GABA Production by Human Intestinal Bacteroides spp.: Prevalence, Regulation, and Role in Acid Stress Tolerance

Abstract

The high neuroactive potential of metabolites produced by gut microbes has gained traction over the last few years, with metagenomic-based studies suggesting an important role of microbiota-derived γ-aminobutyric acid (GABA) in modulating mental health. Emerging evidence has revealed the presence of the glutamate decarboxylase (GAD)-encoding gene, a key enzyme to produce GABA, in the prominent human intestinal genus Bacteroides. Here, we investigated GABA production by Bacteroides in culture and metabolic assays combined with comparative genomics and phylogenetics. A total of 961 Bacteroides genomes were analyzed in silico and 17 metabolically and genetically diverse human intestinal isolates representing 11 species were screened in vitro. Using the model organism Bacteroides thetaiotaomicron DSM 2079, we determined GABA production kinetics, its impact on milieu pH, and we assessed its role in mitigating acid-induced cellular damage. We showed that the GAD-system consists of at least four highly conserved genes encoding a GAD, a glutaminase, a glutamate/GABA antiporter, and a potassium channel. We demonstrated a high prevalence of the GAD-system among Bacteroides with 90% of all Bacteroides genomes (96% in human gut isolates only) harboring all genes of the GAD-system and 16 intestinal Bacteroides strains producing GABA in vitro (ranging from 0.09 to 60.84 mM). We identified glutamate and glutamine as precursors of GABA production, showed that the production is regulated by pH, and that the GAD-system acts as a protective mechanism against acid stress in Bacteroides, mitigating cell death and preserving metabolic activity. Our data also indicate that the GAD-system might represent the only amino acid-dependent acid tolerance system in Bacteroides. Altogether, our results suggest an important contribution of Bacteroides in the regulation of the GABAergic system in the human gut.

Keywords: Bacteroides; GABA; acid stress tolerance; glutamate decarboxylase; gut microbiota.

Figure 1

γ-Aminobutyric acid (GABA) production by intestinal Bacteroides strains in the presence of two different glutamate concentrations. Bacteroides strains were incubated anaerobically at 37°C in mYCFA (8.38 ± 0.27 mM glutamate) and mYCFA-Glu (69.49 ± 1.10 mM glutamate) for 48 h (n = 3). GABA quantified in the culture supernatant is displayed. Parabacteroides and Eubacterium strains were screened identically. Dots represent replicates; bars represent SD. GABA detection limit: 0.03 mM. n.d., not detected.

Figure 2

Genetic characteristics of the glutamate decarboxylase (GAD)-system in Bacteroides strains. (A) Genetic organization of the GAD-system genes (i.e., glutamate decarboxylase, glutaminase, glutamate/GABA antiporter, and potassium channel) in Bacteroides strains tested in vitro compared to distinct GABA-producing taxa. (B) Phylogenetic trees based on GAD-system genes sequences. Trees were generated using the Maximum Likelihood method and the Jukes-Cantor correction model with 1,000 bootstraps. The strains screened in vitro are in bold.

Figure 3

Prevalence of GAD-system genes in 961 Bacteroides genomes and association with species, host, and source of isolation. The phylogenomic tree was generated using 90 single-copy genes (SCG) specific to Bacteroidetes, and metadata were visualized via GToTree. Eubacterium limosum DSM 20543 genome was used to root the tree. All data are available in Supplementary Data File 3.

Figure 4

GABA production kinetic in the presence or absence of glutamine and glutamate in minimal medium (MM). Growth curves (log-scale) of Bacteroides thetaiotaomicron DSM 2079 cultivated in (A) MM, (B) MM-Glu, and (C) MM-Gln (n = 3). Changes in glucose, GABA, glutamine, and glutamate concentrations for 72 h are displayed. (D) Change in pH associated with B. thetaiotaomicron DSM 2079 growth for 72 h in MM, MM-Glu, and MM-Gln. Dots represent replicates; bars represent SD. Significances were calculated by one-way ANOVA test, including Tukey’s test: ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001. Data on metabolic acid production and absolute pH are available in Supplementary Figure S6.

Figure 5

Effect of acid stress on GABA production, pH modulation, and cell viability in B. thetaiotaomicron DSM 2079. B. thetaiotaomicron DSM 2079 cells were incubated in MM, MM-Glu, or MM-Gln at pH 6.3, 5.5, 4.1, or 3.1 for 1 h (n = 3). (A) Concentration of GABA, (B) change in pH, and (C) viability are displayed. Dots represent replicates; bars represent SD. Significances were calculated by one-way ANOVA test, including Tukey’s test: ^****p < 0.0001. Data on glucose consumption and metabolic acid production are available in Supplementary Table S3.