Microbiology and Microbial Pathogenesis Program
Infectious Diseases and Immunity Branch
Division of Basic and Translational Sciences
We are seeking Concept Clearance for a Program Announcement (PA) to solicit proposals to develop new insight into the role of microbes in human oral health and disease through research on the total oral microbial community using a metagenomics approach built upon recent developments in DNA sequencing, gene assembly and bioinformatics.
Background:
Since van Leeuwenhoek first observed dental plaque under a crude microscope in 1683, microbiologists have recognized that the microflora in the mouth is diverse, changing in quantity and composition during health and disease. Despite 321 years of interest in the "animalcules" in oral plaque, many microbes have failed to be identified or cultured in vitro. Recent studies using contemporary molecular biology techniques provide insight into the identification of bacteria in oral biofilms. Ribosomal DNA (rDNA) analyses use the conserved regions of the bacterial genome to classify the organism and indicate its relative relation to other microbes. Although such analyses are useful for identifying the microorganism, rDNA loci constitute only ~0.1% of each microbial genome. The other 99.9% of each genome provides the blueprint for the vast array of metabolic, structural and potential virulence-associated characteristics of each organism. Thus, a more robust form of analysis is needed to study the full genomes of oral microbes.
But how can we study the genomes of bacteria that cannot be cultured? Genomics, the study of the entire genome of an organism, uses DNA extracted from pure cultures of the microbe. Such a procedure is not possible with microbes that cannot be cultured, thus making traditional genomic analyses of as many as 40% of microbes in the mouth impossible. However, recent advances in genomics of bacteria in water and soil have confirmed the utility of metagenomics, a technique for studying the genomes of all microbes, regardless of their ability to be cultured. Metagenomics is a cost-effective, culture-independent approach to identify microbes and analyze microbial genomes. Metagenomics treats the microbial community as a single dynamic entity. It explores the genome content of the community and leads to analysis of changes in content and expression as a function of site, time, and various states of perturbation, e.g., progression towards and regression from disease following treatment.
In metagenomics, microbial samples are collected from an ecological niche such as the subgingival sulcus, tongue, cheek, or tooth (supragingival plaque). The microbes are isolated, lysed and the DNA purified, cut into smaller pieces and cloned into E. coli or other vectors. By sequencing each piece of DNA and storing the information in a large computer database, the entire "genome" of the oral microbiome can then be collected and subsequently analyzed.
Theoretically, a metagenomics database will contain DNA sequences for ALL the genes in the microbial community. With the aid of powerful assembler computer programs, the snippets of DNA sequences will be aligned and reassembled into their original order. Comparative analyses of the genomes will determine whether the genes are similar to those already known to exist. Once genes are identified from the microbiome, their expression under selected conditions will be determined and the functional role of the proteins elucidated. Through gene expression using microarrays and RT-PCR, and proteomic analyses, a rich and robust compendium of information will become available about the genomes of all microorganisms in the oral cavity, regardless of the ability to culture them. Tissue toxicity; aciduricity; factors that stimulate cytokine induction and immunoglobulin degradation; and new virulence factors will be uncovered without the need to grow the microorganisms.
Metagenomics will be used to develop many research projects, including:
- Identification of uncultured bacteria, archaea, bacteriophages, animal viruses and eukaryotic microbes;
- Reconstruction of complete microbial genomes;
- Construction of metabolic pathways of microbes of the oral ecosystem;
- Localization of microbes through the use of fluorescence in situ hybridization (FISH);
- Development of the oral microbial community as humans age from infants to adults;
- Role of perturbation of the (normal) oral microbial community (not just the acquisition or expansion of single species) on health; and,
- Development of new clinical tools using genomics data.
Because of the complexity of such projects, the establishment of collaborative scientific teams, both domestic and international, with diverse scientific disciplines studying polymicrobial diseases, including microbiology, immunology, biochemistry, clinical medicine, pathology, bioengineering, material science, imaging technology, and mathematical modeling will be encouraged.
Portfolio Overview:
A review of the current NIDCR portfolio indicates the Institute is not supporting any projects on this topic.
Recommendations from Workshops and Meetings:
Metagenomics research has been advocated by extramural investigators at several recent American Society for Microbiology (http://www.asm.org/) conferences, including Microbial Communities (May 3-5, 2002), Polymicrobial Diseases (October 19-23, 2003), and Biofilms 2003 (November 1-6, 2003).