Molecular Assembly on the Cell Surface of Actinomyces

描述（由申请人提供）：牙菌斑是已知危害人类的最复杂的微生物群落或生物膜之一。口腔生物膜相关疾病——龋齿、牙龈炎和牙周炎——摧毁了大量人口，并由于缺乏有效的治疗方法而继续造成巨大的经济负担。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靶点的关键参与者。牙菌斑的形成始于早期的细菌附着在牙釉质上，产生一种粘连基质，然后吸引中期和晚期的细菌。放线菌是一种关键的早期定殖菌，由于它不仅能够与牙齿表面直接相互作用，而且能够与许多早期和中期定殖菌直接相互作用，在生物膜的发育中起着突出的作用。因此，我们的研究重点是剖析粘附原理，即菌毛和其他表面蛋白，具体参与这些相互作用及其在细菌表面组装的机制。在过去的资助期间，我们成功地开发了一种简单的新型放线菌基因破坏技术，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种不同菌毛的关键成分。研究人员发现，牙尖菌毛蛋白FimQ具有双重功能，既能促进1型菌毛的组装，又能直接介导细菌粘附在牙齿表面富含脯氨酸的唾液蛋白上。相反，2型纤原的轴纤原纤维FimA介导了受体多糖依赖性的与口服链球菌的共聚集、对红细胞的粘附和生物膜的发育。结构研究揭示了FimA的两个黏附igg样模块对其多价功能至关重要。值得注意的是，我们发现这些纤原蛋白聚合成纤原聚合物需要它们同源的纤原特异性分选酶，这是革兰氏阳性细菌中的一种保守的转肽酶。由此产生的聚合物通过内控分选酶SrtA锚定在细胞壁上，这对于许多具有细胞壁分选信号的表面蛋白的细胞壁锚定也是必不可少的，其中之一，AcaF，被发现在细菌共聚集中起着重要作用。最重要的是，我们发现SrtA的失活极大地扰乱了细菌的形态，并伴有细胞壁和隔膜的异常。因此，分选酶机制在葡萄球菌的细菌发病和适应性中起着关键作用。在这些重大进展和一些新假设的推动下，延续提案有三个主要目标：(1)揭示了分选酶SrtA在细胞表面稳态中的生理功能和调控；(2)剖析了纤维蛋白和非纤维表面蛋白与多种细胞受体的分子相互作用；(3)进一步阐明了放线菌中纤维组装的基本机制，描绘了由纤维蛋白顶端FimQ介导的纤维组装的独特机制，并确定了排序酶介导的纤维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.