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

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.

The investigation addresses the hypothesis 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 mediated through the normally benign oral microbiome. Understanding of the involvement of dysbiosis of the periodontal microbiota in the aetiology of disease has been hindered by the absence of a suitable animal model which allows study of the influence of the microbiome in a well characterised host genetic background. The aims capitalise on recent findings using the mouse model of periodontal disease.
1. Characterise the oral microbiome of the mouse by sequence analysis of 16S rRNA genes representative of orally adapted mouse bacteria using Sanger sequencing of cloned libraries. The data will provide a reference database of the mouse oral microbiome which will be made available to the periodontal research community
2. Use high throughput pyrosequencing (Roche 454 platform) to analyse dysbiosis of the mouse oral microbiome caused by ageing and by the introduction of low levels of Porphyromonas gingivalis, acting as a keystone bacterial pathogen, and to correlate these changes with the development of periodontal bone loss
3. To determine the influence of host genetics on dysbiosis of the mouse oral microbiome by analysis of the microbiota of mouse knockouts in genes shown to increase susceptibility to periodontal bone loss (CXCR2-/-and LFA-1-/-) and also those known to be protective (C5aR-/- and TLR2-/-)
4. To determine the efficacy and influence of transmission of disease-associated and disease-protective microbiomes into germ free animals on periodontal bone loss.
The data will provide the basis for understanding the effects of changes which 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.

Within the body of a healthy adult, microbial cells are estimated to outnumber human cells by a factor of ten to one. These communities, however, remain largely unstudied, leaving almost entirely unknown their influence upon human development, physiology, immunity, and nutrition. To take advantage of recent technological advances and to develop new ones, the Human Microbiome Project is underway with the goal of developing a comprehensive characterization of the human microbiota and analysis of its role in human health and disease.
Study of the human oral microbiome is one of the most advanced in this field in terms of the understanding of the number and types of different species which inhabit this niche in both health and disease. However the interpretation of this information is hindered by the lack of well characterised animal models to enable identification of those bacterial species, or consortia of species, which are functionally important in the maintenance of health and the development of disease. This is particularly important in the case of periodontal disease, where it is well recognised that there is a dramatic shift in the composition of the microbiome but whether this is the cause or the consequence of the disease is unknown.
In this application, we aim to fully characterise the oral microbiome of the mouse in order to study the influence of the microbiome and its dysbiosis on the development of periodontal disease in a genetically well characterised host background. The results of the mouse microbiome analysis will be of benefit to the international periodontal research community, many of whom use the mouse model of periodontal disease as part of the research investigations, and we will therefore make the 16S rRNA data publically available. Moreover, a potential outcome of these investigations will be the identification of bacterial species which have a protective effect on the oral tissues, analogous to the properties of Bifidobacteria in the intestine, and hence future developments may lead to the development of novel therapeutic approaches in collaboration with other academic groups and industry involving a probiotic approach.