Turning on Persistence: Novel Molecular Determinants that Underpin P. gingivalis' Intracellular Survival In Epithelial Cells

开启持久性：支持牙龈卟啉单胞菌在上皮细胞内存活的新型分子决定因素

• 批准号: 10667170
• 负责人: OZLEM YILMAZ
• 金额: $ 8.57万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-10 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667170
• 关键词: Affect; Anti-Bacterial Agents; Antioxidants; Autophagocytosis; Autophagosome; Bacteria; Biological; Buthionine Sulfoximine; Cell Communication; Cell Culture System; Cells; Clinical; Data; Degradation Pathway; Elements; Environment; Enzymes; Epithelial; Epithelial Cells; Equilibrium; Event; Generations; Gingiva; Glutathione; Glycine decarboxylase; Goals; Growth; HSPB1 gene; Homeostasis; Host Defense; Human; Infection; Intervention; Knockout Mice; Knowledge; Label; Life; Lipids; Liquid Chromatography; Mediating; Membrane; Methods; Molecular; NADPH Oxidase; Nutritional; Oral; Oral cavity; Oral mucous membrane structure; Organism; Oxidation-Reduction; Oxidative Stress; PAWR protein; Parents; Pathway interactions; Peptide Signal Sequences; Phenotype; Play; Porphyromonas gingivalis; Production; Proteins; Reactive Oxygen Species; Recombinant Proteins; Research; Role; Secure; Stress; Subcellular structure; System; TXN gene; Testing; Time; Tissues; Translating; Vacuole; Virulence; Work; activation-induced cytidine deaminase; antimicrobial; arm; bactericide; cell type; foot; glutathione peroxidase; inhibitor; microorganism; mouse model; multidisciplinary; mutant; novel; opportunistic pathogen; oral cavity epithelium; oral microbial community; parent grant; pathobiont; pathogen; programs; tandem mass spectrometry; thioredoxin-like protein; trafficking; trait

SUMMARY of The PARENT AWARD A growing multidisciplinary evidence critically underpins that Porphyromonas gingivalis, a leading pathobiont of the oral cavity that successfully remodels oral microbial communities to a pathophysiological state, can live in concert with human gingival epithelial cells (GECs). Epithelial cells are emerged as an integrally important arm of innate defenses in the oral mucosa, while recent observations suggest that these cells can be exploited as privileged growth niches and a reservoir by P. gingivalis, which can intracellularly multiply and remain largely unharmed in GECs. Despite, extensive systems level molecular knowledge exists on the P. gingivalis and GEC interaction, there is considerably little known on the intracellular life of the organism in this central cell type. We recently revealed that formation of autophagosomes is critical for the P. gingivalis’ intracellular replication and evasion of the anti-microbial degradation pathways in the GECs. Our novel preliminary findings also support that lipidation of LC3-C, a key molecule in the ‘selective autophagy’ pathway, which targets intracellular pathogens is significantly modulated by P. gingivalis under the control of an anti-stress molecule, HSP27. Further, glutathione peroxidase (GpX1), a major host redox balance enzyme and a regulator of autophagic flux largely impacted on the global LC3 lipidation state of GECs upon infection. The inhibition of either HSP27 or GpX1 appears to severely affect the intracellular trafficking and viability of the microorganism. The central hypothesis is that P. gingivalis induces a distinct form of selective autophagy, which results in protection of bacterial life and ultimately securing of P. gingivalis’ persistence in the oral mucosa. To test this novel hypothesis, we will pursue two-pronged approach, where we propose the selective autophagy requires tightly coordinated actions of HSP27 and GpX1 to form autophagosomes that fully function as protected replicative niches for P. gingivalis. Aim 1 will define the selective molecular machinery that drives P. gingivalis-containing autophagosome assembly under the control of HSP27 and the mechanisms that disrupt autophagic flux for the evasion of cellular degradation pathways. Aim 2 will establish the role of GpX1 in regulating the selective autophagy in infection via redox homeostasis and suppressing autophagolysosomal machinery. Both aims will employ reductionist primary GECs culture systems to functionally dissect out the mechanisms and phenotypically characterize the molecular events and sub-cellular components. Aim 3 will establish the dual significance of these two components using oral epithelial-tissue-specific knockout mice models. Thus, this proposal aims to fill a significant gap in our fundamental knowledge that is how P. gingivalis, a facultatively intracellular pathogen, establishes a privileged cellular environment and converts nutritionally rich epithelial cells into potentially a central reservoir for bacterial growth and persistence in the oral mucosa. Ultimately, the knowledge gained may translate into molecular strategies that can control or reduce the intracellular colonization and survival methods employed by this important opportunistic pathogen.

父母奖的摘要 不断增长 成功将口腔微生物群落改建为病理生理状态的口腔可以生活 与人类牙龈上皮细胞（GEC）的音乐会。上皮细胞被作为一个积极重要的臂 口腔粘膜中的先天防御措施，而最近的观察结果表明，这些细胞可以探讨为 牙疟原虫的特权生长生位和储层，可以在细胞内繁殖并在很大程度上保持 在GEC中没有受到伤害。尽管在牙龈疟原虫和GEC上仍然存在广泛的系统水平分子知识 相互作用，人们认为在这种中央细胞类型中的生物体细胞内寿命中鲜为人知。我们 最近发现，自噬体的形成对于牙龈疟原虫的细胞内复制至关重要 GEC中的抗微生物降解途径的逃避。我们的小说初步发现也支持 LC3-C的脂化是“选择性自噬”途径中的关键分子，该途径针对细胞内 病原体在抗应力分子HSP27的控制下被牙龈疟原虫显着调节。 此外，谷胱甘肽过氧化物酶（GPX1），主要宿主氧化还原平衡酶和自噬通量调节剂 感染后，在很大程度上影响了GEC的全球LC3脂质状态。抑制Hsp27或 GPX1似乎严重影响微生物的细胞内贩运和生存能力。中央 假设是牙龈疟原虫诱导了选择性自噬的独特形式，这导致 保护细菌寿命，并最终确保牙龈疟原虫在口腔粘膜中的持久性。到 检验这个新颖的假设，我们将采用两管齐的方法，我们提出选择性自噬 需要HSP27和GPX1的紧密协调的动作以形成自噬体，以充分发挥作用 牙龈疟原虫的保护壁ni。 AIM 1将定义驱动P的选择性分子机械。 在HSP27的控制下含牙龈毒素的自噬体组件和破坏机制 自噬通量用于细胞降解途径的演变。 AIM 2将确定GPX1在 通过氧化还原稳态调节感染中的选择性自噬并抑制自噬溶剂体 机械。两种目标都将采用还原主义的基本GECS培养系统来剖析 机制和表型的特征是分子事件和亚细胞成分。目标3意志 使用口服上皮组织特异性敲除小鼠建立这两个组件的双重意义 型号。这就是该提案旨在填补我们的基本知识的显着空白，这就是P.的方式。 牙龈是一种细胞内病原体，建立了特权的细胞环境并转换 营养丰富的上皮细胞可能成为细菌生长和持久性的中心依赖器 口服粘膜。最终，获得的知识可能转化为可以控制或 减少这种重要机会病原体进行的细胞内定植和生存方法。