Depletion of microbiome-derived molecules in the host using Clostridium genetics

Clostridial metabolite production The clostridia are Firmicute bacterial commensals commonly found in the mammalian gut. Clostridia produce a range of metabolites that diffuse into the host's circulation and have been difficult to manipulate genetically, but Guo et al. successfully developed a CRISPR-Cas9 deletion system in Clostridium sporogenes (see the Perspective by Henke and Clardy). The authors used deletion mutants and mass spectrometry to elucidate clostridial synthesis of several different branched short-chain fatty acids (SCFAs), including isobutyrate, 2-methylbutyrate, and isovalerate. Germ-free mice colonized with mutants incapable of synthesizing SCFAs showed altered immunoglobulin A production. This finding potentially links bacterial SCFA production and host responses to the presence of the clostridia. Science, this issue p. eaav1282; see also p. 1309 Genetically knocking out the production of diffusible metabolites from intestinal commensals elucidates the host-bacterial interface. INTRODUCTION The gut microbiota produce hundreds of molecules that are present at high concentrations in circulation and whose levels vary widely among humans. These molecules are a promising starting point for studying the mechanisms of microbiome-host interactions; the few that have been characterized in detail have potent immune or metabolism modulatory activity and are ligands for a G protein–coupled receptor or nuclear hormone receptor. However, in most cases, molecule production has not been linked to specific bacterial strains or metabolic pathways and unraveling the contribution of each molecule to host biology remains difficult. RATIONALE A general system to knock out the production of microbiome-derived molecules in host tissues would enable interrogation of the mechanisms by which they modulate host biology and disease processes. Such a system has been elusive owing to limitations in the genetic manipulability of bacterial species from the microbiome. Clostridium and its relatives, the anaerobic Firmicutes, have been especially difficult to manipulate. Because the anaerobic Firmicutes are the source of many molecules in this pool, their genetic intractability is a central challenge in studying microbiome-derived molecules. RESULTS We describe a CRISPR-Cas9–based method for reliably constructing markerless deletions in a model commensal Clostridium: Clostridium sporogenes. Our system is recyclable, enabling the construction of multiple mutated strains. We demonstrate the utility of this method by using it to knock out the production of 10 C. sporogenes–derived molecules: trimethylamine, 5-aminovalerate, tryptamine, indole propionate, isovalerate, 2-methylbutryate, isobutyrate, isocaproate, propionate, and butyrate; we validated each knockout by verifying the absence of the corresponding metabolite in culture extracts by liquid chromatography–mass spectrometry or gas chromatography–mass spectrometry. We then colonized germ-free mice with wild-type C. sporogenes or one of five metabolite-deficient mutants, showing that the products of these pathways accumulate in host tissues but are depleted one at a time in mice colonized by the corresponding pathway mutant. By comparing mice colonized by wild-type C. sporogenes versus a mutant deficient in the production of the branched short-chain fatty acids isobutyrate, 2-methylbutyrate, and isovalerate, we discovered a previously unknown immunoglobulin A (IgA) plasma cell–modulatory activity of these abundant microbiome-derived molecules. CONCLUSION New genetic systems for abundant bacterial species in the microbiome will enable a variety of mechanistic questions to be addressed. Among these, our method opens the door to interrogating, in a controlled way, the role of highly abundant chemicals from the microbiome in host biology. In addition, it could be useful in future efforts of two varieties. First, its scope could be expanded to other currently inaccessible Firmicutes, such as butyrate producers from Clostridium clusters IV and XIVa (e.g., Faecalibacterium prausnitzii) and the bile acid metabolizers C. scindens and C. hylemonae. Second, it could be used to control the molecular output of the microbiome by editing the genomes of metabolite-producing Firmicutes. Deciphering the contribution of microbiota-derived molecules to host biology remains difficult. To help address this challenge, we introduce a new method for constructing markerless deletions in a model gut Clostridium, using it to knock out the production of 10 C. sporogenes–derived molecules (right). By colonizing germ-free mice with wild-type (WT) C. sporogenes versus a mutant (ΔporA) deficient in the production of branched short-chain fatty acids (SCFAs) (top left), we reveal an immune modulatory function of these molecules (bottom left). Error bars indicate standard deviation. The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp. , the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes–derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A–modulatory activity.