GABA-modulating bacteria of the human gut microbiota

Abstract

The gut microbiota affects many important host functions, including the immune response and the nervous system¹. However, while substantial progress has been made in growing diverse microorganisms of the microbiota², 23-65% of species residing in the human gut remain uncultured^3,4, which is an obstacle for understanding their biological roles. A likely reason for this unculturability is the absence in artificial media of key growth factors that are provided by neighbouring bacteria in situ^5,6. In the present study, we used co-culture to isolate KLE1738, which required the presence of Bacteroides fragilis to grow. Bioassay-driven purification of B. fragilis supernatant led to the isolation of the growth factor, which, surprisingly, is the major inhibitory neurotransmitter GABA (γ-aminobutyric acid). GABA was the only tested nutrient that supported the growth of KLE1738, and a genome analysis supported a GABA-dependent metabolism mechanism. Using growth of KLE1738 as an indicator, we isolated a variety of GABA-producing bacteria, and found that Bacteroides ssp. produced large quantities of GABA. Genome-based metabolic modelling of the human gut microbiota revealed multiple genera with the predicted capability to produce or consume GABA. A transcriptome analysis of human stool samples from healthy individuals showed that GABA-producing pathways are actively expressed by Bacteroides, Parabacteroides and Escherichia species. By coupling 16S ribosmal RNA sequencing with functional magentic resonance imaging in patients with major depressive disorder, a disease associated with an altered GABA-mediated response, we found that the relative abundance levels of faecal Bacteroides are negatively correlated with brain signatures associated with depression.

Fig. 1.. Co-culture assay to isolate KLE1738.

(A) Diluted human fecal sample was plated on Fastidious Anaerobe Agar with 0.5% yeast extract (FAAy), and slower growing, smaller colonies (formed after 4–7 days -- “candidate dependent”, example in blue box) were tested for dependence on their faster growing, larger neighboring colonies (formed after 1–3 days -- “candidate helper”, example in green box) by co-culture. (B) To identify growth factors, the candidate dependent was also tested for growth promotion with Escherichia coli BW25113. Using this method we isolated KLE1738, which grew around the helper (C) Bacteroides fragilis KLE1758, but not Escherichia coli (D). Experiments describing dependency phenotypes were repeated in triplicate. (E) Phylogenetic tree of closely related type and representative genomes belonging to the Ruminococcaceae family. Tree assembled using Randomized Axelerated Maximum Likelihood in PATRIC. Parts of Figure modified from Fenn, 2017⁶; the culture plate and colonies highlighted in (A) is used for an illustration, and is not the source plate for KLE1738. A single stool sample yielded the KLE1738-KLE1758 helper-dependent pair.

Fig. 2.. In vitro and in silico identification of GABA modulating bacteria.

(A) To screen for GABA-producing bacteria, homogenized human stool sample was diluted and mixed with molten FAAy, with or without pH 7.0 MOPS buffer. KLE1738 was then spread on top of the agar and plates were incubated anaerobically for a week. Colonies that KLE1738 grew around were GABA producers (Inset). (B) Identified GABA producers were grown in liquid medium buffered at a pH of 5.0 and 7.0, and GABA levels of the spent medium was quantified using LC/MS and final pH of the medium was tested with pH strips. N=2 independent samples, and error is based on standard error. (C) 1,159 available gut genomes (consisting of 919 species) were analyzed for the genetic potential to produce and/or consume GABA (pathways associated with production or consumption highlighted in Supplemental Information Table 5). (D). The biochemical potential of 533 available gut-related metabolic models in KBase were examined for the capability to produce GABA or consume GABA. Shown are genera that represent at least 0.5% of the 533 models and contain either GABA producers or consumers.

Fig. 3.. Fecal Bacteroides relative abundance inversely correlates with functional connectivity between left DLPFC and DMN structures in patients with Major Depressive Disorder (MDD).

(A) 3-Dimensional plots of the medial surface of the left and right hemispheres in patients with MDD (n=23). Significant cluster of 387 voxels in which fecal Bacteroides relative abundance correlated inversely with functional connectivity between this cluster and the left DLPFC. Colorbar shows Z scores of beta weights of the Bacteroides relative abundance covariate of a multiple linear regression with functional connectivity as the dependent variable. (B) Scatter plot of the average functional connectivity (Z score) over a sphere of radius 5mm centered at the voxel of peak significance (+12, −57, 0 in MNI coordinates) and abundance of fecal Bacteroides (Pearson r = −0.67, p=.0005). n=23.