Yano et al Page 10 promote GI motility by activating TGr5 G-protein-coupled receptors on ECs( Alemi et al 013), which is consistent with our finding that Sp-induced metabolites raise 5-HT levels in ECs and that Sp colonization improves Gl motility. Particular Clostridium species are nown to possess high 7a-dehydroxylation activity required for the production of deoxycholate from cholate(Kitahara et al., 2001; Narushima et al., 2006), which is in line levels. Deoxycholate concentrations are substantially higher in the colon versus small e with our finding that Sp microbes, comprised largely of Clostridia, increase deoxychola intestine(Sayin et al., 2013), which, coupled to the finding that bacterial load and diversity is greater in the colon versus small intestine( Sekirov et al., 2010), could contribute to the egional specificity of microbiota-mediated increases in 5-HT synthesis to colonic ECs Phylogenetic analysis of 16S rDNA sequences reveals that a subset of microbes recovered from Sp-colonized mice cluster taxonomically with known 7a-dehydroxy lating Clostridia (Figures 6G and S7). Notably, there are striking phylogenetic commonalities between taxa 于93 identified in Sp-and hSp-colonized mice(Figure S7), consistent with their very similar luminal metabolomic profiles(Figure 6C)and ability to promote 5-HT synthesis from colonic ECs(Figure S3) e also reveal that the metabolites a-tocopherol. tyramine and paba are elevated in feces by Sp. hSp or SPF colonization, co-vary with fecal 5-HT levels, and sufficiently induce 5- HT in vitro and in vivo(Figures 6 and S6 and Table S1). a-tocopherol is a naturally abundant form of vitamin E, with reported therapeutic effects for several diseases(Brigelius- depressive-like behavior in pre-clinical models(Lobato et al., 2010), suggesting alin g 9 Flohe and Traber, 1999). Interestingly, patients with depression exhibit decreased plasma a- tocopherol (Maes et al., 2000, Owen et al., 2005), and treatment with a-tocopherol reduces between a-tocopherol and 5-HT-related disease. Tyramine is a trace amine that acts as a neurotransmitter and catecholamine-releasing agent. Particular bacteria can produce tyramine by decarboxylation of tyrosine in the gut, where tyramine is reported to stimulat fast ileal contractions and neuropeptide Y release(Marcobal et al., 2012). PABA is an intermediate of folic acid synthesis and essential nutrient for some bacteria. Particular species can generate PABA from chorismate(de Crecy-Lagard et al., 2007), but physiological roles for PABA in the GI tract are unclear. Subsets of microbes from Sp-and hSp-colonized mice relate phylogenetically to Clostridia with putative genes for a- tocopherol and tyrosine metabolism(Figures 6G and S7). Screening Sp microbes for target metabolic functions could serve as a tractable approach for further parsing the Sp consortium into the minimal species required for increasing 5-HT biosynthesis by ECs While there is increasing evidence for a bi-directional relationship between the gut microbiota and gut sensorimotor function, the particular microbes and mechanisms involved are unclear. The microbiota is required for normal IPAN excitability(Mc Vey Neufeld et al 2013), and recent studies reveal that changes in the microbiota can alter levels of neuroactive molecules. such as nitric oxide. substance P and endocannabinoids which have the potential to influence gut motor activity(Quigley, 2011). Mucosal immune responses ( Collins, 1996), including key interactions between macrophages and enteric neurons (Muller et al., 2014), also modulate gl motility via the gut microbiota. It will be interesting to determine whether 5-HT-mediated effects on immunity(Baganz and Blakely, 2013) Cell. Author manuscript; available in PMC 2016 April 09promote GI motility by activating TGR5 G-protein-coupled receptors on ECs (Alemi et al., 2013), which is consistent with our finding that Sp-induced metabolites raise 5-HT levels in ECs and that Sp colonization improves GI motility. Particular Clostridium species are known to possess high 7α-dehydroxylation activity required for the production of deoxycholate from cholate (Kitahara et al., 2001; Narushima et al., 2006), which is in line with our finding that Sp microbes, comprised largely of Clostridia, increase deoxycholate levels. Deoxycholate concentrations are substantially higher in the colon versus small intestine (Sayin et al., 2013), which, coupled to the finding that bacterial load and diversity is greater in the colon versus small intestine (Sekirov et al., 2010), could contribute to the regional specificity of microbiota-mediated increases in 5-HT synthesis to colonic ECs. Phylogenetic analysis of 16S rDNA sequences reveals that a subset of microbes recovered from Sp-colonized mice cluster taxonomically with known 7α- dehydroxylating Clostridia (Figures 6G and S7). Notably, there are striking phylogenetic commonalities between taxa identified in Sp- and hSp-colonized mice (Figure S7), consistent with their very similar luminal metabolomic profiles (Figure 6C) and ability to promote 5-HT synthesis from colonic ECs (Figure S3). We also reveal that the metabolites α-tocopherol, tyramine and PABA are elevated in feces by Sp. hSp or SPF colonization, co-vary with fecal 5-HT levels, and sufficiently induce 5- HT in vitro and in vivo (Figures 6 and S6 and Table S1). α-tocopherol is a naturally abundant form of vitamin E, with reported therapeutic effects for several diseases (Brigelius￾Flohe and Traber, 1999). Interestingly, patients with depression exhibit decreased plasma α- tocopherol (Maes et al., 2000; Owen et al., 2005), and treatment with α-tocopherol reduces depressive-like behavior in pre-clinical models (Lobato et al., 2010), suggesting a link between α-tocopherol and 5-HT-related disease. Tyramine is a trace amine that acts as a neurotransmitter and catecholamine-releasing agent. Particular bacteria can produce tyramine by decarboxylation of tyrosine in the gut, where tyramine is reported to stimulate fast ileal contractions and neuropeptide Y release (Marcobal et al., 2012). PABA is an intermediate of folic acid synthesis and essential nutrient for some bacteria. Particular species can generate PABA from chorismate (de Crecy-Lagard et al., 2007), but physiological roles for PABA in the GI tract are unclear. Subsets of microbes from Sp- and hSp-colonized mice relate phylogenetically to Clostridia with putative genes for α- tocopherol and tyrosine metabolism (Figures 6G and S7). Screening Sp microbes for target metabolic functions could serve as a tractable approach for further parsing the Sp consortium into the minimal species required for increasing 5-HT biosynthesis by ECs. While there is increasing evidence for a bi-directional relationship between the gut microbiota and gut sensorimotor function, the particular microbes and mechanisms involved are unclear. The microbiota is required for normal IPAN excitability (McVey Neufeld et al., 2013), and recent studies reveal that changes in the microbiota can alter levels of neuroactive molecules, such as nitric oxide, substance P and endocannabinoids, which have the potential to influence gut motor activity (Quigley, 2011). Mucosal immune responses (Collins, 1996), including key interactions between macrophages and enteric neurons (Muller et al., 2014), also modulate GI motility via the gut microbiota. It will be interesting to determine whether 5-HT-mediated effects on immunity (Baganz and Blakely, 2013) Yano et al. Page 10 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript