Characterization of the effect of a newly identified gene encoding the lipid A deacylase on Porphyromonas gingivalis virulence

表征新鉴定的编码脂质 A 脱酰酶的基因对牙龈卟啉单胞菌毒力的影响

• 批准号: 9763953
• 负责人: Richard Peters Darveau
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9763953
• 关键词: Active Sites; Amino Acids; Anaerobic Bacteria; Attenuated; Bacteria; Bacteroides thetaiotaomicron; Biology; Characteristics; Chronic; Complement; Data; Dental; Diagnostic; Disease; Environment; Environmental Risk Factor; Enzymes; Foundations; Future; Gene Expression; Genes; Genetic Transcription; Goals; Health; Hydrophobicity; Immune Evasion; Immune response; Immunologic Receptors; In Vitro; Infection; Inflammasome; Inflammatory; Inflammatory Response; Innate Immune Response; Knowledge; Ligands; Link; Lipid A; Location; Measures; Mediating; Membrane; Molecular; Operon; Pathogenesis; Pentas; Peptide Hydrolases; Periodontal Diseases; Periodontitis; Phenotype; Play; Porphyromonas gingivalis; Process; Production; Property; Protein-arginine deiminase; Proteins; Regulation; Reporting; Risk; Role; Sampling; Site; Structure; TLR4 gene; Testing; Transcription Initiation Site; Transcriptional Regulation; Vesicle; Virulence; Virulence Factors; Work; attenuation; deacylation; design; differential expression; dysbiosis; gingipain; human subject; in vivo; insight; microbial; mouse model; mutant; pathogen; response; subgingival microbiome; tool

Abstract The Gram-negative sub-gingival anaerobic bacterium Porphyromonas gingivalis is closely associated with periodontitis, a chronic inflammatory disorder. It is found in greater abundance in periodontal diseased sites than in healthy sites suggesting it is well equipped to survive hostile inflammatory conditions. In addition, mouse models of infection have demonstrated that infection with P. gingivalis can tilt the sub-gingival microbiome to a disease-inducing dysbiotic state. Lipid A, the hydrophobic anchor of lipopolysaccaharide, is the bacterial ligand of the host innate immune receptor, TLR4. In P. gingivalis, lipid A is modified by deacylation, from an initially synthesized penta- acylated structure to tetra-acylated structures, a step essential for evading the TLR4-mediated pro- inflammatory response. Tetra-acylated lipid A is also known to contribute significantly to bacterial virulence properties such as outer membrane vesicle (OMV) formation, and attenuation of inflammasome activation. OMV's in P. gingivalis are distinct from the outer membrane (OM) in that they contain more under- acylated lipid A, more Type IX secreted proteins (T9SS), and more anionic A-LPS, the last known to be an OM tether for T9SS proteins. T9SS proteins comprise many virulence proteins including gingipain proteases and peptidylarginine deiminase. OMVs, hence, are considered as long distance carriers of a heavy virulence payload. Since lipid A molecules of OMVs have been shown to be tetra-acylated, and OMVs are a favored environment for T9SS virulence proteins, we hypothesize that the OM of a deacylase deficient strain is, conversely, attenuated for these virulence factors due to absence of tetra-acylated lipid A. Identification of the as yet unknown lipid A deacylase, required for a key step in the lipid A biology of P. gingivalis, was the focus of our previous R21 application. Our work led to identification of PGN_1123, a highly conserved gene, as the deacylase-encoding gene in P. gingivalis 33277. In this application, our goal is to study the effect of lipid A deacylation on different aspects of P. gingivalis pathogenesis. In Aim 1, we will compare wild-type 33277 with the ∆PGN_1123 mutant for OMV production, amount of A-LPS, gingipain activity and inflammasome activation, all of which contribute to P. gingivalis virulence. In Aim 2, we will investigate whether expression of PGN_1123 is up-regulated during disease. PGN_1123 is the last gene of a three gene operon. We will compare in vivo expression of all three genes of the operon from healthy vs. diseased sub-gingival plaque samples obtained from human subjects. A decrease in potency of virulence properties in the ∆PGN_1123 mutant will be followed by an in depth study of the enzyme to better understand the deacylation process, and to devise ways of inhibiting it. Increased PGN_1123 expression during disease will imply the gene is regulated. Future studies will include investigating its regulation, and evaluating use of its expression as a diagnostic tool for measuring extent of disease.

摘要 革兰氏阴性龈下厌氧菌牙龈卟啉单胞菌与 牙周炎是一种慢性炎症性疾病它在牙周病患者中的含量更高 这表明它在恶劣的炎症条件下能够很好地存活。此外，本发明还提供了一种方法， 感染的小鼠模型已经证明牙龈卟啉单胞菌感染可以使龈下倾斜 将微生物组转化为致病的生态失调状态。 脂质A是脂多糖的疏水性锚，是宿主先天性巨噬细胞的细菌配体。 免疫受体TLR 4。在牙龈卟啉单胞菌中，脂质A通过脱酰作用从最初合成的五聚体修饰。 酰化结构到四酰化结构，这是逃避TLR 4介导的促凋亡的关键步骤。 炎症反应。四酰化脂质A也被认为是细菌毒力的重要因素 例如外膜囊泡（OMV）形成和炎性小体活化的减弱。 牙龈卟啉单胞菌中的OMV与外膜（OM）不同，因为它们含有更多的下层- 酰化脂质A、更多的IX型分泌蛋白（T9 SS）和更多的阴离子A-LPS，最后一种已知是OM T9 SS蛋白系链。T9 SS蛋白包含许多毒力蛋白，包括牙龈卟啉菌蛋白酶和 肽基精氨酸脱亚胺酶因此，OMV被认为是重毒力的远距离携带者 有效载荷。由于OMV的脂质A分子已被证明是四酰化的，因此OMV是一种有利的脂质A分子。 T9 SS毒力蛋白的环境，我们假设脱酰基酶缺陷型菌株的OM是， 相反，由于缺乏四酰化脂质A，这些毒力因子减弱。 目前尚不清楚的脂质A脱酰酶的鉴定，需要一个关键步骤，在脂质A生物学的P。 牙龈，是我们以前的R21应用的重点。我们的工作导致鉴定了PGN_1123，其是一种高度 保守基因，作为牙龈卟啉单胞菌33277中的脱酰基酶编码基因。在这个应用程序中，我们的目标是研究 脂质A脱酰作用对牙龈卟啉单胞菌发病机制不同方面的影响。 在目标1中，我们将比较野生型33277与PGN_1123突变体的OMV产生、OMV的量、OMV的量和OMV的量。 A-LPS、牙龈蛋白酶活性和炎性小体活化，所有这些都有助于牙龈卟啉单胞菌的毒力。 在目的2中，我们将研究PGN_1123的表达在疾病期间是否上调。 PGN_1123是三基因操纵子的最后一个基因。我们将比较所有三种基因的体内表达， 来自健康与患病的龈下菌斑样品的操纵子，所述龈下菌斑样品得自人类受试者。 在PGN_1123突变体中毒力特性的效力降低之后，将进行深入的研究。 研究酶，以更好地了解脱酰过程，并设计抑制它的方法。 疾病期间PGN_1123的表达将意味着该基因受到调控。未来的研究将包括调查 其调控，并评估其表达作为测量疾病程度的诊断工具的用途。