Affecting roughly half a million Americans each year, bacterial infections caused by Clostridioides difficile—commonly known as C. diff—are a serious and persistent problem for patients and hospitals alike. The bacterium can cause severe diarrhea, life-threatening inflammation of the colon, and recurring illness that dramatically reduces quality of life—especially for older adults, who face the highest risk of complications and death.

C. diff remains difficult to control for a combination of factors. The bacterium survives many disinfectants, allowing it to easily spread in health care settings, where it is the most common cause of infectious diarrhea. After entering the body through the mouth, the bacterium travels to the colon, where it colonizes and starts releasing toxins that damage tissues. About one in nine patients treated for C. diff will develop another infection within weeks or months—often unpredictably—with the risk of a repeat infection increasing from there. And some strains of the bacterium have become resistant to the first-line antibiotics used to treat it.

Researchers at Tufts University School of Medicine are tackling these challenges by studying C. diff at multiple levels, from how individual bacterial cells behave inside the gut to the molecular switches that help them survive and spread. Together, these approaches are revealing hidden vulnerabilities that could lead to better ways to prevent new or recurrent infections, predict severe disease, or stop the bacterium before it causes harm.

Watching infections unfold, cell by cell
“C. diff is everywhere,” said Aimee Shen, an associate professor of molecular biology and microbiology at Tufts School of Medicine. “But infections can look very different from one patient to the next.”

Some people carry the bacterium without ever getting sick. Others develop severe, life-disrupting illness—typically after being treated for another illness with antibiotics that wipe out beneficial gut bacteria that may have otherwise warded off an infection.

“A bad C. diff infection is reportedly incredibly painful, like glass shards moving through your intestine,” said Shen. “And there’s some research that shows that C. diff toxins actually act on neurons in the gut.”

To better understand why the spectrum of disease severity varies so widely, Shen and her collaborators developed a new imaging approach that lets them track what individual C. diff cells were doing inside the body. They applied fluorescent “reporters”— microscopic glowing tags that mark gene activity—to track which genes are turned on in individual C. diff cells in tissue samples from infected mice. This allowed them to see where the bacteria hide in the gut, which cells switched on toxin genes, and how activity differed from cell to cell during infection.

Their study, recently published in Nature Communications, showed that C. diff spread throughout the entire gut, including closer to the gut’s vulnerable lining than previously thought.

However, toxin production didn’t depend on the bacteria’s location and only some cells made toxins at any given time. Shen said this suggests that disease may be driven by a small, hard-to-detect subpopulation rather than simply how many bacteria are present.

The imaging study also revealed other unexpected findings, including that a strain of toxin-overproducing bacteria formed unusually long, filament-like shapes in the gut during the acute phase of infection. “These were not observed in later stages of an infection,” said Shen. “This suggests that bacteria producing the most destructive amounts of toxins may be particularly susceptible to certain stresses encountered during infection.”

By illuminating how infections unfold cell by cell in this way, the new imaging method may provide information that could someday help doctors predict which patients are likely to develop severe or recurrent disease. It also may help researchers develop new treatments that better target harmful subpopulations of C. diff bacteria while sparing beneficial gut microbes.

Finding a potential weak spot
One reason C. diff spreads so effectively is its ability to form tough, dormant spores that act like microscopic seeds sealed in armor. Transmitted via trace amounts of fecal matter, these spores can survive for long periods, stubbornly resisting heat and many common disinfectants, including hand sanitizers. Once ingested, C. diff spores germinate—springing back to life and thus able to spawn toxins.

This is a pivotal moment scientists hope to block.

Shen’s lab has long studied how the bacterium recognizes it has reached the right place to reawaken. Most spore-forming bacteria rely on the same standard molecular sensors, but C. diff uses a different system. Its spores respond to bile acids found in digestive fluids, along with other signals that together flip the bacterium’s switch from dormancy to active growth.

In a study recently published in PLOS Biology, Shen, Tufts’ School of Medicine professor Ekaterina Heldwein, and their collaborators identified a key part of that switch. They found that two proteins, CspC and CspA, lock together to form a signaling hub that helps spores interpret environmental cues. By mapping the structure of this protein pair and testing how it functions, the team showed the combined complex controls how sensitive spores are to germination signals.

“It’s like we’ve identified a central control panel for deciding when the spore comes back to life,” Shen says. “If we understand how that panel works, scientists someday may be able to design new drugs to keep it switched off.”

Searching for more precise targets
Together, the studies offer a clearer picture of both how C. diff causes disease and when it becomes dangerous.

Now, in addition to continuing their work on single-cell imaging and spore germination, Shen’s lab is working to uncover other hidden rules that govern C. diff’s behavior. This includes how it reproduces using a division mechanism unlike those seen in other well-studied bacteria—the focus of a 2023 study published by Shen and collaborators in Nature Communications.

“The hope is the aspects that make C. diff unique—how it spreads, reproduces, and damages tissue—will allow researchers to design ways to target it much more specifically, while keeping the rest of the gut microbiome healthy and intact,” she said.

Citation: Research reported in the fluorescent imaging study was supported by the National Institutes of Health’s National Institute of Allergy and Infectious Diseases under award numbers R21AI168849, T32AI007422, and F31AI191587, as well as by a Burroughs Wellcome Fund Investigators in the Pathogenesis of Disease grant. Nicholas DiBenedetto, M. Lauren Donnelly-Morell, and Carol Kumamoto from the Department of Molecular Biology and Microbiology at Tufts University School of Medicine were co-authors on the study.

The germination study was supported by the National Institutes of Health’s National Institute of General Medical Sciences under awards numbers R01GM108684, K12GM133314, and T32GM007310; the National Institutes of Health’s National Institute of Allergy and Infectious Diseases under award numbers F31AI186517 and F31AI154814; and a Burroughs Wellcome Fund Investigators in Pathogenesis National Institute of General Medical Sciences grant. Morgan McNellis, Gonzalo González-Del Pino, Juan Serrano-Jiménez, Emily Forster, Anca Ioana Stoica, and Ekaterina Heldwein from the Department of Molecular Biology and Microbiology at Tufts University School of Medicine were co-authors on the study.

Complete information on methodology, limitations, and conflicts of interest is available in the two published papers.

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