Tobacco-induced alterations to P. gingivalis-host interactions

描述（由申请人提供）：牙周炎是一种破坏性的传染病，与多种致命和使人衰弱的慢性全身性疾病相关，在美国每年消耗数十亿美元的医疗费用。吸烟者表现出对牙周炎的易感性增加;更有可能感染关键的牙周病原体牙龈卟啉单胞菌;并且相对于非吸烟者，该细菌物种的数量更高。尽管如此，吸烟者始终表现出减少的临床炎症。我们表明，当牙龈卟啉单胞菌暴露于香烟烟雾提取物（CSE）时，纯化的人单核细胞的促炎反应被显著抑制，并且当细胞被传代培养回到不含CSE的新鲜培养基中时，牙龈卟啉单胞菌的炎症诱导潜力被恢复。然而，烟草对编码牙龈卟啉单胞菌毒力决定因子的基因调控的影响基本上是未知的。 我们假设CSE代表了一种环境压力，牙龈卟啉单胞菌通过改变基因表达来适应这种压力。我们认为，这种适应性反应可能涉及胁迫相关基因的诱导，毒力决定因素和细菌表面成分的改变。我们还假设，这种香烟烟雾诱导的牙龈卟啉单胞菌的生理变化将包括编码关键病原体识别决定簇的基因的活性改变，这将解释烟雾暴露的牙龈卟啉单胞菌的炎症反应降低。我们的目标是在原发性人类单核细胞炎症模型中鉴定、分离和测试CSE改变的基因产物。 通过转录组分析和几种微生物决定因素的结构表征产生的初步数据强烈支持我们的假设。我们已经确定了几个基因，基因产物和细菌结构，在CSE暴露时失调，可能在牙龈卟啉单胞菌参与先天免疫系统中发挥关键作用。这些包括菌毛蛋白、LPS、主要外膜抗原和已知潜在炎症的其他牙龈卟啉单胞菌组分，如dnaJ、grpE和ragA。 这些研究代表了一个新的研究领域，将提供一些第一批信息，说明牙龈卟啉单胞菌如何在分子水平上对香烟烟雾作出反应，并可能为吸烟者的疾病发病机制提供独特的见解。因此，这些研究将开辟新的领域，并为未来的研究提供基础，将阐明烟草如何影响病原体-宿主反应相互作用的机制。 公共卫生相关性：革兰氏阴性厌氧菌牙龈卟啉单胞菌是慢性牙周炎的病原体，慢性牙周炎是一种破坏性的烟草诱导和/或加重的疾病。吸烟者更容易被牙龈卟啉单胞菌感染，并且比非吸烟者拥有更多的这些细菌细胞。然而，吸烟者也表现出减少的临床炎症，并且与此一致，暴露于烟雾的牙龈卟啉单胞菌表现出降低的从免疫细胞诱导炎症反应的能力。这种降低的炎症潜力的机制是完全未知的。因此，我们将通过对在香烟烟雾提取物（CSE）条件培养基中培养的牙龈卟啉单胞菌的微阵列分析来鉴定烟草暴露引起的牙龈卟啉单胞菌基因失调。我们还将研究CSE对已知在先天免疫细胞（如LPS和菌毛）活化中起关键作用的细胞结构特征的影响。烟草改变的细菌结构和CSE失调基因的产物的炎症潜力将在新鲜分离的人单核细胞中进行测试。这项研究将提供一些第一批信息，说明牙龈卟啉单胞菌如何在分子水平上响应香烟烟雾，并可能提供独特的见解吸烟者的疾病发病机制。

DESCRIPTION (provided by applicant): Periodontitis is a destructive, infectious disease that has been associated with multiple fatal and debilitating chronic systemic diseases and consumes billions of dollars in health costs annually in the US. Tobacco smokers exhibit increased susceptibility to periodontitis; are more likely to be infected with the key periodontal pathogen Porphyromonas gingivalis; and to harbor higher numbers of this bacterial species relative to non-smokers. Despite this, smokers consistently exhibit reduced clinical inflammation. We show that the pro-inflammatory response of purified human monocytes is significantly suppressed when P. gingivalis is exposed to cigarette smoke extracts (CSE) and that the inflammation inducing potential of P. gingivalis is restored when cells are sub-cultured back into fresh medium without CSE. However, the influence of tobacco on the regulation of genes encoding P. gingivalis virulence determinants is essentially unknown. We hypothesize that CSE represents an environmental stress and that P. gingivalis responds by altering gene expression to adapt to this stress. We believe that this adaptive response may involve the induction of stress-related genes, virulence determinants and alterations of bacterial surface components. We also hypothesize that such cigarette smoke-induced physiological changes in P. gingivalis will include altered activities of genes encoding key pathogen recognition determinants, which will explain the reduced inflammatory response of smoke-exposed P. gingivalis. It is our aim to identify, isolate and test CSE-altered gene products in a primary human monocyte model of inflammation. Preliminary data generated through transcriptome analyses and the structural characterization of several microbial determinants are strongly supportive of our hypotheses. We have identified several genes, gene products, and bacterial structures that are dysregulated on CSE exposure that may play pivotal roles in the engagement of the innate immune system by P. gingivalis. These include fimbrial proteins, LPS, major outer membrane antigens, and other P. gingivalis components of known inflammatory potential, such as dnaJ, grpE and ragA. These studies represent a novel area of investigation and will provide some of the first information illustrating how P. gingivalis responds at the molecular level to cigarette smoke and may provide unique insights into disease pathogenesis in smokers. Thus, these studies will break new ground and provide the foundation for future studies that will elucidate mechanisms explaining how tobacco influences pathogen-host response interactions. PUBLIC HEALTH RELEVANCE: The Gram negative anaerobic bacterium, Porphyromonas gingivalis, is a causative agent of chronic periodontitis, a destructive tobacco-induced and/or exacerbated disease. Smokers are more likely to be infected by P. gingivalis and to harbor higher numbers of these bacterial cells than non-smokers. However, smokers also exhibit reduced clinical inflammation and, in keeping with this, smoke-exposed P. gingivalis exhibits a reduced capacity to illicit an inflammatory response from immune cells. The mechanisms underlying this reduced inflammatory potential are entirely unknown. Therefore, we will identify P. gingivalis genes that are dysregulated by tobacco exposure by microarray analysis of P. gingivalis cultured in cigarette smoke extract (CSE)-conditioned medium. We will also examine the effects of CSE on the characteristics of cellular structures known to play critical roles in the activation of innate immune cells, such as LPS and fimbriae. The inflammatory potential of tobacco-altered bacterial structures and the products of CSE-dysregulated genes will be tested in freshly isolated human monocytes. This study will provide some of the first information illustrating how P. gingivalis responds at the molecular level to cigarette smoke and may provide unique insights into disease pathogenesis in smokers.