Physiological Interaction Between Probiotic Bacteria and Porphyromonas gingivalis

描述(申请人提供)：本提案的目标是确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。这一应用的意义涉及到全球对广泛和日益增长的抗生素耐药性的担忧，以及对更多生态上可接受的治疗方法的日益支持，包括使用拮抗细菌或其产品的益生菌和益生菌方法。最常用于胃肠道健康的益生菌是乳酸菌(LAB)，但人们对它们在口腔中的使用或它们如何抑制病原体知之甚少。人们对其他非实验室细菌的潜在益生菌影响知之甚少，这些都构成了益生菌领域的重大知识空白。在我们的初步数据中，我们 已经确定了潜在的实验室和非实验室口腔共生菌，可以抑制关键的口腔病原体牙龈假单胞菌。在这项提案中，我们将使用新技术来确定这些细菌是如何相互作用的，并探索它们作为益生菌的潜力。在目标1中，我们将识别参与牙龈假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假单胞菌转座子。 与益生菌共培养的突变体库，并通过大规模并行测序方法(TN-SEQ)确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选择压力下的存活率。为了补充TN-SEQ鉴定益生菌的靶基因，我们还将通过RNA-SEQ测定在加入和不加入益生菌的野生型牙龈假单胞菌中的基因表达模式，以确定受影响最大的功能。目的鉴定部分益生菌产生的生长抑制分子，首先建立TN-seq和RNA-seq所确定的受益生菌高度影响的牙龈假单胞菌基因的靶向缺失和反式互补菌株的测试系统，以确认益生菌的抑制作用。候选益生菌菌株上清液中的代谢物将通过质谱仪进行鉴定，并在突变菌株和互补突变菌株上进行测试。在目标3中，我们将测试牙龈假单胞菌与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，以量化牙龈假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。本项目中鉴定的抑制菌或其产物可能是预防牙周炎的候选益生菌。作为口腔共生细菌和LAB益生元靶点的基因/途径也可能成为控制牙龈假单胞菌毒力的药物开发的主要靶点。

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.