Global Systems Analysis of the Antibody Response to Gingival Bacteria in Periodontitis using Immunometagenomics

One of the most surprising results from 20th Century science was that 90% of the cells in the human body are bacteria, and that the average human lives with his/her own zoological garden of bacteria. Current estimates are that the mouth, and the gut, each contain upwards of 1,000 different (non-overlapping) bacterial species. These bacteria are called the microbiota. Now, this finding is surprising, because we normally associate bacteria with disease. The fact that we contain so many bacteria is beginning to colour how we think about human diseases that currently have no apparent causation. There is emerging evidence that major human diseases such as rheumatoid arthritis, psoriasis, Crohn's disease, and even some forms of cancer, may be a response to members of the microbiota.
A disease which is known to be a direct response to members of the microbiota (in this case the bacteria present on the gingivae (gums)) is the most common chronic inflammatory and destructive disease of humans - periodontitis. One of the mysteries of periodontitis is whether this disease is due to a small number of specific bacteria or is a general response to all of the bacteria living on the gums. It is very difficult to test which bacteria are involved in periodontitis because: (i) there are so many different types of bacteria involved and (ii) 50% or more of the bacteria present on the gums cannot be cultivated in the laboratory, and therefore, cannot be studied. Thus, using conventional means, it is virtually impossible to decide if periodontitis is due to the actions of selected bacteria or is a general response to all the bacteria on the gums. This also creates difficulties in terms of trying to improve anti-bacterial treatments for periodontitis.
However, within the last few years, methods have been developed to allow the study of all the bacteria associated with diseased gums. This methodology is known as functional genomics, and allows the DNA from all of the bacteria on the gums to be used to produce all the proteins that such bacteria make, under controlled conditions. Thus bacteria that cannot be grown in the lab can be directly examined using this methodology. Using a fairly simple screening system it is possible to determine which of these proteins are stimulating the immune system and, therefore, likely to be causing disease. Thus we can identify both the bacteria and the bacterial proteins responsible for causing periodontitis. This information should allow much better treatments of this very common human disease to be developed. In addition, as periodontitis is now recognised to be a potential causative factor in serious systemic conditions, such as heart disease and diabetes the information obtained in this study could impinge on our understanding of these conditions.

Periodontitis is a highly prevalent, chronic immunological disease of the gingivae, causing destruction of the periodontal ligament and alveolar bone, and is driven by undefined immune responsiveness to the supra- and sub-gingival microbiota. Determining the immune stimuli driving this disease has proved impossible using standard techniques because of the huge numbers of potential bacteria involved and the fact that many of the bacteria of the gingival microbiota cannot be cultured.
It has been suggested that the causation/perpetuation of periodontitis could be explained by two competing hypotheses: (i) the disease is due to immunity to selected bacteria and; (ii) no specific bacteria are involved and any organism can induce pathological immunity. This preliminary proposal aims to test these two competing hypotheses using a systems biology approach. We have collected the gingival bacteria and plasma from a large cohort of periodontal patients and from healthy (non-periodontitis) patients (controls). The bacteria will be used to generate a metagenomic library in the phage vector Lambda-ZAP thus producing a library of clones expressing the total population of proteins present in the gingival bacteria. This library will be screened with Cy-3/Cy-5 labelled IgG and IgA prepared from pooled plasma from patient groups and from controls in a format designed to identify clones binding to immunoglobulins from patients as opposed to healthy controls. This global analysis of the immune response to the gingival microbiota should clearly determine if the pathological immunity in periodontitis is focused on a small number of organisms or is a generalised response to all members of this microbiota. Further, this analysis may identify specific immunogens which could determine future treatment for this complex disease.

Chronic inflammation now seems to be a central risk factor in major diseases such as cardiovascular disease and diabetes. Periodontitis is the most common chronic inflammatory disease of humanity. In addition to the obvious oral sequelae of periodontitis: tooth loss, oral malodour and loss in quality of life, which can be devastating for a large number of people, there is also the growing belief that this condition is a predisposing factor in systemic disease, such as coronary artery disease and type II diabetes. Periodontitis is a disease caused by members of the oral bacterial microbiota. There is also accumulating evidence that the microbiota may have a role to play in certain idiopathic diseases such as Crohn's disease, psoriasis and rheumatoid arthritis. Thus, understanding the response of the immune system in periodontitis to the oral microbiota may provide important clues not just to this disease but to other human conditions for which the causation is unknown.
Thus to address the question of the potential beneficiaries of this research - the first answer is: those individuals who suffer from periodontitis. If this pilot study identified a small number of bacteria, or bacterial proteins as key immunogens in periodontitis, then this could, immediately, focus clinical trials into the removal of these specific bacteria or the tolerisation of patients to these specific proteins. Thus from a one year project it could, in theory, be possible to have a clinical output in 3-5 years. Now if this immunometagenomic strategy works then it could be applied to other diseases such as Crohn's disease or psoriasis to identify the major organisms driving immunopathology. Again, if this led to the discovery of specific bacteria or bacterial antigens, then it could provide novel therapeutic targets and treatments which could be specified in a 3-5 year time span, all depending on the state of the World's economy and how much money is being committed to medical research.