Bacterial Peptidoglycan Serves as a Critical Modulator of the Gut-Immune-Brain Axis in Drosophila.

Metabolites released by gut-associated bacteria can modulate many physiological processes in the host, including its behavior. By employing a model of oral bacterial infection, we recently uncovered that bacterial peptidoglycan (PGN) influences fly female egg-laying behavior by activating the NF-kB cascade in a subset of brain neurons. While shedding light on PGN as a potential mediator of communication between the microbiota and the brain in Drosophila, these findings prompt us to study the impact that PGN can exert on all brain cells. Using high-resolution mass spectrometry, we demonstrate that the PGN released by gut-associated bacteria can rapidly be detected in the central nervous system (CNS), hence demonstrating that following translocation from the gut to the hemolymph, the insect blood, it can be directly perceived by brain cells. By combining whole-genome transcriptome analyses, comprehensive genetic assays and reporter gene systems, we show that bacterial infection primarily elicits a PGN dose-dependent NF-kB immune response in glial cells forming the outer cell layer of the blood-brain barrier. Overall, our findings establish that gut-derived PGN is a critical mediator of the gut-brain axis in Drosophila. In addition to triggering behavioral changes, sensing of circulating PGN by the CNS also induces a local immune response of the BBB. We have gathered evidence indicating that peptidoglycan (PGN) originating from the microbiota can access the Drosophila brain, thereby influencing the transcription of specific genes. To investigate this phenomenon, we subjected adult female Drosophila to a 24-hour diet comprising either bacteria or sucrose (as a control condition). Subsequently, we dissected the brains to compare the gene expression profiles under both dietary conditions. This experimental protocol was conducted on both wild-type animals and a mutant strain known as PGRP-LB, which encodes a PGN-degrading enzyme. In the PGRP-LB mutant, the levels of microbiota-derived PGN are elevated in the fly body cavity. Each biological condition will be replicated three times to ensure robustness and reliability of the results