How interactions of oral bacteria enhance resistance to innate immunity

描述（由申请人提供）：放线菌聚集菌（Aa）生长在人类口腔中，是牙周炎的病原体。在口腔中，Aa必须在口腔微生物菌群附近生长，其中数量最多的通常是口腔链球菌，如戈登链球菌（Sg），它能够将糖发酵成有机酸并释放过氧化氢（H2O2）。我们的初步结果表明，Aa通过上调一个参与防御先天免疫的基因（aplA）来响应来自Sg的H2O2。因此，Aa似乎利用H2O2作为预期信号来增加对宿主先天免疫的抵抗力。我们的目标是了解在宿主先天免疫因子存在的情况下，与口腔链球菌共培养如何影响Aa存活，我们提出了两个目标来实现这一目标。我的第一个目的是测试与Sg共培养是否会减少Aa的活化和吞噬。我假设共培养会减少调理和吞噬作用，并计划通过免疫荧光定量调理蛋白Aa细胞表面以及测量吞噬细胞对Aa和Sg的摄取水平来验证这一点。我们的第二个目标是测试共培养生物膜是否能增加对先天免疫的保护，无论是通过生物膜结构还是细胞的空间接近来诱导参与免疫防御的基因。我们假设共培养的生物膜将更能抵抗先天免疫因子。为了验证这一假设，我们将通过平板计数法和显微镜检测暴露于活性血清和吞噬细胞后，Aa和Sg细胞在共培养与纯培养生物膜中的存活率。我们还将利用荧光报告子与Aa aplA启动子的融合来确定其表达是否依赖于共培养生物膜中与Sg的接近程度，以及这是否会影响两种物种在接近时的生存。了解了这些事件，我们就能更好地了解氨基酸与邻近细胞之间的相互作用，从而抵抗人类的免疫系统。公共卫生相关性：国家牙科和颅面研究所（NIDCR）的使命是通过研究改善口腔、牙科和颅面健康。我们通过确定口腔病原体在其自然环境中必须具有的关键关系以及它们如何影响宿主免疫的生存来完成这一使命。细菌感染经常在纯培养物中进行研究，尽管人体中经常发生多物种感染。我们希望进一步描述这一独特的观察结果，即不同的细菌物种会影响另一种细菌对人类免疫系统的抵抗力，并导致抗菌治疗的新策略。

DESCRIPTION (provided by applicant): The bacterium Aggregatibacter actinomycetemcomitans (Aa) grows in the human oral cavity and is a causitive agent of periodontitis. In the oral cavity, Aa must grow in close proximity to oral microbial flora, the most numerous of which are often oral streptococci, such as Streptococcus gordonii (Sg), that are able to ferment sugars into organic acids and release hydrogen peroxide (H2O2). Our preliminary results show that Aa responds to H2O2 from Sg by upregulating a gene (aplA) involved in defense against innate immunity. Thus, Aa appears to be using H2O2 as an anticipatory signal to increase resistance to host innate immunity. The goal of our proposal is to understand how co-culture with oral streptococci impacts Aa survival in the presence of host innate immune factors, and we propose two aims to accomplish this goal. My first aim tests if co-culture with Sg diminishes opsonization and phagocytosis of Aa. I hypothesize that co-culture will reduce opsonization and phagocytosis and plan to test this by quantifying opsonizing proteins Aa cell surface by immunofluoresence as well as measuring levels of uptake of Aa and Sg by phagocytic cells. Our second aim tests if co-culture biofilm increases protection from innate immunity, whether by biofilm architecture or spatial proximity of cells to induce genes involved in immune defense. We hypothesize that co-culture biofilms will be more resistant to innate immune factors. To test this hypothesis we will measure Aa and Sg cell survival in co-culture vs pure culture biofilms by plate count assay and microscopy after exposure to cxjmplement active serum and phagocytic cells. We will also utilize fluorescent reporter fusions to the Aa aplA promoter to determine if its expression is dependent on proximity to Sg in the co-culture biofilm and if this impacts survival of either species while in close proximity. Understanding these events provides us with a better knowledge of interactions Aa must have with its neighbors that allow it to resist the human immune system. Public Health Relevance: The mission of the National Institute of Dental and Craniofacial Research (NIDCR) is to improve oral, dental and craniofacial health through research. We fulfill that mission by determining key relationships that an oral pathogen must have in its natural environment and how they impact survival from host immunity. Bacterial infections are often studied in pure culture despite the fact that multi-species infections occur often in the human body. We wish to further characterize the unique observation that different bacterial species impact another's resistance to the human immune system and my lead to new strategies for antimicrobial therapy.