In the last year, the National Institute of Dental Research awarded two separate research grants that, together, set a new standard for studying oral pathogens. Over the next three years, these studies will sequence the genomic DNA of the periodontal bacterium Actinobacillus actinomycetemcomitans (Aa) and the opportunistic fungus, Candida albicans. The sequencing data produced by these studies will be instrumental in understanding the genetic mechanisms responsible for virulence and drug resistance in these pathogens, and may lead to novel approaches for preventing or eradicating infections.
The National Institute of Dental Research awarded a grant to Dr. David W. Dyer at the University of Oklahoma to determine the sequence of the 2.2 million nucleotide base pairs that make up the genome of Aa.
Aa was selected for the project primarily because of its important role in periodontal disease. Aa is thought to be the cause of localized juvenile periodontitis (LJP), a condition that affects 70,000 children and adolescents each year in the United States. LJP is a chronic inflammatory infection of the gingiva and underlying bony tissues, and there is no effective prevention. Treatment involves surgical or mechanical removal of diseased tissues ("scaling and planing"), coupled with antibiotic therapy. Despite such extensive treatment, the condition can still recur. Without treatment, LJP can result in tooth loss by the age of 20.
The pathology of Aa is poorly understood. It is known that Aa attaches to and invades human epithelial cells and produces toxins that can kill or maim human cells. However, little is known about other virulence factors harbored by the organism, and current NIDR-supported projects focus on specific factors rather than the total spectrum of pathologic mechanisms. Determining the complete nucleotide sequence of Aa will eventually yield a more complete picture of pathogenesis.
Aa will join the ranks of eight other microorganisms whose genetic blueprints have been determined, and several others that will be completed in the near future. The most recent organism to be completely sequenced is Helicobacter pylori, the bacterium that causes stomach ulcers. Other notable organisms that have been sequenced include the brewer's yeast Saccharomyces cerevisiae, the laboratory workhorse and sometimes human pathogen Escherichia coli, and Haemophilus influenza, the major cause of bacterial meningitis in children and a close relative of Aa. The relationship between these two pathogens is another reason Aa was considered an important choice for DNA sequencing. Though similar by taxonomic standards, these organisms have important biological differences that will be reflected at the genomic level. H. influenza causes a rapidly invasive disease, while Aa participates in a slowly developing chronic condition. Determining the DNA sequence of Aa will allow scientists to compare these organisms at the molecular level. This information will provide valuable insights into the basic mechanisms used by these bacteria to destroy human tissues.
Sequencing the Aa genome will also contribute to the overall bacterial DNA database. This growing collection of coded messages will ultimately be the key to finding and attacking soft spots in bacterial defense systems. Identifying the genes in various bacterial pathogens, determining the proteins they code for, and how genes vary from species to species will assist in developing new drugs to counter antibiotic resistance, or to select candidate proteins for vaccine development.
Aa sequencing data will be made available during the course of the study. The data will be accessible through the Internet at http://www.genome.ou.edu.
NIDR and the Burroughs Wellcome Fund are cofunding a sequencing project at Stanford University to identify the genes of the fungus, Candida albicans. Candida joins a list of pathogenic microbes, including the world's three leading killers--malaria, cholera, and tuberculosis--that are now targets of international gene-mapping efforts.
Candida is one of the most commonly encountered human pathogens and has significant medical consequences for both oral and systemic disease. It causes a wide variety of infections ranging from mucosal infections in generally healthy persons to life-threatening systemic infections in individuals with impaired immunity. Candidiasis is one of the earliest and most common opportunistic infections to occur in the oral cavity of HIV-infected individuals. Oral candidiasis causes pain and discomfort and can lead to potentially deadly infections in AIDS patients and other individuals with compromised immune systems. Because of the limited number of safe and effective antifungal drugs, along with what appears to be the development of increased resistance to the most common drugs used to treat oral candidiasis, it is of paramount importance to understand the genetic makeup of Candida in order to develop new and effective therapeutic agents.
Dr. Ronald Davis, principal investigator on the project, was a member of the research team that recently sequenced the genome of the brewer's yeast, Saccharomyces cerevisiae. His approach in this study is aimed at identifying the genes of Candida rather than sequencing the complete genome. This sequencing strategy is a large-scale method of gene discovery that will quickly and economically provide key research tools to the entire Candida albicans research community.
The project uses a shotgun sequencing approach in which the genomic DNA, consisting of about 16 million nucleotide base-pairs and 6,000 genes, is digested into fragments a few thousand base-pairs in length. Each of the DNA fragments is sequenced from both ends towards the middle until a gene sequence is recognized. The partial gene sequences are then compared to computer databases filled with the DNA sequences of genes from many different organisms. Similarities to well-characterized genes will provide clues as to the function of the Candida genes.
The project has already identified over 100 new Candida genes and will eventually produce at least partial sequence data for approximately 95 percent of the total number of genes. Genes that appear to have interesting functions related to reproduction, drug resistance, or pathogenicity will be completely sequenced. Results will be updated periodically on the Internet ( http://alces.med.umn.edu/Candida.html).
It is expected that the number of studies aimed at sequencing the genomes of oral pathogens will continue to increase. A recent addition is an NIDR-supported project at Forsyth Dental Center that has just begun to sequence the genome of the periodontal pathogen, Porphrymonas gingivalis. Similar projects dealing with other oral microorganisms are anticipated in the near future.