Physiological Interaction Between Probiotic Bacteria and Porphyromonas gingivalis

益生菌与牙龈卟啉单胞菌之间的生理相互作用

• 批准号: 8685401
• 负责人: Margaret J Duncan
• 金额: $ 65.8万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-25 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8685401
• 关键词: 3-Dimensional; Address; Affect; Antibiotic Resistance; Area; Bacteria; Cells; Coculture Techniques; Collaborations; Complement; Conditioned Culture Media; Data; Dental; Development; Drug Controls; Epithelium; Equilibrium; Gene Expression Profile; Gene Proteins; Gene Targeting; Genes; Genetic; Gingiva; Goals; Growth; Health; Human; Immune system; Infection; Inflammation; Inflammatory; Intervention; Knowledge; Laboratories; Lead; Libraries; Mass Spectrum Analysis; Massive Parallel Sequencing; Measures; Metabolic; Modeling; New Agents; Oral; Oral cavity; Oral health; Organism; Parents; Pathway interactions; Periodontitis; Physiological; Population; Porphyromonas gingivalis; Prevention; Probiotics; Qualifying; Research; System; Testing; Tissue Model; Tissues; Tooth Loss; Tooth structure; Virulence; Work; bone loss; commensal microbes; cost; fitness; gastrointestinal bacteria; inflammatory marker; inhibitor/antagonist; innovation; insight; lactic acid bacteria; member; microbial community; monolayer; mutant; new technology; oral bacteria; oral commensal; oral pathogen; pathogen; prebiotics; pressure; prevent; public health relevance; transcriptome sequencing

DESCRIPTION (provided by applicant): The goal of this proposal is to identify potential probiotic strains and their inhibitory mechanisms in order to develop new therapies to prevent periodontitis. The significance of this application relates to global concerns about widespread and increasing antibiotic resistance and the growing support for more ecologically acceptable therapies including pro- and prebiotic approaches using antagonistic bacteria or their products. The most frequently employed probiotic bacteria for gastrointestinal health are lactic acid bacteria (LAB), but little is known about their use in the oral cavity or how they might suppress pathogens. Even less is known about the potential probiotic effects of other non-LAB bacteria and these constitute significant knowledge gaps in the probiotic field. In our preliminary data, we have identified potential LAB and non-LAB oral commensal bacteria that can suppress the key oral pathogen Porphromonas gingivalis. In this proposal, we will use novel technologies to identify how these bacteria interact and explore their potential as probiotics. In Aim 1 we will identify genes involved in interactions between P. gingivalis and selected probiotic bacteria. A key innovation here is that we will utilize a recently developed saturated P. gingivalis transposon mutant library in co-culture with the probiotic bacteria and identify critical genes in the interacion by a massively parallel sequencing approach (Tn-seq) to quantitatively assess survival of mutants under selective pressure of probiotic activity. To complement identification probiotic target genes by Tn-seq, we will also determine gene expression patterns by RNA-seq in wild-type P. gingivalis cultured with and without probiotics to identify functions most affected. Aim 2 addresses identification of growth inhibiting molecules produced by selected probiotics, first by setting up a test system of targeted deletions and in trans complemented strains of P. gingivalis genes identified by Tn-seq and RNA-seq as highly affected by probiotic bacteria to confirm probiotic inhibition. Metabolites in supernatants of candidate probiotic strains will be identifiedby mass spectrometry and tested on the mutant and complemented mutant strains. In Aim 3 we will test combinations of P. gingivalis and either probiotic bacteria or synthetic inhibitor derivatives for activity in a 3-D gingival infection model to quantify survival of both P. gingivals and probiotics and measure markers of inflammation in gingival cells. The information obtained upon completion of this work will provide new insights into how metabolic actions between bacteria can be exploited in order to maintain a healthy microbiota. Inhibitory bacteria or their products identified in our project may be candidate probiotic agents for the prevention of periodontitis. Genes/pathways that are targets of prebiotics from oral commensal bacteria and LAB may also become lead targets for development of drugs that control P. gingivalis virulence.

描述（由申请人提供）：本提案的目标是鉴定潜在的益生菌菌株及其抑制机制，以开发预防牙周炎的新疗法。该申请的重要性涉及全球对广泛和日益增加的抗生素耐药性的关注，以及对更生态可接受的疗法的日益支持，包括使用拮抗细菌或其产品的益生元和益生元方法。最常用于胃肠道健康的益生菌是乳酸菌（LAB），但对它们在口腔中的用途或它们如何抑制病原体知之甚少。关于其他非LAB细菌的潜在益生菌作用的了解甚至更少，这些构成了益生菌领域的重大知识空白。在我们的初步数据中， 已经确定了潜在的LAB和非LAB口腔细菌，可以抑制关键的口腔病原体牙龈卟啉单胞菌。在这项提案中，我们将使用新技术来确定这些细菌如何相互作用，并探索它们作为益生菌的潜力。在目标1中，我们将鉴定参与牙龈卟啉单胞菌和所选益生菌之间相互作用的基因。这里的一个关键创新是，我们将利用最近开发的饱和牙龈卟啉单胞菌转座子 突变体文库与益生菌共培养，并通过大规模平行测序方法（Tn-seq）鉴定相互作用中的关键基因，以定量评估突变体在益生菌活性的选择压力下的存活。为了补充通过Tn-seq鉴定益生菌靶基因，我们还将通过RNA-seq确定在有和没有益生菌的情况下培养的野生型牙龈卟啉单胞菌中的基因表达模式，以鉴定最受影响的功能。目的2解决了由所选益生菌产生的生长抑制分子的鉴定，首先通过建立由Tn-seq和RNA-seq鉴定为受益生菌高度影响的牙龈卟啉单胞菌基因的靶向缺失和反式互补菌株的测试系统来确认益生菌抑制。候选益生菌菌株上清液中的代谢产物将通过质谱法鉴定，并在突变株和补充突变株上进行测试。在目标3中，我们将测试牙龈卟啉单胞菌和益生菌或合成抑制剂衍生物的组合在3-D牙龈感染模型中的活性，以量化牙龈卟啉单胞菌和益生菌的存活率并测量牙龈细胞中的炎症标志物。这项工作完成后获得的信息将为如何利用细菌之间的代谢作用以维持健康的微生物群提供新的见解。本研究所鉴定的抑制性细菌或其产物有可能成为预防牙周炎的候选益生菌制剂。作为来自口腔粘膜细菌和LAB的益生元的靶标的基因/途径也可能成为开发控制牙龈卟啉单胞菌毒力的药物的主要靶标。