Thermal Proteome Profiling Adapted To Bacterial Research

An existing technique has been adapted to study the melting behaviour of proteins so that it can be used for the study of bacteria.

Thermal proteome profiling (TPP) was developed in 2014 (Savitski et al., Science 2014) and enables scientists to compare the melting behavior of all proteins in a cell or organism before and after a perturbation, such as the administration of a drug. By adapting TPP to bacteria, it can now be used to study the activity and architecture of most proteins in a bacterial cell while it’s alive.

With antibiotic resistance spreading worldwide, there is a strong need for new technologies to study bacteria. André Mateus, a postdoc working in the Savitski and Typas groups at European Molecular Biology Laboratory (EMBL), led the study.

Bacteria Take The Heat

While human bodies cease to function at temperatures above 42°C, E. coli bacteria still grow regularly up to 45°C.

“We discovered that proteins in the middle of a bacterial cell are less tolerant to heat than those at the cell surface. Surprisingly, a protein’s location is more predictive for its melting behavior than which other proteins it interacts with,”

said Mikhail Savitski.

With thermal proteome profiling, researchers can also investigate the effects of drugs on bacteria. Protein-drug interactions typically increase the proteins’ heat tolerance, resulting in higher melting points.

Therefore, comparing the heat tolerance of drug-treated and untreated bacterial cells helps to identify targets of antimicrobial drugs, but also to decipher how the bacterial cell succumbs to the drug or tries to bypass its action.

Drug Resistance

The researchers found that subunits of protein complexes located in a single compartment melt similarly, while protein complexes spanning compartments often have their subunits melting in a location‐wise manner.

“In one particular case, we were able to elucidate a novel drug resistance mechanism,” says André Mateus. “Cells use proteins to pump antibiotics out of the cell. After genetically removing one such efflux pump from their chromosome, bacteria became more sensitive to many drugs, but curiously more resistant to one specific antibiotic called aztreonam. Using TPP, we found that this was due to dramatically reduced levels of a specific porin – a protein that acts as a pore – used by aztreonam to enter the cell.”

Thermal proteome profiling in Escherichia coli

Thermal proteome profiling in Escherichia coli
Credit: André Mateus, et al CC-BY

Compared to other techniques, TPP allows scientists to investigate the effects of perturbations on thousands of individual proteins in a short timeframe. Most of the obtained insights – like the changes in the activity of proteins in vivo – would be impossible with other conventional techniques and for so many proteins simultaneously, showing TPP’s potential to study bacteria in detail.

“TPP in bacteria provides a powerful tool to understand protein–protein, protein–metabolite, and protein–drug interactions. Obtaining protein abundance and stability levels in a single measurement offers an informative global view of the cell, particularly when probed in distinct chemical, environmental, or genetic perturbations,”

the authors conclude.

André Mateus, Jacob Bobonis, Nils Kurzawa, Frank Stein, Dominic Helm, Johannes Hevler, Athanasios Typas, View ORCID ProfileMikhail M Savitski
Thermal proteome profiling in bacteria: probing protein state in vivo
Molecular Systems Biology (2018) 14, e8242

Top Image: Aleksandra Krolik / EMBL