Biosynthesis and Function of Treponema denticola Lipooligosaccharide

描述(申请人提供)：齿密螺旋体是一种原产于口腔的革兰氏阴性人类螺旋体；它被认为是与牙周炎的发生和发展密切相关的红色复合体细菌群落中的三个关键细菌物种之一。牙周病在人类中很普遍，并与包括心血管疾病和早产在内的各种全身疾病有关。然而，人们对齿纹夜蛾的毒力知之甚少，因为这种有机体很难生长和 遗传转化体系还不完善。我们的目标是确定参与齿状毛滴虫致病的毒力因子。在革兰氏阴性菌中，被广泛研究的主要毒力属性之一是脂多糖(LPS)。关于口服密螺旋体是否含有内毒素的问题一直存在争议。尽管已有关于齿状毛滴虫外膜脂多糖活性的研究，但最近的一项结构研究确定了齿状毛滴虫具有一种不同于内毒素的新型外膜脂类。基因组分析还表明，基因组中不存在合成经典内毒素所必需的基因。这种新型OM脂被归类为脂寡糖(LOS)。LOS是齿状毛滴虫的重要表面成分，其结构与重要的革兰氏阳性菌毒力因子脂磷壁酸中的结构基序非常相似。使用纯化的LOS进行的体外研究表明，齿状毛滴虫LOS可以促进破骨细胞的形成，并改变软组织的完整性。到目前为止，还没有人试图评估差异修饰的LOS在体内细菌毒力中的重要性，因为还没有构建明确的突变体来描述LOS的生物合成途径。重要的是，我们在齿纹夜蛾的遗传转化方面取得了进展，并确定了一个可能负责合成和组装齿纹夜蛾的遗传位点。此外，我们还为一个基因产生了TDE1418突变体，该基因编码了一个假定的K5抗原合成酶；TDE1418突变体削弱了LOS的修饰。有趣的是，TDE1418突变体未能在牙髓感染的小鼠模型中促进骨吸收，这表明齿状毛滴虫在体内参与了细菌的毒力。我们现在能够确定LOS生物合成的遗传基础，并检查其生物活性；从而验证我们的假设，即由齿状毛滴虫组装的LOS结构介导细菌毒力。我们将通过实现两个具体目标来实现这一目标：(1)确定TDE1418突变体中LOS的生物合成和组装是如何调控的。(2)利用TDE1418突变体，在体内评价LOS对齿状毛滴虫诱导骨吸收的功能贡献。这一建议将使我们能够进一步开发遗传转化方案，并调查未知的毒力因子，如LOS，这将有助于确定口腔螺旋体在牙周和牙髓疾病发病中的作用。

DESCRIPTION (provided by applicant): Treponema denticola is a Gram-negative human spirochete indigenous to the oral cavity; it has been recognized as one of three key bacterial species from the "red complex" bacterial community that is strongly associated with onset and progression of periodontitis. Periodontal disease is prevalent in humans, and has been associated with a variety of systemic diseases including cardiovascular disease, and preterm birth. However, little is known of T. denticola virulence, as the organism is difficult to grow and the genetic transformation system is not well established. Our goal is to identify virulence factor involved in T. denticola pathogenesis. One of the major virulence attributes that has been extensively studied in Gram-negative bacteria is lipopolysaccharide (LPS). There has been controversy over the issue of whether oral treponemes contain LPS. Despite previous investigations regarding LPS activity in T. denticola outer membrane (OM), a recent structural study has determined that T. denticola possesses a new type of OM lipid, different from LPS. Genomic analysis of T. denticola has also revealed that none of the genes essential for synthesis of classical LPS exist in the genome. The new type of OM lipid has been classified as a lipooligosaccharide (LOS). LOS is a prominent surface component of T. denticola, whose structure is very similar to the structural motifs found in lipoteichoic acid, an important Gram-positive bacteria virulence factor. In vitro studies using purified LOS have revealed T. denticola LOS can enhance osteoclastogenesis and alter soft tissue integrity. To date there has been no effort to evaluate the importance of differentially modified LOS in bacterial virulence in vivo as no defined mutants have been constructed to delineate the LOS biosynthetic pathway. Importantly, we have made progress in the genetic transformation of T. denticola, and have identified a genetic locus that is potentially responsible for the synthesis and assembly of T. denticola LOS. Furthermore, we have generated a TDE1418 mutant for a gene that encodes a putative K5 antigen synthase in the locus; the TDE1418 mutant impaired modification of LOS. Interestingly, the TDE1418 mutant failed to promote bone resorption in a mouse model for endodontic infection, suggesting T. denticola LOS is involved in bacterial virulence in vivo. We are now in a position to determine the genetic basis of LOS biosynthesis and examine its biological activity; thereby testing our hypothesis that the LOS structure assembled by T. denticola mediates bacterial virulence. We will achieve this goal by carrying out two specific aims: (1) Determine how LOS biosynthesis and assembly is modulated in the TDE1418 mutant. (2) Assess the functional contribution of LOS to T. denticola-induced bone resorption in vivo-utilizing the TDE1418 mutant. This proposal will permit us to further develop genetic transformation protocols, and investigate uncharacterized virulence factors such as LOS that will help define the role of oral spirotchetes in the pathogenesis of periodontal and endodontic diseases.