Senior Member of the Staff
Department of Molecular Genetics
Professor, Department of Oral and Developmental Biology
Harvard School of Dental Medicine
P.Gingivalis website
email:
University of California at Santa Barbara, B.S., 1969, Chemistry
University of California at Santa Barbara, M.A., 1971, Biology
University of California at San Francisco, D.D.S., 1973, Dentistry
University of Rochester, Ph.D., 1979, Pharmacology
To prevent and control bacterial diseases, it is first essential to identify which species of bacteria are responsible for causing the disease. After this information has been obtained, it is then possible to investigate how each organism exerts its pathogenic effects. The Dewhirst laboratory has used analysis of bacterial DNA to fingerprint over 2,000 different bacteria, including almost all that can live in the human mouth. These fingerprints are being used in a number of clinical studies to determine which bacteria are most important in causing periodontal and other oral diseases.
Linking a particular bacterium to a particular disease requires the capacity to identify bacterial species quickly and reliably. Until the 1980s, the identification and taxonomy of bacteria were based on phenotypic analysis. With the development of macromolecule sequencing methods, it became possible to base bacterial identification and taxonomy on much more robust sequence information. 16S rRNA rapidly became the molecule of choice for sequence analysis, and for the first time, it was possible to reconstruct the phylogeny of bacteria. Our laboratory was one of the first to determine large numbers of bacterial 16S rRNA sequences and has contributed over 1,000 sequences to GenBank, in collaboration with the Paster laboratory. We have studied numerous genera, including: Actinobacillus, Arcobacter, Atopobium, Bacteroides, Campylobacter, Capnocytophaga, Cardiobacterium, Eikenella, Fusobacterium, Selenomonas, Veillonella, Haemophilus, Helicobacter, Kingella, Neisseria, Pasteurella, Pasteurellaceae, Porphyromonas, Prevotella, Streptococcus and Treponema. The sequence information has enabled us to develop highly specific DNA probes for the rapid identification of over 250 bacterial species. We are using these probes in clinical studies to identify organisms associated with root caries, adult periodontitis, refractory periodontitis, acute necrotizing ulcerative gingivitis (ANUG), noma (a particularly virulent oral gangrene), necrotizing ulcerative periodontitis (NUP), alveolar osteitis, oral cancer, as well as organisms that invade or adhere to oral epithelial cells. These studies seek to identify the bacteria most directly involved in the pathogenesis of oral diseases.
While microbiologists have made great advances in culturing techniques, 40% of the organisms in the human oral cavity and over 99% of the bacteria in the environment are currently uncultivable in the laboratory. 16S rRNA sequencing has been extremely useful in the identification of uncultivable organisms. Our laboratory has been in the forefront of isolating DNA from complex bacterial populations, amplifying the 16S rRNA genes by PCR, and building a 16S rRNA library by cloning the genes into E. coli. With these methods, we have identified uncultured organisms from termite guts, hamsters with ileitis, shrimp with necrotizing hepatopancreatitis, clams with intranuclear pathogens, and over 700 species from the human oral cavity and stomach. We have identified organisms that are only remotely related to the major cultivable bacterial groups and some that are similar to organisms found in extreme environments, such as hydrothermal vents on the ocean floor. Clinical studies using 16S rRNA based DNA probes are in progress to evaluate the role of these previously unknown organisms in human oral diseases.
In addition to our work on oral bacteria, we have collaborated with Dr. James G. Fox of MIT to identify Helicobacter species that can cause stomach, gallbladder, liver, and intestinal diseases in humans and animals. At the present time, Helicobacter species are the only bacteria that have been found to be associated with cancer in both humans and animals. In one study, conducted in Chile to investigate an increasing incidence of gallbladder and extrahepatic biliary tract cancer, we have confirmed an association of both of these diseases with the presence of bile-resistant Helicobacter species.
Porphyromonas gingivalis is the organism most strongly associated with adult forms of periodontal disease. To gain a full understanding of this pathogen, we undertook a genome project to determine the 2.4 million base sequence of its DNA with The Institute for Genomic Research (TIGR) in Rockville, Maryland. The sequence has been completed and annotated and is available on the TIGR Comprehensive Microbial Resource web site as well as via the BROP. The genome information provides the sequence of every protein, antigen, and virulence factor. Using this information, it will be possible to develop a comprehensive understanding of the organism, and to develop therapeutic approaches to eliminate this periodontal pathogen from the mouths of patients.
The Dewhirst laboratory currently has a grant from the National Institute for Dental and Craniofacial Research (DE-016937) to characterize all of the cultivable and currently uncultivated microorganisms present in the human oral cavity (microbiome) and all of their genes (metagenome) in order to develop an integrated view of host-microbiome interactions in health and disease. The objectives of this project are to define the unnamed and uncultured oral taxa and to obtain broad metagenomic sequence information that is representative of the phylogenetic diversity of these microorganisms. Studies have identified almost 800 species in the oral cavity, of which from this and other laboratories only 35% are named, 11% unnamed but represented by strains, and 54% are unnamed and known only as 16S rRNA sequence phylotypes. There is currently no naming scheme for the unnamed 65% of the microbiome, thus it is nearly impossible to link sequence, phenotypic, prevalence, disease association, and bibliographic information. In this project we will link these important types of data by naming the unnamed phylotypes, and establishing a curated, Internet-accessible Human Oral Microbiome Database for all prokaryotic oral taxa. There are species from 121 genera in the oral cavity and only minimal genomic information exists for half of these genera. We will next obtain partial genome sequences for 100 phylogenetically diverse human oral bacteria. Genomic libraries for each species will be constructed and 384 clones will be sequenced from each end, yielding 10–20% genome coverage for each species. This will add information on 600 genes and proteins for each species and over 60,000 new gene sequences to the oral metagenome. These phylogenetically diverse sequences will facilitate attribution of contigs to specific genera or species in other metagenome projects. Currently, 65% of oral prokaryotes are known only as phylotypes based on 16S rRNA sequences. Meaningful biological characterization requires having living organisms to manipulate on the bench. Finally, we will identify and deposit reference strains of approximately 200 unnamed oral phylotypes. Strains from phenotypically characterized investigator culture collections will be identified by 16S rRNA sequence analysis and novel reference strains deposited with the American Type Culture Collection. This research will help us to better understand the several hundred bacteria that live in the human oral cavity and their genes. These bacteria can cause tooth decay, periodontal disease, a variety of other oral diseases and infections elsewhere in the body. Knowing more about these many bacteria will allow scientists to develop better methods for treating and preventing oral diseases
Colombo APV, Boches SK, Cotton SL, Goodson JM, Kent R, Haffajee AD, Socransky SS, Hasturk H, Van Dyke TE, Dewhirst F, Paster BJ. (2009) Comparisons of subgingival microbial profiles of refractory perio-dontitis, severe periodontitis, and periodontal health using the human oral microbe identification microarray. J. Periodontol. 80:1421-1432.
Paster BJ, Dewhirst FE. (2009) Molecular microbial diagnosis. Periodontol. 2000 51:38-44.
Fox JG, Shen Z, Muthupalani S, Rogers AR, Kirchain SM, Dewhirst FE. (2009) Chronic hepatitis, hepartic dysplasia, fibrosis, and billary hyperolasia in hamsters naturally infected with a novel Helicobacter classified in the H. bilis cluster. J. Clin. Microbiol. 47:3673-3681
Aas JA, Barbuto SM, Alpagot T, Olsen I, Dewhirst FE, Paster BJ. (2007) Subgingival plaque microbiota in HIV positive patients. J. Clin. Periodontol. 34(3):189-195.
Nichols FC, Riep B, Mun J, Morton MD, Kawai T, Dewhirst FE, Smith MB. (2006) Structures and biological activities of novel phosphatidylethanolamine lipids of Porphyromonas gingivalis. J. Lipid Res. 47(4):844–853.
Fox JG, Taylor NS, Howe S, Tidd M, Xu S, Paster BJ, Dewhirst FE. (2006) Helicobacter anseris sp. nov. and Helicobacter brantaesp. sp. nov., isolated from feces of resident Canada geese in Greater Boston. Appl. Environ. Microbiol.72(7):4633-4637.
Dewhirst FE, Shen Z, Scimeca MS, Stokes LN, Boumenna T, Chen T, Paster BJ, Fox JG. (2005) Discordant 16S rDNA and 23S rDNA phylogenies for the genus Helicobacter: Implications for phylogenetic inference and systematics. J. Bacteriol. 187(17):6106–6118.
Rachel Anderson, B.S.
