New technologies for studying microbes and antimicrobial mechanism of action are currently warranted due to the rise of antibiotic resistance. Thermal proteome profiling (TPP) is based on the principle that proteins become more resistant to heat-induced unfolding when bound to a ligand. Here, we used TPP in Escherichia coli to identify the mechanisms by which chemical and genetic perturbations affect it. We established TPP in E. coli and used it to identify the targets and mechanism of action of two potential new antibiotics: darobactin, a BamA inhibitor; and SCH79797, a molecule that inhibits folate metabolism and affects membrane integrity. We then profiled 121 knockout mutants to study the functional proteome of E. coli. This revealed that essential proteins are rarely regulated in their abundance, but commonly change in their thermal stability and thus their activity. We found that functionally associated proteins have coordinated abundance and thermal stability changes across mutants, which are a result of their co-regulation and physical interactions (with metabolites, co-factors or other proteins). This allowed us to suggest the function of uncharacterized proteins in a guilt-by-association manner. We further observed that enzyme thermal stability was correlated with metabolite levels, as measured by targeted metabolomics. Finally, our proteome measurements were able to explain the molecular mechanisms of growth phenotypes in different chemical and environmental perturbations. In conclusion, TPP provides a way to systematically phenotype the cell. This platform can be used to improve our understanding of basic bacterial biology by gaining insights into protein structure, protein complex formation, metabolic activity, and drug-protein interactions in microbial species.

Mapping the functional proteome landscape of bacteria with thermal proteome profiling

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ACS Fall 2022

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