Scientists have revealed how toxins produced by common gut bacteria enter colon cells, solving a mystery that has puzzled researchers for more than 15 years. The discovery not only explains how toxins begin to damage the colon, but also suggests new ways to block their effects before they cause colorectal cancer.
The study results come from a multicenter team led by researchers from the Johns Hopkins Kimmel Cancer Center, the Bloomberg Kimmel Institute for Cancer Immunotherapy, and the Johns Hopkins University School of Medicine. Published in natureresearch has shown that BFT is a toxin produced by. Bacteroides fragilismust first bind to a host protein called claudin-4 before damaging colon cells. This research was supported in part by the National Institutes of Health.
“This is an exciting moment because we’ve made many attempts over time to identify the receptor,” says Cynthia Sears, MD, Bloomberg Kimmel Professor of Cancer Immunotherapy and senior author at Johns Hopkins University School of Medicine. “Understanding how bacterial toxins work could open the door to new approaches for the detection and treatment of related diseases such as diarrhea, colorectal cancer, and bloodstream infections.”
Hidden receptors allow enterotoxin access to colon cells
The research team’s findings have already led to promising strategies for blocking toxins. Researchers have developed a molecular decoy that successfully blocks bluefin tuna and prevents damage to the colon in animal models.
Bacteroides fragilis Although present in up to 20% of healthy people, certain strains can cause inflammation in the colon and promote tumor growth. Previous research from Sears’ lab has shown that BFT causes chronic inflammation by cleaving E-cadherin, a protein that helps maintain the colon’s protective barrier. What happened just now natural medicine Studies also demonstrated that the toxin’s activity promoted colon tumor formation.
One big question remained unanswered. BFT does not appear to bind directly to E-cadherin, suggesting that another molecule helped the toxin reach its target in the first place.
CRISPR screen reveals missing links
To identify that missing piece, Maxwell White, MD/PhD, a candidate in the Sears lab, led a genome-wide CRISPR screening effort in collaboration with Matthew Waldo’s lab at Harvard Medical School.
The researchers systematically disabled individual genes in colonic epithelial cells to determine which genes are required for the toxin to work. One protein immediately stood out: claudin-4. When claudin-4 was removed, BFT was unable to attach to cells and E-cadherin remained intact.
“It took some time to get the assay working and validate the approach, but once we were able to screen, claudin-4 was clearly a top hit,” White says. “It was an exciting moment.”
This discovery surprised researchers. Professor Sears said many scientists had expected this receptor to be a signaling protein like a G-coupled protein receptor, but claudin-4 belonged to a different class of proteins. A review of previous studies also failed to find other toxins that behaved similarly. Most protease toxins bind directly to the molecules they attack, rather than first binding to another receptor.
Scientists identify molecular target of toxin
To test this interaction, Johns Hopkins researchers collaborated with structural biologists F. Xavier Gomis Roos and Ulrich Eckhardt of the Barcelona Institute of Molecular Biology.
Using biophysical techniques, White and his team in Barcelona showed in laboratory experiments that BFT and claudin-4 form a tightly bound, one-to-one complex. This provided the first direct physical evidence that toxins attach to receptors before damaging colon cells.
The researchers then tested their findings in biological systems through a collaboration with Min Dong’s lab at Harvard Medical School. They teamed up with Kang Wang and colleagues to examine how the toxin behaves in mouse models.
Molecular decoy protects mice from intestinal toxins
The research team created a soluble version of claudin-4 that acts as a decoy by displaying the part of the receptor normally recognized by the toxin. BFT did not bind to colon cells, but instead bound to decoy proteins.
This strategy successfully protected mice from BFT-induced colon injury.
“This approach can be repeated using small molecules and other biologics with better pharmacological properties,” White says. The research team is currently investigating which types of treatments are most effective at blocking toxins.
doubts still remain
Although researchers have identified the receptor and demonstrated that it binds strongly to BFT, one important question remains unanswered. They have not yet captured the exact experimental structure that shows exactly how the toxin and claudin-4 are combined.
Current artificial intelligence modeling tools, including AlphaFold, have not been able to fully resolve this interaction.
Other authors of the paper include Jason Chen, Xiaoguan Wu, Abby L. Geis, and Jessica Queen of Johns Hopkins University, and Hailong Zhang, Karthik Hulahari, and Ji Zhang of Harvard Medical School.
This 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 UpToDate. This arrangement will be managed by Johns Hopkins University in accordance with its conflict of interest policy.
