Scientists finally solved how a common gut bacterium triggers colon cancer

Scientists have uncovered how a toxin produced by a common gut bacterium gains access to colon cells, solving a mystery that has puzzled researchers for more than 15 years. The discovery not only explains how the toxin begins damaging the colon, but also points to a possible new way to block its effects before they contribute to colorectal cancer.

The findings come from a multi-institutional team led by researchers at the Johns Hopkins Kimmel Cancer Center Bloomberg~Kimmel Institute for Cancer Immunotherapy and the Johns Hopkins University School of Medicine. Published in Nature, the study shows that the toxin, known as BFT and produced by Bacteroides fragilis, must first attach to a host protein called claudin-4 before it can injure colon cells. The research was supported in part by the National Institutes of Health.

"We've made several attempts over time to identify the receptor, so this is an exciting moment," says senior author Cynthia Sears, M.D., Bloomberg~Kimmel Professor of Cancer Immunotherapy and professor of medicine at Johns Hopkins. "Understanding how bacterial toxins work can open doors to new approaches for detection and therapy for associated diseases, including diarrhea, colorectal cancer and bloodstream infections."

Hidden Receptor Gives Gut Toxin Access to Colon Cells

The team's findings have already inspired a promising strategy to block the toxin. Researchers developed a molecular decoy that successfully intercepted BFT in animal models, preventing it from damaging the colon.

Bacteroides fragilis is found in up to 20% of healthy people, but certain strains can trigger inflammation in the colon and promote tumor growth. Earlier research from Sears' laboratory showed that BFT causes chronic inflammation by cutting E-cadherin, a protein that helps maintain the colon's protective barrier. That earlier Nature Medicine study also demonstrated that the toxin's activity drives colon tumor formation.

One major question remained unanswered. BFT did not appear to bind directly to E-cadherin, suggesting another molecule first helped the toxin gain access to its target.

CRISPR Screen Reveals the Missing Link

To identify that missing piece, Maxwell White, an M.D./Ph.D. candidate in the Sears lab, led a genomewide CRISPR screening effort in collaboration with the laboratory of Matthew Waldor at Harvard Medical School.

The researchers systematically disabled individual genes in colon epithelial cells to determine which ones were required for the toxin to work. One protein stood out immediately: claudin-4. When claudin-4 was removed, BFT could no longer attach to the cells, leaving E-cadherin unharmed.

"It took a while to get the assay working and validate the approach, but once we were able to do the screen, claudin-4 was a clear, resounding top hit," says White. "That was an exciting moment."

The discovery surprised the researchers. Sears says many scientists had expected the receptor to be a signaling protein, such as a G-coupled protein receptor, but claudin-4 belongs to a different class of proteins. A review of previous research also failed to uncover another toxin that behaves in the same way. Most protease toxins bind directly to the molecules they attack instead of first attaching to a separate receptor.

Scientists Confirm the Toxin's Molecular Target

To verify the interaction, the Johns Hopkins researchers teamed up with structural biologists F. Xavier Gomis-Rüth and Ulrich Eckhard at the Molecular Biology Institute of Barcelona.

Using biophysical techniques, White and the Barcelona team showed that BFT and claudin-4 form a tightly bound one-to-one complex in laboratory experiments. This provided the first direct physical evidence that the toxin attaches to the receptor before damaging colon cells.

The researchers then tested their findings in living systems through a collaboration with the laboratory of Min Dong at Harvard Medical School. Working with Kang Wang and colleagues, they examined how the toxin behaved in mouse models.

Molecular Decoy Protects Mice From Gut Toxin

The team created a soluble version of claudin-4 that acted as a decoy by displaying portions of the receptor normally recognized by the toxin. Rather than binding to colon cells, BFT attached to the decoy proteins instead.

This strategy successfully protected mice from BFT-induced colon damage.

"This approach could be iterated upon with small molecules or other biologics that have better pharmacological properties," says White. The team is now investigating which types of therapies may be most effective at blocking the toxin.

Questions Still Remain

Although the researchers identified the receptor and demonstrated that it binds tightly to BFT, one important challenge remains unresolved. They have not yet captured the precise experimental structure showing exactly how the toxin and claudin-4 fit together.

Current artificial intelligence modeling tools, including AlphaFold, were unable to fully resolve the interaction.

Additional authors on the paper include Jason Chen, Shaoguang Wu, Abby L. Geis and Jessica Queen at Johns Hopkins and Hailong Zhang, Karthik Hullahalli and Jie Zhang at Harvard Medical School.

The research was supported 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.

Sears receives royalties for writing and reviewing for UpToDate. This arrangement is managed by The Johns Hopkins University in accordance with its conflict-of-interest policies.