Researchers uncover how bacterial toxin may trigger colorectal cancer

For years, scientists suspected that a bacterial toxin produced by microbes in the human gut played a role in the development of colorectal cancer. Now, researchers have directly observed how the toxin damages DNA, providing new insight into how it may contribute to the disease.

The study, published in Science, is the first to reveal the structure of the DNA lesion created by colibactin, a toxin produced by certain strains of gut bacteria. The research was led by Emily Balskus of Harvard’s Department of Chemistry and Chemical Biology and Victoria D’Souza of the Department of Molecular and Cellular Biology.

“This molecule has been really challenging to study because it’s very chemically unstable,” said Emily Balskus, the Thomas Dudley Cabot Professor of Chemistry.

The findings provide some of the strongest evidence to date linking colibactin to colorectal cancer and suggest that the toxin could become a future target for disease prevention strategies.

A unique form of DNA damage

Most carcinogens damage a single strand of DNA. Colibactin, however, causes a far more serious type of injury known as an inter-strand cross-link.

DNA normally exists as a double helix made up of two intertwined strands. Colibactin chemically binds both strands together, preventing them from separating properly.

“An inter-strand cross-link means that your DNA-damaging agent reacts with both strands of DNA. It links the two strands of DNA together, creating a particularly toxic form of DNA damage to the cell,” Balskus explained.

Such damage can interfere with normal cellular processes, resulting in chromosome breaks, faulty DNA repair, and potentially cancer-causing mutations.

“Every time the cell is trying to make a DNA copy out of its genome, it needs to unwind the two strands. The cross-link causes a major hurdle for replication,” said Victoria D’Souza, professor of molecular and cellular biology.

Tracking the toxin’s preferred targets

The research team set out to answer a fundamental question: Does colibactin damage DNA randomly, or does it target specific genetic sequences?

Colibactin is produced by certain strains of Escherichia coli (E. coli) and other gut bacteria that carry a biosynthetic gene cluster known as pks or clb.

Because the toxin rapidly breaks down outside living cells, researchers had to generate it directly from living bacteria during their experiments.

Using colibactin-producing bacteria, the team exposed short DNA fragments to the toxin and carefully analyzed where DNA cross-links formed.

The researchers combined traditional gel-based DNA sequencing with advanced mass spectrometry techniques to identify the toxin’s preferred binding sites.

Why colibactin targets specific DNA sequences

The analysis revealed that colibactin strongly prefers DNA regions rich in adenine (A) and thymine (T), two of the four chemical building blocks of DNA.

To understand why, researchers turned to structural biology techniques. D’Souza’s laboratory used nuclear magnetic resonance (NMR) spectroscopy to build a detailed structural model of the DNA lesion created by colibactin.

“I think the biggest hurdle for solving a structure by NMR is the need for a lot of material. The team was able to produce enough and overcome that major challenge,” D’Souza said.

The resulting model showed that AT-rich regions of DNA contain a narrow channel known as the minor groove, which possesses the ideal shape and electrical charge for colibactin binding.

“The cool thing about colibactin is that it perfectly fit in terms of shape and charge complementarity,” D’Souza said.

The discovery helps explain why mutations linked to colibactin consistently appear at specific locations within the genome.

Connecting colibactin to colorectal cancer

Previous studies had already shown that exposure to colibactin leaves behind a distinctive pattern of mutations in DNA. These mutation signatures have been detected in colorectal tumors.

The new findings provide a molecular explanation for why those mutations occur in specific DNA regions, strengthening the connection between colibactin-producing bacteria and colorectal cancer development.

Researchers also noted that mutations associated with colibactin appear more frequently in tumors from younger patients.

This observation is particularly intriguing because E. coli, one of the primary producers of colibactin, is especially abundant in the gut microbiome during infancy. Scientists believe early-life exposure to environmental risk factors may contribute to the growing incidence of early-onset colorectal cancer.

A promising target for prevention

The researchers say the study opens the door to future efforts aimed at preventing colorectal cancer by targeting colibactin-producing bacteria or blocking the toxin’s activity.

By understanding exactly how colibactin damages DNA, scientists may eventually develop strategies to identify high-risk individuals or intervene before cancer-causing mutations accumulate.

Collaboration across disciplines

The project involved scientists from multiple institutions and disciplines, including collaborators at the University of Minnesota.

Researchers say the complexity of studying colibactin required expertise spanning chemistry, molecular biology, structural biology, genetics, and analytical chemistry.

“When a project is as difficult as colibactin has been, you can’t expect one group to have all the expertise that’s needed to solve problems,” Balskus said. “At Harvard, we’ve benefited so much from having great access to experts across a wide range of different disciplines.”

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