Studies on virulence regulation in Porphyromonas

描述（申请人提供）：牙龈卟啉单胞菌是一种黑色革兰氏阴性厌氧菌，是牙周病的重要病原体，也与心血管疾病和其他全系统疾病有关。牙龈疼痛被认为是导致其致病性的主要因素。然而，这些牙龈疼痛是如何被调节/激活的，人们知之甚少。我们研究计划的长期目标是阐明牙龈卟啉卟啉毒力调节的分子机制，作为开发新的治疗干预措施的先决条件，以帮助控制和预防牙周病和其他牙龈卟啉卟啉相关疾病（如心血管疾病）。在本应用中需要解决的具体假设是，新的vim（毒力调节）基因参与了牙龈蛋白酶成熟/激活所必需的特定糖基化修饰。这一假设是基于我们在之前的资助期所做的观察，我们已经确定了三个新基因，vimA， vimE和vimF，当它们失活时，会抑制牙龈痛和其他毒力因子的成熟/激活。这些研究是牙龈卟啉卟啉菌翻译后蛋白酶活性调节的第一个证据。此外，无活性的原酶牙龈蛋白酶的存在，也显示出糖基化与牙龈蛋白酶成熟/激活之间的联系。在这个项目中，我们希望扩展这些发现，并更充分地阐明牙龈蛋白酶生物发生的维生素依赖机制。具体目的是利用我们独特的模型系统对牙龈卟啉菌的牙龈蛋白酶生物发生进行全面评估。具体目标是：1。目的：研究牙龈卟啉单胞菌中牙龈蛋白酶的糖基化特征及其对结构/功能的影响。我们将评估特定的碳水化合物缺陷（包括n -链和o -链糖）在原酶牙龈蛋白酶种类的维生素缺陷等基因突变体。我们将确定糖附着的特定位点，并将它们对牙龈痛物理性质的影响联系起来。2. 确认VimF的糖基转移酶功能，并评价其在糖基化中的特异性作用。由于VimF是一种假定的糖基转移酶，我们将把它的功能与从vwnf缺陷突变体中观察到的牙龈中的特定碳水化合物缺陷联系起来。这将使我们能够验证牙龈疼痛区域需要特定的糖基化来促进激活/成熟的假设。3. 目的探讨牙龈卟啉卟啉与VimA蛋白的相互作用。我们的数据表明，vimA可能是参与牙龈蛋白酶糖基化/激活的复合物的一部分。为了验证这一假设，我们将通过定义其相互作用的动力学和鉴定其特定的结合域来评估VimA与gingpain相互作用的能力。将进行位点特异性突变以确认其对牙龈蛋白酶糖基化的影响，牙龈蛋白酶活性水平及其对生物体毒力潜力的影响。

DESCRIPTION (provided by applicant): Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is an important etiological agent of periodontal disease and is also linked to cardiovascular disease and other systemic diseases. The gingipains are considered the major factor that contributes to its pathogenicity. How these gingipains are regulated/activated however is poorly understood. The long term objective of our research program is to elucidate the molecular mechanism(s) for virulence regulation in P. gingivalis as a prerequisite to the development of novel therapeutic interventions to aid in the control and prevention of periodontal disease and other P. gingivalis-associated diseases (e.g. cardiovascular disease). The specific hypothesis to be addressed in this application is that the novel vim (virulence modulating) genes are involved in specific glycosylation modifications that are essential for gingipain maturation/activation. That hypothesis is based on the observations made in the previous funding period where we have identified three novel genes, vimA, vimE and vimF, which when inactivated inhibit the maturation/activation of the gingipains and other virulence factors of P. gingivalis. Those studies were among the first evidence of posttranslational regulation of protease activity in P. gingivalis. Further, the presence of the inactive proenzyme gingipain species which also displayed altered carbohydrate modification in the wim-defective isogenic mutants link glycosylation with gingipain maturation/activation. In this project, we wish to extend these findings and to more fully clarify the vim-dependent mechanism(s) of gingipain biogenesis. The specific aims are designed to use our unique model system for a comprehensive assessment of gingipain biogenesis in P. gingivalis. The specific aims are: 1. To characterize the glycosylation of the gingipains in the vim-defective isogenic mutants of P. gingivalis and correlate the effects on structure/function. We will evaluate the specific carbohydrate defects (including N-linked and O-linked sugars) in the proenzyme gingipain species of the vim-defective isogenic mutants. We will identify the specific sites for the attachment of the sugars and correlate their effects on the physical properties of the gingipain. 2. To confirm the glycosyltransferase function of VimF and to evaluate its specific role in glycosylation. Because VimF is a putative glycosyl transferase we will correlate its function with the specific carbohydrate defect observed in gingipains from the vwnF-defective mutant. This will allow us to test the hypothesis that regions of the gingipain require specific glycosylation to facilitate activation/maturation. 3. To characterize the interaction of the gingipains and the VimA protein in P. gingivalis. Our data suggest that vimA may be part of a complex that is involved in gingipain glycoslylation/activation. To test this hypothesis, we will assess the ability of VimA to interact with the gingpains by defining the kinetics of its interaction and the identification of its specific binding domain. Site-specific mutations will be performed to confirm its effect on gingipain glycosylation, level of gingipain activity and its impact on the virulence potential of the organism.