Tiny terrors: Researcher works to uncover how bacteria develop antibiotic resistance through efflux

by Susan Szuch

There are many ways that bacteria can develop antibiotic resistance—through acquiring resistance from another bacterium, or through the function of protein complexes known as efflux pump that expel antibiotics out of the bacterial cell.

These protein structures are extremely small—if the period at the end of this sentence is one millimeter, then 100,000 pumps will fit inside the period, according to Fatemeh Khalili-Araghi, assistant professor in physics at the University of Illinois at Chicago. Khalili-Araghi is working to understand how the efflux pumps function in gram-negative bacteria like E. coli.

“It is a fascinating protein complex. I have been studying membrane proteins and pumps for the past several years, all at the individual protein level,” Khalili-Araghi says. “We know a lot about the function of its individual proteins or domains in the complex structure, but there is little known about the function of the complex as a whole and how these domains are structurally coupled together.”

The research focuses solely on the way that efflux pumps function.

“We wanted to get the full picture and study how this complex structure transports the drugs out of bacteria to understand one of the mechanisms by which bacteria become resistant to antibiotics,” Khalili-Araghi says.

Khalili-Araghi is using the Blue Waters supercomputer at the National Center for Supercomputing Applications at the University of Illinois at Urbana-Champaign to run multiple simulations at the same time, something that she wouldn’t be able to do on other computers.

“We had to have many, many simulations running at the same time in parallel,” Khalili-Araghi says. “If you were going to do that in any other machine, you would have to run them in series, which would mean that we’d have to wait forever for them to finish.”

While previous research was present that allowed researchers to have access to the atomic structure of individual domains, or the way that atoms are arranged, the atomic structure of the entire complex was not known except through very low-resolution electron microscopy images. Using the low-resolution images of the complex and molecular dynamics simulation, Khalili-Araghi was able to put together a high quality map of the system as a whole with atomic resolution.

In contrast to other membrane systems, the protein complex or efflux pumps used by bacteria to expel unwanted material are complex, according to Khalili-Araghi. They span the bacterial membrane by having a channel in the outer membrane and a proton pump in the inner membrane of bacteria that are connected by a fusion protein in the middle. The fusion protein creates a tunnel between the inner and outer membrane proteins. The inner membrane pump uses the proton gradient between the two sides of the membrane to actively transport the drugs out of the cell and into the tunnel, which in turn transports the drugs to the outer membrane channel. The channel will then expel the drugs out of the cell if it is open.

Their results so far have shown that in contrast to the existing model, the fusion protein is the only connection between the inner membrane pump and outer membrane channel. As such, the fusion protein plays an active role in transferring conformational changes of the inner pump to the outer membrane that results in opening of the channel to the extracellular side.

Khalili-Araghi and her team are studying the transport pathway of two antibiotics to understand how the shape of the molecule affects how it is transported, seeing as the pump is not very selective and “allow the majority of these compounds that look like these molecules to go through them,” says Khalili-Araghi.

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