essential for the cell wall anchoring of many surface proteins with a cell wall sorting signal One of these, AcaF, is found to play a significant role in bacterial coaggregation. Most importantly, we discovered that inactivation of SrtA greatly perturbs bacterial morphology accompanied with abnormal cell wall and septa. Thus, the sortase machinery is a key player of bacterial pathogenesis and fitness in A. oris. Driven by these major advancements and several new hypotheses, the continuation proposal has three major aims: (1) Uncover the physiological function and regulation of sortase SrtA in cell surface homeostasis, (2) Dissect the molecular interactions of fimbrillins and non-fimbrial surface proteins with various cellular receptors, (3) Further elucidate the fundamental mechanisms of fimbrial assembly in Actinomyces, delineate the distinct mechanism of fimbrial assembly mediated by the tip fimbrillin FimQ, and identify trans- acting factors required for sortase-mediated fimbrial assembly.