From in silico screening to in vivo validation in zebrafish – a framework for reeling in the right psychobiotics†

The potential of gut bacteria to interact with the nervous system is now well known. Therefore, the characterization of bacterial strains that can modulate signalling pathways of the nervous system is a topic of growing interest, as it represents a potential alternative therapeutic target to treat central nervous system disorders. However, a streamlined screening framework is required to guide the rational identification and selection of such bacteria, known as psychobiotics. In this work, we introduce a framework that integrates in silico, in vitro and in vivo approaches to identify psychobiotic candidates capable of both metabolising prebiotics of interest and producing neuroactive molecules. To prove the effectiveness of the approach, we characterized a bacterial strain, Lactiplantibacillus plantarum APC2688, for its capacity to modulate the GABAergic system and alter the stress-related behaviour of zebrafish larvae. In brief, in silico analyses of the genomic content of APC2688 identified it as capable of degrading different prebiotics and producing neuroactive compounds known to modulate the stress response in animal models. Then, in vitro results confirmed the ability of this strain to produce GABA, tryptophan and acetate, while growing with the candidate prebiotics of interest, fructooligosaccharides (FOS), galactooligosaccharides (GOS) and inositol. In vivo experiments demonstrated that the administration of bacterial supernatants induced changes in the expression of gad1 and gabra1 in zebrafish larvae, two essential genes in the GABAergic signalling pathway, and altered the anxiety-like behaviour of the larvae. These results highlight the efficiency of our framework in integrating orthogonal approaches to discover and characterise bacteria capable of modulating the microbiome–gut–brain axis.