Manipulation and quantification of the levels of Autoinducer-2 quorum sensing signal in the mouse gut

Quorum Sensing is a signalling mechanism used by bacteria to regulate gene expression as a function of population density, enabling them to engage in group behaviours. Autoinducer-2 (AI-2) is the most ubiquitously produced quorum sensing signal among bacterial species and it is unique in its capability of fostering cell-cell signalling across species from different phyla in multispecies communities. Alterations of AI-2 levels in the mouse gut can change the composition of the major gut microbiota phyla, but given the chemical instability of this signal, its quantification in intestinal samples and in vivo manipulation are challenging. Here, we optimised a Gas Chromatography - Mass Spectrometry (GC-MS) method to detect and quantify AI-2 in intestinal samples. Using a newly synthesised deuterated AI-2 molecule as the internal standard for AI-2 quantification, we quantified the levels of AI-2 in the cecum of conventionally raised mice with a complex microbiota. Moreover, we used a commensal Klebsiella sp. with probiotic potential to manipulate AI-2 levels in the mouse gut. We showed that mice colonised with mutants of this commensal Klebsiella sp. can be used to manipulate the levels of AI-2 in the mouse gut in both mono-colonised animals and those with a complex microbiota following recovery from antibiotic treatment. Overall, our results show that the analytical approach proposed here allows for robust and specific direct measurements of AI-2 in mouse caecal samples and can also be applied to other complex biological samples containing AI-2 aiming to study the role of quorum sensing signalling in microbiota communities.