Modulating microbiome metabolites in vivo.

Decades of microbiome studies, using a combination of multiomics approaches, have revealed strong associations between microbiota and human health. The next step is to identify the causal molecular mechanisms behind this association: Which microbes, genes, and their metabolites, if any, are responsible for a host phenotype? And if these molecules do affect us, how can we modulate their concentrations in the host to promote gut health? Uncovering the causal relationship between a gut microbe and its host's biology at the level of molecular mechanisms is challenging, largely because we lack an efficient way to manipulate gut microbial genes. More than 90,000 gut bacterial metagenomes have been identified ([ 1 ][1]), and thousands of bacteria have been isolated and their genomes sequenced, but less than 5% of the species are genetically tractable. New tools are in great need for these non-model gut microbes. The gut microbiota modulates host biology in multiple ways. A substantial contribution to this regulation comes from microbiome-derived metabolites. Like a hormone released by an endocrine gland, these molecules have three notable features: They are exclusively released by the gut microbes, some are highly abundant and circulatory, and some are ligands for host receptors. My research focuses on dissecting the molecular mechanisms behind the interaction between microbiota metabolites and human health. I have a strong interest in developing new genetic tools for human gut Clostridium isolates, toggling the expression of microbiota genes and concentrations of metabolites in the host, and studying their effects on human health and diseases. My long-term goal is to understand and eventually reprogram the molecular “language” of host-microbe interactions for therapeutic applications. Previous efforts to study a microbiome-derived molecule in the host used two main strategies: introducing a compound by injection or gavage, or modulating a bacterial species that produces the target metabolite. Both approaches provide insight into the mechanism of biological function but lack a clean background or control (i.e., the physiological concentration of the target metabolite, or other biological activities of the added bacteria). In my lab, we believe the most precise format for investigating a microbiome-derived metabolite in the host is to compare two organisms that differ only in its production. Such an experiment requires knowledge of the metabolic genes responsible for the metabolite and genetic tools for a robustly producing gut microbe. However, a key technical barrier limiting the method's generalization is that many high-abundance gut-derived molecules are produced by Clostridium and its relatives, which have been notoriously resistant to genetic manipulation. In this work, we started by introducing the CRISPR-Cas9 system into a gut microbe, Clostridium sporogenes ([ 2 ][2]). C. sporogenes is a gut commensal commonly found in healthy humans that produces many fermentation metabolites entering the host's circulation. Although many CRISPR-Cas9 systems have been established in mammalian cells and in bacteria like Escherichia coli , the genetics of which are well characterized, building such a system in C. sporogenes is not at all straightforward. Clostridium and its relatives are notoriously resistant to genetic manipulation, largely because there is no efficient approach for introducing extracellular DNA and most Clostridium have a very low endogenous homologous recombination frequency, which further limits the applicability of a CRISPR-Cas9 system. After struggling for over 9 months with no viable colonies, I found that the guide RNA and Cas9 components needed to be introduced separately into C. sporogenes to promote efficient extracellular DNA uptake and homologous recombination. This allowed us to identify and mutate the metabolic genes of more than 10 C. sporogenes –fermented metabolites. By monocolonizing germ-free mice with the C. sporogenes mutants and the wild-type strain, we can modulate the amount of these metabolites in the host. Although it is well known that microbiome metabolites like propionate and butyrate are host GPCR (G protein–coupled receptor) ligands that modulate immune functions by inducing intestinal regulatory T cells ([ 3 ][3]), little is known about structurally similar branched short-chain fatty acids (BSCFAs) and their biology. The system that we developed enabled us to interrogate BSCFA biology in two groups of mice that differed only in their BSCFAs and the responsible metabolic gene. We found that BSCFAs suppress intestinal immunoglobulin A (IgA) plasma cells as well as the concentrations of IgA bound to the surface of a variety of innate immune cells, suggesting a previously unrecognized role for the BSCFAs in regulating IgA-related immune cell biology. A healthy human gut is filled with trillions of microbes, but less than 5% of them can be genetically manipulated. We plan to build a general workflow that facilitates the development of genetic tools for these previously genetically inaccessible gut microbes. Considering that many microbiome-modulated biological phenotypes require the colonization of a complex microbiota, we further aim to assemble a genetically tractable yet phylogenetically diverse gut consortium. If successful, this system will greatly expedite the characterization of the causal molecular mechanisms behind microbiota-human interaction and will also lay the basis for engineering a gut microbiome to promote human gut health and to prevent and cure diseases. FINALIST Chun-Jun (CJ) Guo Chun-Jun (CJ) Guo received undergraduate degrees from Fudan University and a Ph.D. from the University of Southern California. After completing his postdoctoral fellowship at the University of California, San Francisco, and Stanford University in the Fischbach group, CJ started his laboratory in the Jill Roberts Institute for Research in Inflammatory Bowel Disease at Weill Cornell Medicine in 2018. His research uses CRISPR-based microbial genetics, state-of-the-art analytical chemistry, and healthy and diseased mouse models to mechanistically dissect how microbiome-derived genes, pathways, and metabolites affect host biology. [ www.sciencemag.org/content/369/6500/153.2 ][4] 1. [↵][5]1. A. Almeida et al ., Nature 568, 499 (2019). [OpenUrl][6][CrossRef][7] 2. [↵][8]1. C.-J. Guo et al ., Science 366, eaav1282 (2019). [OpenUrl][9][Abstract/FREE Full Text][10] 3. [↵][11]1. M. G. Rooks, 2. W. S. Garrett , Nat. Rev. Immunol. 16, 341 (2016). 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