Thermal proteome profiling in bacteria: probing protein state in vivo

Increasing antibiotic resistance urges for new technologies for studying microbes and antimicrobial mechanism of action. We adapted thermal proteome profiling (TPP) to probe the thermostability of Escherichia coli proteins in vivo. E. coli had a more thermostable proteome than human cells, with protein thermostability depending on subcellular location-forming a high-to-low gradient from the cell surface to the cytoplasm. While subunits of protein complexes residing in one compartment melted similarly, protein complexes spanning compartments often had their subunits melting in a location-wise manner. Monitoring the E. coli meltome and proteome at different growth phases captured changes in metabolism. Cells lacking TolC, a component of multiple efflux pumps, exhibited major physiological changes, including differential thermostability and levels of its interaction partners, signaling cascades, and periplasmic quality control. Finally, we combined in vitro and in vivo TPP to identify targets of known antimicrobial drugs and to map their downstream effects. In conclusion, we demonstrate that TPP can be used in bacteria to probe protein complex architecture, metabolic pathways, and intracellular drug target engagement.