Mechanisms of probiotic inhibition of pathogenic oral bacteria

 描述(申请人提供)：益生菌是一种微生物，当被食用时，会对宿主产生积极或有益的影响。在口腔中使用益生菌作为预防龋齿和牙周病的治疗方法才刚刚开始探索。牙龈卟啉单胞菌(Pg)是牙周病发生发展过程中的重要病原菌。目前PG相关性牙周炎的治疗主要是基于症状的治疗，而不是预防性治疗。我们从乳制品中分离到了几株能在体外抑制PG诱导的细胞毒性和PG生长的益生菌。此外，我们还证明了这些菌株的培养上清对PG生长有抑制作用。初步测试表明，抑制分子 似乎是一种相对分子质量在30-100 kDa之间的蛋白质。我们建议用阴阳离子交换层析和质谱法分离和鉴定德氏乳杆菌的分泌因子。利用这些数据，我们将确定编码抑制因子的基因组位置。我们将克隆和纯化一种重组抑制因子，以进行进一步的检测。利用先前在PG中构建的转座子文库，我们将使用大规模并行测序技术快速、定量地筛选PG中与抑制上清液和/或重组抑制蛋白相互作用的基因，以快速确定潜在的作用机制。最后，我们将检测pg临床分离株的药敏情况。 以及其他口腔细菌对德氏乳杆菌抑制上清和/或纯化的重组抑制蛋白的作用，以确定德氏乳杆菌抑制物对临床分离的PG菌株的作用谱。完成本申请中概述的目标可能确定一种新的种间抑制机制，并证明我们的德氏乳杆菌益生菌菌株和/或其分泌产品临床应用的可行性。

 DESCRIPTION (provided by applicant): Probiotics are microorganisms that when consumed confer a positive or beneficial effect on the host. Use of probiotics in the oral cavity as preventative therapies are just beginning to be explored for dental caries and periodontal disease. Porphyromonas gingivalis (Pg) is an important organism in the onset and progression of periodontal disease. Current treatment for Pg associated periodontitis is largely based on treatment of symptoms rather than preventative therapies. We have isolated several probiotic strains of Lactobacillus delbreuckii from dairy products that inhibit Pg-induced cytotoxicity and Pg growth in vitro. Furthermore, we have demonstrated that supernatants derived from these strains of L. delbreuckii inhibit Pg growth. Initial testing indicates that the inhibitory molecule appears to be a protein with a molecular weight between 30-100 kDa. We propose to isolate and identify the secreted factor from L. delbreuckii by anion/cation exchange chromatography and mass spectrometry. Using this data, we will identify the genomic location encoding the inhibitory factor. We will clone and purify a recombinant inhibitory factor to carry out further testing. Using a previously constructed transposon library in Pg, we will use massively parallel sequencing technology to quickly and quantitatively screen for genes in Pg that interact with the inhibitory supernatant and/or recombinant inhibitory protein to quickly identify potential mechanisms of action. Lastly, we will examine the susceptibility profile of clinical isolates of Pg and other oral bacteria to L. delbreuckii inhibitory supernatants and/or purified recombinant inhibitory protein in an effort to determine spectrum of action of the L. delbreuckii inhibitor against clinical Pg isolates. Completion of the aims outlined in this application may identify a novel interspecies inhibitory mechanism and demonstrate the feasibility of a clinical application for our L. delbreuckii probiotic strains and/or their secreted products.