Environmental Uncultivable Bacteria Model for Understanding Human Periodontitis

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DESCRIPTION (provided by applicant): I025, a novel uncultivable bacteria group, has been associated with periodontal disease, which affects 18% of the adult population in the industrialized world. Neither the natural reservoir nor the complete diversity of this group is known. I025 belongs to the candidate phylum TM7, which was first discovered in diverse natural environments including seawater, soil, and activated sludge, and is made entirely of uncultivable species. Recent independent investigations have detected TM7 ribosomal DNA (rDNA) gene sequences in the human oral microbial community of both healthy and diseased individuals, whereas I025 was detected primarily in diseased individuals. rDNA sequence comparison between environmental and human TM7 strains indicate some are nearly identical, suggesting they are close relatives, if not the same species. We hypothesize that an environmental I025-like bacteria can serve as model for better understanding the I025 role in human periodontitis. Our goal is to establish such an I025 model. Resistance to cultivation, however, has kept TM7 species undescribed beyond their rDNA sequence and few morphological features, despite their conspicuous filaments of up to 70 ¿m in length in the human oral plaque. Undergraduate and Master's students at SJSU will apply modern and highly sensitive culture-independent molecular techniques to investigate the function of I025-like bacteria in their natural habitat. The specific aims of this project are: Aim 1: Identify ecological sites with TM7 and then characterize the diversity of TM7 in those environmental samples related to human periodontal-associated TM7 species through PCR amplification of 16S rDNA gene and sequence analysis. Aim 2: Quantify environmental TM7 closely related to the periodontitis-associated TM7 (I025) through real-time quantitative PCR and fluorescence in situ hybridization. Aim 3: Characterize the metabolic function of TM7 cells in their natural environment through innovative methods that combine microautoradiography and in situ hybridization to measure nutrient uptake by single cells, without the need of cultivation. Ecological environments serve as the reservoir for many human pathogens. I025 is a potential emerging human pathogen, and once its environmental source is defined and the nutrient requirements understood, we can proceed to establish a model for better understanding its role in humans.