P Gingivalis LPS: Hemin-induced lipid A structural remodelling

P Gingivalis LPS：血红素诱导的脂质 A 结构重塑

• 批准号: 8471010
• 负责人: Richard Peters Darveau
• 金额: $ 52.52万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471010
• 关键词: Adult; Affect; Animal Disease Models; Animal Model; Bacteria; Bacterial Infections; Binding; Biochemical; Biological Assay; Culture Media; Deletion Mutation; Detection; Disease; Epitopes; Escherichia coli; Evaluation; Fractionation; Funding; Generations; Genes; Genetic; Goals; Hemin; Heterogeneity; Host Defense; Human; Immune response; Immune system; In Vitro; Infection; Inflammation; Label; Lipid A; Lipopolysaccharides; Location; Measures; Membrane; Membrane Proteins; Modeling; Modification; Molecular; Molecular Profiling; Mono-S; Oryctolagus cuniculus; Pathogenesis; Peptides; Periodontitis; Phenotype; Phosphoric Monoester Hydrolases; Polymyxin B; Porphyromonas gingivalis; Procedures; Proteins; Recombinant Proteins; Regulation; Reporting; Resistance; Serum; Signal Transduction; Structure; Substrate Specificity; System; Testing; Thin Layer Chromatography; Validation; antimicrobial; antimicrobial peptide; bacteria characteristic; bone loss; cytokine; defense response; in vivo; killings; microbial; microbial community; mutant; novel; oral bacteria; pathogen; periopathogen; protein expression; public health relevance; response; toll-like receptor 4; uptake

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.

描述（由申请人提供）：牙龈卟啉单胞菌是一种重要的革兰氏阴性牙周病病原体，与成人型牙周炎密切相关。牙龈卟啉单胞菌脂多糖（LPS）显示出不寻常的脂质A结构异质性，我们假设这可能是先天宿主防御反应的潜在调节剂。在上一个资助期间，我们发现了牙龈卟啉单胞菌逃避和破坏人类先天免疫系统TLR 4组分的新分子机制。脂多糖（LPS）与Toll样受体4（TLR 4）结合后的信号转导是宿主对革兰氏阴性病原体感染的先天免疫应答的重要方面。我们发现牙龈卟啉单胞菌利用内源性脂质A1-和4 '-磷酸酶活性来修饰其LPS，产生免疫沉默的非磷酸化脂质A。这种独特的脂质A提供了这种细菌所采用的高度有效的机制，以逃避TLR 4传感并抵抗阳离子抗微生物肽的杀伤。因此，我们对该更新申请的总体假设是：“牙龈卟啉单胞菌通过调节脂质A磷酸酶活性来调节其与宿主先天防御系统的相互作用”。具体地，我们已经发现氯化血红素调节牙龈卟啉单胞菌的脂质A结构组成，使得低氯化血红素浓度产生TLR 4沉默LPS，而在高氯化血红素浓度下发现TLR 4拮抗剂脂质A。我们的结果表明，脂质A磷酸酶活性的氯化血红素浓度调节将牙龈卟啉单胞菌脂质A活性从TLR 4回避转变为TLR 4抑制，可能改变这种细菌、当地微生物群落和宿主先天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'磷酸酶的酶活性（Aim 1）、表征脂质A1和4'磷酸酶蛋白表达（Aim 2）和遗传调节（Aim 3）来检验。此外，目的4将检查TLR 4逃避和抑制性脂质A结构改变牙周炎兔模型中与疾病和宿主先天免疫系统相关的局部微生物群落的能力。这些研究将阐明牙龈卟啉单胞菌调节其脂多糖与先天宿主防御系统相互作用的机制，并在动物疾病模型中测试脂质A结构调节的贡献。