Spatial metabolomics of in situ host–microbe interactions at the micrometre scale

Spatial metabolomics describes the location and chemistry of small molecules involved in metabolic phenotypes, defence molecules and chemical interactions in natural communities. Most current techniques are unable to spatially link the genotype and metabolic phenotype of microorganisms in situ at a scale relevant to microbial interactions. Here, we present a spatial metabolomics pipeline (metaFISH) that combines fluorescence in situ hybridization (FISH) microscopy and high-resolution atmospheric-pressure matrix-assisted laser desorption/ionization mass spectrometry to image host–microbe symbioses and their metabolic interactions. The metaFISH pipeline aligns and integrates metabolite and fluorescent images at the micrometre scale to provide a spatial assignment of host and symbiont metabolites on the same tissue section. To illustrate the advantages of metaFISH, we mapped the spatial metabolome of a deep-sea mussel and its intracellular symbiotic bacteria at the scale of individual epithelial host cells. Our analytical pipeline revealed metabolic adaptations of the epithelial cells to the intracellular symbionts and variation in metabolic phenotypes within a single symbiont 16S rRNA phylotype, and enabled the discovery of specialized metabolites from the host–microbe interface. metaFISH provides a culture-independent approach to link metabolic phenotypes to community members in situ and is a powerful tool for microbiologists across fields. This work combines mass spectrometry imaging at high resolution with FISH for the visualization and identification of microorganisms. The authors develop a sample preparation and imaging pipeline called metaFISH to colocalize metabolite patterns with community members and apply it to a host–microbe symbiosis (mussel and its symbionts) to identify symbiosis-specific metabolites.