For nearly two decades, scientists have known that a common gut bacteria can produce a toxin that damages the colon and contributes to tumor formation. What they couldn’t explain is how the toxin first attaches to intestinal cells and begins its attack. Now researchers have discovered this missing step, revealing a surprising vulnerability that could lead to new ways to protect the colon.
The breakthrough comes from a team led by scientists at the Johns Hopkins Kimmel Cancer Center, its Bloomberg~Kimmel Institute for Cancer Immunotherapy and the Johns Hopkins University School of Medicine. Their findings, published in Natureidentify a protein called claudin-4 as an essential docking point for a bacterial toxin known as BFT.
Even more promising, researchers have already used this discovery to develop a molecular decoy that intercepts the toxin before it can damage cells in the colon. The strategy successfully protected mice from the harmful effects of the toxin, suggesting a possible new approach to preventing bacterial damage associated with colorectal cancer. The research received partial support from the National Institutes of Health.
“We’ve made several attempts over time to identify the receptor, so this is an exciting moment,” says lead author Cynthia Sears, MD, Bloomberg~Kimmel Professor of Cancer Immunotherapy and professor of medicine at Johns Hopkins. “Understanding how bacterial toxins work may pave the way for new approaches to detecting and treating associated diseases, including diarrhea, colorectal cancer and bloodstream infections.”
How a common gut bacteria may promote colon cancer
The investigation focuses on Bacteroides fragilisa bacteria found in the intestines of up to 20% of healthy people. Although the presence of this bacteria does not automatically mean a person will develop cancer, some strains produce a powerful toxin that can disrupt the protective lining of the colon, causing inflammation and promoting tumor growth.
The toxin, known as B. fragile toxin or bluefin tuna, has been under investigation for years. In a landmark 2009 study published in Natural medicineSears and his colleagues demonstrated that the toxin could promote the development of colon tumors. Their work established an important link between toxin-producing gut bacteria, chronic inflammation and colorectal cancer.
Previous experiments also found that BFT damages a protein called E-cadherin, which helps hold neighboring cells together and maintain the colon’s protective barrier. When this protein is broken, the barrier is compromised, potentially allowing harmful substances and bacteria to interfere with surrounding tissues.
However, a crucial question remained unanswered. Although BFT can break down E-cadherin, the toxin does not appear to attach directly to this protein. Researchers suspected another molecule helped the toxin reach its target, but repeated attempts to identify it failed.
Scientists identify the missing link using CRISPR
To solve the mystery, researchers turned to CRISPR, a powerful gene-editing technology that allows scientists to selectively turn off genes and study what they do.
Maxwell White, MD/Ph.D. candidate working in the Sears lab, led a genome-wide CRISPR screening effort in collaboration with Matthew Waldor’s lab at Harvard Medical School. The team systematically turned off genes in colon epithelial cells, the cells that form the inner lining of the colon, to determine which ones were necessary for the toxin to attach and cause damage.
One gene stood out dramatically. It produces claudin-4, a protein that helps neighboring cells form tight junctions, seals that restrict the movement of substances between cells and help maintain the intestinal barrier.
When the researchers turned off the gene responsible for claudin-4, BFT was no longer able to bind to cells in the colon. Without this initial attachment, the toxin was unable to attack E-cadherin.
The discovery revealed that claudin-4 acts as a bacterial toxin receptor. Indeed, the toxin must first attach to this protein before it can exert its harmful activity.
“It took a while to get the test running and validate the approach, but once we were able to do the screening, claudin-4 was a clear and resounding success,” White says. “It was an exciting moment.”
Unexpected discovery reveals new bacterial attack strategy
The identity of the receptor surprised scientists.
Sears and other researchers expected that BFT would interact with a protein involved in cell signaling, such as a G protein-coupled receptor. These receptors act somewhat like communications switches, allowing cells to respond to signals from their environment.
Claudin-4, however, serves a very different purpose. Rather than functioning primarily as a signaling receptor, it helps maintain physical connections between neighboring cells.
The discovery also revealed an unusual mode of action for a bacterial toxin. BFT belongs to a class of enzymes called proteases, which cut other proteins. Most proteases act directly on their targets, but BFT appears to require a distinct receptor before it can reach and damage E-cadherin.
When the team reviewed previous scientific research, they could not identify another toxin known to use the same mechanism.
To establish that BFT and claudin-4 truly bind, the Johns Hopkins researchers collaborated with structural biologists F. Xavier Gomis-Rüth and Ulrich Eckhard of the Barcelona Institute of Molecular Biology.
Using techniques that measure how the molecules interact, White and the Barcelona team showed that BFT and claudin-4 form a tightly bound complex in laboratory experiments. Each toxin molecule is associated with a receptor molecule, providing direct physical evidence of the connection.
Clever molecular decoy blocks dangerous gut toxin
Identifying the receptor did more than explain how the toxin worked. This also gave scientists an idea to stop it.
If BFT requires claudin-4 to attach to colon cells, perhaps a substitute could move the toxin away from its target.
To test this possibility, the team developed a molecular decoy. It was a soluble protein carrying claudin-4 sequences, designed to attract bluefin tuna without allowing it to attack the protective lining of the colon.
The researchers then worked with Min Dong’s lab at Harvard Medical School, including Kang Wang and colleagues, to study this approach in mice. These experiments allowed scientists to examine how the toxin behaved in the more complex environment of a living gut.
The results were encouraging. Instead of binding to claudin-4 receptors on colon cells, BFT attached itself to decoy proteins. By intercepting the toxin, the decoys successfully protected the mice from BFT-induced damage.
The results suggest it might be possible to interfere with the first step of the toxin rather than trying to repair the damage after it has occurred.
“This approach could be repeated with small molecules or other biologics with better pharmacological properties,” says White. The team is currently exploring which molecular approaches might be most effective in blocking the toxin.
Small molecules are compounds that can sometimes be made into drugs, while biologics are treatments made from or involving biological materials, such as proteins. Future versions of the decoy strategy could potentially be designed to work more effectively in the body or be easier to administer.
What this discovery could mean for colon cancer prevention
Although the results identify a promising new target, important questions remain.
Scientists have established that BFT attaches to claudin-4 and that blocking this interaction can protect mice from toxin-induced damage. However, they have not yet captured the exact experimental three-dimensional structure showing how the two molecules fit together.
Even advanced artificial intelligence tools, including AlphaFold, have failed to completely resolve the interaction. Understanding its precise structure could help researchers design more effective compounds that prevent the toxin from binding.
Further experiments will also be needed to determine whether the decoy approach can safely and effectively prevent toxin-related illnesses in humans. Mouse experiments demonstrated protection against tissue damage, but they did not establish that the strategy prevents colorectal cancer.
Nonetheless, this discovery resolves a major question that has persisted since the link between BFT and colon tumor formation was established in 2009. By identifying the toxin’s previously unknown point of attachment, researchers have discovered a potential way to interrupt a harmful biological process before it begins.
The results could ultimately support new strategies for detecting or treating conditions associated with bacterial toxins, including colorectal cancer, diarrhea and bloodstream infections.
Study authors, funding and disclosures
Other researchers who contributed to the study include Jason Chen, Shaoguang Wu, Abby L. Geis and Jessica Queen of Johns Hopkins, as well as Hailong Zhang, Karthik Hullahalli and Jie Zhang of Harvard Medical School.
Funding was provided by the Bloomberg ~ Kimmel Institute for Cancer Immunotherapy, Janssen Research and Development, Cancer Research UK, the National Institutes of Health (grant numbers R01 AI042347, R01 NS080833, R01 NS117626, R01 AI170835, and R01 AI189789), and the Howard Hughes Medical Institute.
Gn Health
