Thursday, November 21, 2024
11/21/2024
DESCRIPTION (provided by applicant): Background. Human health is intimately connected with the presence and activities of a wide range of microbial species that live on and within us (the microbiota). Characterization of these microbes will help us to understand how they influence human health. The Human Microbiome Project (HMP) aims to sequence the genomes from a large number of the thousands of bacterial species to be found within our microbiota for this purpose. When considering our gut microbiota, a major difficulty encountered is that the majority (~75%) of the many hundreds of bacterial species that reside there are as-yet uncultured, severely restricting the amount of research that can be done to characterize them fully in terms of their contributions to health. There is thus an urgent need to develop tools and methods to specifically access these as-yet uncultured species. Hypothesis. Within the human distal gut microbiota, approximately 5-10% of the species present account for ~99% of the total content. I hypothesize that many of the uncultured microbial species within the human gut are minority species that are numerically rare within the microbiota consortium, and that their rarity is controlled by growth constraints placed on them by the more dominant members of the consortium. I propose that manipulating this population by sorting the rare away from the more dominant species, will relieve this growth suppression, and that a combination of genome sequencing and semi-high throughput screening for optimal growth requirements will allow in vitro culture of these potentially medically-relevant minority species. Preliminary Studies. My laboratory has already provided the HMP with genomic DNA from over 100 bacterial isolates from the human gut, several of which are novel, previously uncultured species which were recovered simply by careful microbiological screening, paying close attention to the strict anaerobic environments and fastidious diets required by most of the gut microbiota species. We routinely use a specialized culture technique to model the gut bacterial community in vitro, and we have used spent culture media from these models to demonstrate the concept of in vitro growth suppression on culturable members of the community. Specific Aims. I propose to develop an innovative method to enrich for as-yet uncultured bacterial species from the human gut, using Fluorescence Activated Cell Sorting (FACS) combined with tailored magnetic antibodies. In addition, I propose a novel diffusion plate technique to rapidly screen for optimal growth conditions for recovered, enriched populations of as-yet uncultured organisms. Work Proposed. We will use fluorescent DNA probes to bind to molecular signatures of bacterial species within the gut microbiota, and FACS to sort the targeted strains. Recovered bacterial cells will be used to immunize mice, and resulting antibodies will magnetically labeled and used to enrich for live target bacterial species within the population. Enriched target populations will be subjected to genome sequencing and in vitro culture attempts using diffusion plates formulated with a wide range of potential growth substrates. PUBLIC HEALTH RELEVANCE: The bacterial community that resides within the human gut (the gut microbiota) is a highly complex consortium of hundreds of species that as a whole is poorly understood. Characterizing the bacterial species that comprise the gut microbiota is a current focus for research, as it is expected that this microbial community plays a pivotal role in human health. Since the majority of bacterial species within the gut microbiota have not yet been cultured in vitro, they remain largely unstudied. The ultimate goal of this research proposal is to develop techniques to enrich for and culture the as-yet uncultured bacterial species of the human gut microbiota, to render them accessible to detailed, health-driven research.
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