Each of us harbors an enormous microbial community. In the gut, these microbes form a metabolic organ whose cells outnumber our own by 10-fold, whose genes outnumber those in the human genome by 100-fold, and whose composition can be transformed overnight. It is becoming increasingly clear that variation in these communities has important consequences for health.
The central hypothesis that guides our research is that resident human-associated microbes play critical roles in our response to nutrients, toxins, and pathogens. We use genomics and biochemistry to study the process of selection and competition that shapes these communities.
Extensive Research Description
How do the enormous microbial
populations that accompany us through life influence our health? Much of what
we consume, both beneficial and noxious, is first encountered and processed by
the human gut microbiota, a metabolic organ of 10-100 trillion cells that
encodes an enormous genetic repository strikingly different from our human
genomes in its content, extent of inter-individual variation, and plasticity. A
lifelong course of environmental exposures, selection, and competition shapes
the structure and function of these communities: this is a central issue in
interpreting microbial diversity, a major challenge in human microbiome
projects, and a process that we know almost nothing about.
Due to the availability of massively parallel DNA sequencing and new techniques for high-throughput metabolite profiling, our ability to describe these communities has exploded: this surge of data far outpaces existent strategies to mechanistically dissect the relationship between microbiota composition and function. Consequently, fundamental questions connecting microbial genome content to function remain unexplored. For example, do human symbiotic microbes, like their pathogenic counterparts, possess dedicated mechanisms critical for survival in vivo or is their existence largely passive or based on highly redundant, interchangeable pathways? Does community structure influence this map of genetic requirements? What host or environmental factors play dominant roles in this selective process? These questions are timely because a role for the gut microbiota is an emergent theme in our understanding of many metabolic disorders and infectious diseases.
To this end, we developed a generally applicable, new experimental approach (insertion sequencing, or INSeq) for functional genome-wide analysis of organisms for which a genome sequence (and potentially little else) is known. This approach is based on modification of the broad host-range, randomly integrating transposable element mariner to capture short fragments of adjacent genomic DNA. Massively parallel sequencing of these captured genomic fragments from tens of thousands of mariner insertion strains of a given bacterial species produces a high-resolution map of the precise location and relative abundance of each transposon in the population. Selection changes the representation of transposon mutants, thereby allowing identification of genes and pathways that are key to fitness under the conditions being examined. We first applied this system to the prominent human gut symbiont Bacteroides thetaiotaomicron, in vitro and in vivo in wildtype and genetically manipulated gnotobiotic mice, in the presence or absence of defined communities of other human gut symbionts. The results clearly indicated that symbiont survival in the gut is in no way a ‘passive’ affair; instead, we identified hundreds of genes that are absolutely required for fitness in vivo. These genes (i) include, but extend far beyond, those required for maximal growth in vitro; (ii) are not a random sampling of the genome, but instead are enriched in specific functional categories; and (iii) often display a community context-dependent impact on fitness, such that they are required by B. thetaiotaomicron as a member of some defined microbial communities but not others. These studies also highlighted an unexpected aspect of selection and competition in this system—the vitamin B12 and its analogs (corrinoids), studied for over 50 years for their role in human health, may be key mediators of microbial community structure in the mammalian gut. Future studies are focused on understanding corrinoid exchange in the gut and applying INSeq to other prominent human gut symbionts.
For many microbial communities, the great majority of organisms have not been cultured in the laboratory. We’re developing high-throughput approaches for assembling large human gut culture collections from single healthy or diseased individuals, with the goal of re-uniting discrete components of these communities in germfree mice. These personalized culture collections provide a new platform for studying resource sharing and competition in the gut.
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- Dantas, G., Sommer, M.O.A., Degnan, P.H., and Goodman, A.L. Experimental approaches for defining functional roles of microbes in the human gut. Annual Reviews Microbiology 67 p459-75 (2013).