GoKawiilBacterial growth and generation of in vitro bacterial metabolite libraries
Based on phylogenetic diversity, 100 bacterial strains were picked from the HMP bacterial culture collection and our laboratory’s internal human gut bacterial culture collections (Supplementary Table 1). Anaerobic bacteria were cultured in a vinyl anaerobic chamber (Coy Laboratory Products), containing 4% H 2 , 10% CO 2 and 86% N 2 . Aerobic bacteria were cultured in an aerobic microbiological incubator. All bacteria were cultured in triplicate at 37 °C for 48 h. To extract bacterial metabolites with an acetonitrile-methanol-water (2:2:1) mixture (ACN-MeOH-water), the bacterial cultures were dried overnight at room temperature under vacuum (GeneVac). Then, the dried matter was resuspended in ACN-MeOH-water with intermittent sonication. The suspensions were centrifuged at 4,000g for 10 min, and the supernatant was dried overnight at room temperature under a vacuum (GeneVac). The dried extracts were dissolved in sample buffer (PBS containing 10% DMSO, v/v). To extract bacterial metabolites with butanol or ethyl acetate, the bacterial cultures were mixed with two volumes of butanol or ethyl acetate and shaken vigorously for 10 min in a horizontal shaker at room temperature. The organic phase was collected and dried overnight at room temperature under a vacuum (GeneVac), and the dried extracts were dissolved in sample buffer. The three types of extracts comprise the bacterial in vitro metabolome libraries.
Mouse experiments
All animal protocols were approved by the Yale University Institutional Animal Care and Use Committee (IACUC Protocol 11513). Mice were on a dark–light cycle of 12 h–12 h with temperature (22 °C) and humidity (50%) controlled.
Gnotobiotic mice and generation of in vivo bacterial metabolite libraries
GF C57BL/6 mice were maintained in flexible film isolators (Class Biologically Clean) with all bedding, chow (Teklad, 2018S) and water autoclaved before import. All GF breeding isolators were regularly monitored for bacteria (culture-dependent and -independent techniques). For the monoassociation gnotobiotic experiment, male GF mice (aged 6 to 8 weeks) were transferred from the isolators to positive pressure ventilated microisolator cages (Tecniplast, ISO72P) and gavaged with 0.2 ml of bacterial culture. Bacterial inocula from all 100 strains were freshly prepared and aliquoted in 2 ml screw cap tubes (Sarstedt, 72.693.005). After 2 weeks of colonization, the caecal contents were collected after euthanization. To prepare the bacteria in vivo metabolome libraries, the caecal contents were resuspended using ACN-MeOH-water solvent. After vigorously shaking in the horizontal shaker at room temperature, the supernatant was collected after centrifuging at 4,000g for 10 min. The supernatant was divided equally into three parts. They were directly dried overnight at room temperature under a vacuum, and the dried extracts were resuspended in sample buffer. One of them was saved as ACN-MeOH-water extracted metabolomes. The other two parts were further extracted using either butanol or ethyl acetate, respectively, followed by drying and dissolving using the same method described for preparing the in vitro metabolome libraries. These three types of extracts comprise the bacteria in vivo metabolome libraries.
Chemical supplementation in mouse drinking water
To support B. breve WT (NWP289) and Δchat colonization and ACh production, mice in these experiments received drinking water supplemented with 1.5% (w/v) HMOs (LayerOrigin SuperHMO Prebiotic Mix) and 0.25% choline (Sigma-Aldrich, C1879). To enhance the potential impacts of commensal ACh, the AChEi rivastigmine (TCI America, R0093) was supplemented in the mouse drinking water (40 mg l−1). Atropine (Cayman Chemical, 12008) and mecamylamine (Cayman Chemical, 14602) were provided in the drinking water at 25 mg l−1. Water was replenished weekly.
Serum sample preparation
To prepare serum samples, blood was collected in serum tubes (SAI Infusion Technology, PMTS-SG-1.1). After centrifugation at 4,000g for 5 min, serum samples were collected and stored at −80 °C until use. To collect brain and liver samples, mice were perfused with 10 ml of ice-cold PBS after euthanization. The liver and brain samples were then collected, flash–frozen and stored at −80 °C until use.
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