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Breakthrough in Understanding Bacterial Antibiotic Resistance

A team from Cornell University has made significant strides in understanding the mechanisms that allow bacteria to withstand antibiotic treatment. Their findings reveal a shuttling mechanism involving a complex of proteins that actively expels a wide variety of antibiotics, along with other substances, from bacterial cells.

Following the identification of this resistance mechanism, researchers are now exploring methods to disrupt it, potentially enhancing the effectiveness of antibiotics.

The project was spearheaded by Peng Chen, a professor of chemistry at Cornell. The co-lead authors include Wenyao Zhang, who earned a Ph.D. in 2023, and Christine Harper, a Ph.D. graduate of 2022.

Understanding Bacterial Resistance

Among the different types of bacteria, gram-negative species are notably more resistant to antibiotic treatment due to their additional protective membrane structure. These bacteria utilize a complex protein system known as MacAB-TolC, which facilitates the expulsion of harmful substances from the cell. This efflux system comprises three distinct proteins: TolC, located on the outer membrane; MacB, positioned on the inner membrane; and MacA, which is anchored in the periplasmic space.

This tripartite protein complex functions as a multidrug efflux pump, transporting not only antibiotics but also virulence factors—substances that enable bacteria to infect host organisms.

The Complex Assembly

For the efflux mechanism to function effectively, the proteins must assemble in a precise ratio: two MacB proteins for every six MacA proteins and three TolC proteins. While this ratio is well established, researchers have been curious about the entry point for molecules in the periplasm and how they interact with the assembled structure.

Chen’s research team employed single-molecule imaging techniques on Escherichia coli to gain insights into these protein dynamics. Their measurements revealed an unexpected imbalance in protein concentrations, with an excess of MacB proteins and even more TolC proteins than needed for the functional complex. They also observed that the adaptor protein MacA could separate from the assembly, challenging previous understandings of the system’s stability.

Significance of the Imbalance

Chen noted, “Having these extra MacB proteins without an accompanying MacA creates openings for substrates to access the channel. This allows substrates to bind to the surplus MacB proteins, prompting some MacA proteins to relocate and enable assembly.” This mechanism facilitates the export of various substances out of the cell.

Innovative Methodology to Disrupt Resistance

To verify whether this mechanism could be obstructed, Chen’s group partnered with researchers at the University of California, San Francisco. Under the guidance of former Cornell professor Christopher Hernandez, the team developed a microfluidic device that employs mechanical stress to alter a bacterium’s resistance to toxins.

Through experiments, the researchers determined that compressing E. coli within this device deformed the bacterial cell, disrupting the assembled complex and subsequently diminishing its resistance to antibiotics.

Future Directions in Research

Having established a correlation between the imbalanced protein stoichiometry and cellular function, Chen is eager to investigate its implications in other biological systems. “This type of stoichiometric imbalance is likely present in various protein complexes, but its functional significance is often overlooked,” he stated. “Our findings support the notion that a thorough analysis of protein ratios in different complexes is essential for understanding cellular function.”

The collaborative research involved co-authorship from Hernandez, postdoctoral researcher Junsung Lee, former postdoctoral researcher Bing Fu, and Malissa Ramsukh, a Cornell graduate. The study received funding from the National Institutes of Health, the Army Research Office, and the National Science Foundation.

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