P Gingivalis LPS: Hemin-induced lipid A structural remodelling

描述(申请人提供)：牙龈卟啉单胞菌是一种重要的革兰氏阴性周围病原菌，与成人型牙周炎密切相关。牙龈假单胞菌脂多糖(LPS)显示出不寻常的脂类A结构异质性，我们推测这可能是一种潜在的天然宿主防御反应的调节器。在之前的资助期间，我们发现了牙龈假单胞菌利用一种新的分子机制来逃避和颠覆人类先天性免疫系统的TLR4成分。内毒素(LPS)与Toll样受体4(TLR4)结合后的信号转导是宿主对革兰氏阴性菌感染的先天免疫反应的一个重要方面。我们发现，牙龈假单胞菌利用内源性脂质A1和4‘-磷酸酶活性来修饰其内毒素，产生免疫沉默的非磷酸化类脂A。这种独特的类脂A提供了一种高效的机制，该细菌利用这种机制来逃避TLR4感应，并抵抗阳离子抗菌肽的杀伤。因此，我们对这一更新应用的总体假设是：“牙龈假单胞菌通过调节类脂A磷酸酶活性来调节其与宿主天然防御系统的相互作用”。具体地说，我们发现氯化高铁血红素调节牙龈假单胞菌的类脂A结构组成，从而在低浓度下产生TLR4沉默的脂多糖，而在高浓度时发现TLR4拮抗剂类脂A。我们的结果表明，氯化血红素浓度调节类脂A磷酸酶活性将牙龈假单胞菌类脂A活性从TLR4逃避转变为TLR4抑制，潜在地改变了该细菌、当地微生物群落和宿主天然免疫系统之间的关键相互作用。我们的假设将通过直接测定脂质A1和4‘磷酸酶活性(目标1)、表征脂质A1和4’磷酸酶蛋白表达(目标2)和遗传调节(目标3)来验证。此外，目标4将在兔牙周炎模型中检测TLR4逃避和抑制类A结构改变与疾病相关的局部微生物群落和宿主先天性免疫系统的能力。这些研究将阐明牙龈假单胞菌调节其内毒素与天然宿主防御系统相互作用的机制，并在动物疾病模型中检验A类脂结构调节的作用。 公共卫生相关性：牙龈卟啉单胞菌是一种口腔细菌，被认为是影响很大比例成年人的牙周病的原因。这种细菌致病的机制还不是很清楚。这项提案将研究这种细菌的一个独特特征，它允许细菌激活或抑制一种形式的炎症。我们怀疑宿主免疫反应的调节有助于其致病能力。

DESCRIPTION (provided by applicant): Porphyromonas gingivalis is an important gram-negative periopathogen strongly associated with adult type periodontitis. P. gingivalis lipopolysaccharide (LPS) displays an unusual amount of lipid A structural heterogeneity which we hypothesized may be a potential modulator of the innate host defense response. During the previous funding period we discovered a novel molecular mechanism used by P. gingivalis to evade and subvert the TLR4 component of human innate immune system. Signal transduction following binding of lipopolysaccharide (LPS) to Toll-like receptor 4 (TLR4) is an essential aspect of host innate immune responses to infection by Gram negative pathogens. We found that P. gingivalis, uses endogenous lipid A 1- and 4'-phosphatase activities to modify its LPS, creating immunologically silent, nonphosphorylated lipid A. This unique lipid A provides a highly effective mechanism employed by this bacterium to evade TLR4 sensing and to resist killing by cationic anti- microbial peptides. Therefore our overall hypothesis for this renewal application is: "P. gingivalis modulates its interactions with the host innate defense system through regulation of lipid A phosphatase activity". Specifically we have found that hemin regulates the lipid A structural composition of P. gingivalis such that a low hemin concentration a TLR4 silent LPS is made whereas at high hemin concentrations a TLR4 antagonist lipid A is found. Our results indicate that the hemin concentration regulation of lipid A phosphatase activity shifts P. gingivalis lipid A activity from TLR4 evasive to TLR4 suppressive, potentially altering critical interactions between this bacterium, the local microbial community, and the host innate immune system. Our hypothesis will be examined by directly determining lipid A 1 and 4' phosphatase enzymatic activity (Aim 1), characterizing lipid A 1 and 4' phosphatase protein expression (Aim 2), and genetic regulation (Aim 3). Furthermore, Aim 4 will examine the ability of the TLR4 evasive and suppressive lipid A structures to alter the local microbial community associated with disease and the host innate immune system in a rabbit model of periodontitis. These studies will elucidate the mechanisms by which P. gingivalis regulates its lipopolysaccharide interactions with the innate host defense system and test the contribution of lipid A structural regulation in an animal model of disease. PUBLIC HEALTH RELEVANCE: Porphyromonas gingivalis is an oral bacterium that is believed to contribute to the form of gum disease that affects a large percentage of adults. The mechanism's that this bacterium employs to induce disease are not well understood. This proposal will examine a unique characteristic of this bacterium that allows the bacterium to either activate or suppress a form of inflammation. We suspect the modulation of the host immune response contributes to its ability to cause disease.