Biosynthesis and Function of Treponema denticola Lipooligosaccharide

描述（由申请人提供）：齿垢密螺旋体是一种口腔固有的革兰氏阴性人类螺旋体;它已被认为是与牙周炎的发作和进展密切相关的“红色复合体”细菌群落的三种关键细菌物种之一。牙周病在人类中普遍存在，并且与多种全身性疾病（包括心血管疾病和早产）相关。然而，很少有人知道T。由于微生物难以生长， 遗传转化系统还没有很好地建立起来。我们的目标是鉴定T.齿垢发病机制在革兰氏阴性菌中已被广泛研究的主要毒力属性之一是脂多糖（LPS）。关于口腔密螺旋体是否含有LPS的问题一直存在争议。尽管以前的研究表明T. denticola外膜（OM），最近的结构研究已经确定，T。齿垢菌具有一种不同于LPS的新型OM脂质。T. Denticola还揭示了基因组中不存在合成经典LPS所必需的基因。新型OM脂质已被归类为脂寡糖（LOS）。LOS是T.其结构与脂磷壁酸（一种重要的革兰氏阳性细菌毒力因子）中发现的结构基序非常相似。使用纯化LOS的体外研究显示T.齿垢LOS可增强破骨细胞生成并改变软组织完整性。到目前为止，还没有努力评估差异修饰的LOS在体内细菌毒力中的重要性，因为还没有构建确定的突变体来描述LOS生物合成途径。重要的是，我们在T.并确定了一个可能负责T.齿垢此外，我们已经产生了一个TDE 1418突变体的基因编码一个假定的K5抗原合酶的基因座; TDE 1418突变体受损的LOS的修改。有趣的是，TDE 1418突变体在牙髓感染的小鼠模型中不能促进骨吸收，提示T。齿垢LOS参与细菌体内毒力。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T.齿垢介导细菌毒力。我们将通过实施两个具体目标来实现这一目标：（1）确定LOS生物合成和组装在TDE 1418突变体中是如何被调节的。(2)评估LOS对T的功能贡献。利用TDE 1418突变体在体内进行龋齿诱导的骨吸收。这项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spirotchetes在牙周和牙髓疾病的发病机制中的作用。

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.