Dysbiosis of the oral microbiome in periodontal disease: host gene and pathogen effects

人体支持胃肠道和泌尿生殖道以及皮肤表面大量微生物群落的生长。总而言之，这些细菌群落被称为人类微生物群。人们普遍认为，人体微生物群通过对人体发育、生理、免疫和营养等方面的影响，在人类生物学中发挥着重要作用。尽管近年来人类微生物群的组成受到了相当大的关注，但微生物群落调节疾病或预防疾病的确切机制在很大程度上仍然不确定。然而，最近的研究表明，胃肠道的几种慢性疾病与肠道微生物组的组成改变有关：即微生物组各组成部分的相对丰度与健康中发现的丰度相比发生了变化。此外，动物研究表明，一些与疾病相关的微生物群在转移到健康的接受者身上时可以复制疾病，这表明微生物群的失调在疾病的病因中很重要。在这项调查中，口腔微生物群的失调将与牙周病的发展相关。牙周病是人类最常见的炎症性疾病之一，在大约20%的人口中会导致牙齿脱落，这给NHS带来了巨大的成本。它也被认为是其他疾病发展的危险因素，包括心血管疾病和II型糖尿病。本研究的假设是，由宿主的遗传状态或牙周病原体的引入引起的口腔微生物群的失调会导致炎症性牙周病的发展，并通过正常良性的口腔微生物群介导骨丢失。为了验证这一假设，这项研究将使用小鼠模型来检查口腔微生物群的失调和疾病的发展。首先，将使用非培养方法来表征小鼠口腔微生物组，该方法涉及从本研究使用的实验室小鼠口腔细菌中分离出的16S rRNA基因的DNA序列分析。每个16S rRNA序列都是对单个细菌物种的诊断，因此它们将提供一个参考数据库，可用于高通量调查微生物组的变化，其中只对16S rRNA基因的一部分进行了测序。然后，我们将检查将牙龈卟啉单胞菌(一种经常存在于人类疾病中的牙周细菌)引入小鼠口腔微生物组的效果。初步的培养研究表明，只引入低水平的这种微生物会导致小鼠口腔微生物总数的大幅增加，并导致存在的主要细菌类型的变化，但使用非培养方法将更好地了解总体变化。宿主遗传状态对微生物组的影响也将通过对小鼠进行基因工程来检测，这些小鼠已经移除了对牙周组织免疫状态重要的基因。这些转基因小鼠中的一些更容易患牙周病，而另一些似乎受到保护，初步证据表明，这些动物的微生物群不同。在最终目标中，将检查与疾病相关和具有疾病保护作用的微生物向以前的无菌动物传播的效果，以直接确定生态失调对疾病发展的影响。这些信息将为了解已知的变化对人类牙周病微生物群的影响提供基础，并可能确定那些导致疾病的细菌物种和那些可能具有保护作用的细菌物种。

The human body supports the growth of a wide array of microbial communities in the gastro-intestinal and urogenital tracts and on the surface of the skin. Together, these communities of bacteria are referred to as the human microbiome. It is widely acknowledged that the human microbiome plays a significant role in human biology through its influence on human development, physiology, immunity and nutrition. Although the composition of the human microbiome has received considerable attention in recent years, the precise mechanisms whereby the microbial communities mediate disease or protection from it remain largely uncertain. However, recent studies have shown that several chronic diseases of the gastro-intestinal tract are associated with alterations to the composition of the intestinal microbiome: referred to as dysbiosis wherein there are shifts in the relative abundancies of individual components of the microbiome compared to the abundancies found in health. Furthermore, it has been shown in animal studies that some disease-associated microbiomes can reproduce the disease when they are transferred to healthy recipients suggesting that dysbiosis of the microbiome is important in the causation of disease. In this investigation, dysbiosis of the oral microbiome will be examined in relation to the development of periodontal disease. Periodontal disease is one of the most common inflammatory diseases of humans leading to tooth loss in approximately 20% of the population and a significant cost to the NHS. It is also thought to be a risk factor for the development of other diseases including cardiovascular disease and type II diabetes. The hypothesis for this study is that dysbiosis of the oral microbiome, induced by either the genetic status of the host or by the introduction of periodontal pathogens leads to the development of inflammatory periodontal disease and bone loss mediated through the normally benign oral microbiome. To test the hypothesis, this study will examine dysbiosis of the oral microbiome and the development of disease using a mouse model. Initially, the mouse oral microbiome will be characterised using a non-cultural approach involving DNA sequence analysis of 16S rRNA genes isolated from oral bacteria of the laboratory mice used in this study. Each 16S rRNA sequence is diagnostic for an individual bacterial species and hence they will provide a reference database which can be used for high throughput investigations of changes to the microbiome where only part of the 16S rRNA gene is sequenced. The effect of the introduction of Porphyromonas gingivalis, a periodontal bacterium frequently present in human disease, into the mouse oral microbiome will then be examined. Preliminary cultural studies have shown that introduction of only low levels of this organism leads to a major increase in the total amount of the mouse oral microbiome and changes to the major types of bacteria present but the use of a non-cultural approach will gain a better understanding of the overall changes. The influence of the host genetic status on the microbiome will also be examined using mice which have been genetically engineered to remove genes important in the immune status of the periodontal tissues. Some of these genetically engineered mice are far more susceptible to periodontal disease whereas others appear to be protected and there is preliminary evidence to suggest that the microbiomes of these animals are different. In the final aim, the effect of transmission of disease-associated and disease-protective microbiomes into previously germ free animals will be examined to directly determine the influence of dysbiosis on the development of disease. This information will provide the basis for understanding the effects of changes which are known to occur to the microbiome in human periodontal disease and will potentially identify those bacterial species which are responsible for the disease and those which may be protective.