For years, copper has been used ubiquitously in health care to stave off bacterial infections—from doorknobs in hospital rooms to catheters and IUDs—and yet how the element does so is unclear. In a new publication in Molecular Microbiology, researchers from the University of Illinois Urbana-Champaign School of Molecular & Cellular Biology have shed new light on how copper poisons microbes.

The use of copper as an antibacterial agent dates back to ancient civilizations, with the oldest known reference in Egyptian medical texts written between 2600 and 2200 B.C. Copper is also deployed by our immune system to fight off pathogens and nowadays, copper mugs are a popular option for a vodka cocktail known as a mule. Despite its historical significance, researchers have not yet identified all the cellular targets that copper can disrupt. 

“We can see a growth defect in Escherichia coli with low microbial doses of copper,” said first author Stefanie Eben, a PhD candidate in microbiology in Dr. Jim Imlay's lab. “That fact is exploited for a lot of antimicrobial applications. But microbes have evolved defense mechanisms to protect themselves against such doses.” 

Clearly microbes consider copper to be a threat, as evidenced by their efflux pumps that remove copper from the cell. Indeed, previous studies from the Imlay lab have shown that copper can damage dehydratases, which play key roles in metabolism. Yet, the bacteria still struggle when they are grown in media that do not require these enzymes, indicating that additional targets exist.

“Our hypothesis was that copper poisons cells by causing adventitious disulfide bonds in cytoplasmic proteins,” Eben explained. “The reason we thought it does is because copper likes to stick to thiols, which contain sulfur, and it’s also redox active, meaning it carries out chemical reactions.” 

To test this hypothesis, Eben overloaded the cells with copper. She employed copper efflux mutants, which are incapable of removing copper from their cells. Using alkaline phosphatase (AP) as a sensitive proxy of disulfide bond formation, Eben was able to test the effects of copper. The enzyme requires disulfide bonds to become activated, a reaction that copper can help with. She discovered that copper was unable to oxidize the AP when it was located in the cytoplasm, but it was still able to do so when the AP cysteine residues were in vitro and in the E. coli periplasm. 

Eben was surprised by the results. At first, the lab questioned whether they had sufficiently overloaded the cells with copper. They then tested whether other cytoplasmic copper targets are damaged by the concentrations used previously and indeed copper damaged the same dehydratases that were previously investigated. 


Stefanie Eben holds up a Petri dish. The dish is in soft focus, with Stefanie in sharp focus behind it.
Stefanie Eben, PhD candidate in microbiology.

“The big takeaway is that the cytoplasm has around 40 mM thiols and around 30 µM of copper. That is a big difference; in order for copper to oxidize a significant fraction of the protein thiols, it needs to be a rapid catalyst,” Eben said. “However, my data has shown that this is not the case and thiol oxidation is inefficient.”

They have ruled out cytoplasmic thiols as a potential copper target; however, copper can still impact the few thiol-containing proteins in the periplasm. Looking ahead, Eben said she and other researchers in the Imlay Lab are interested in exploring a similar hypothesis with other chemicals that can interact with cytoplasmic thiol such as hydrogen peroxide and iron, which have been the focus of the Imlay lab for several years. Eben said she is eager to see where the research goes next, and she has enjoyed diving into her investigation of copper’s interactions with microbes. 

“It’s really cool when the project finally comes together and you find something that might interest other people, or when you realize that even with small findings you can bring the entire field forward,” Eben enthused.

Photo credit: Fred Zwicky, University of Illinois Urbana-Champaign

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