The pathogen Clostridioides difficile (C. difficile) is the most common cause of healthcare-associated infectious diarrhea. A team led by investigators at Vanderbilt University Medical Center has now found that C. difficile can use a compound that kills the human gut’s resident microbes to survive and grow, giving it a competitive advantage in the infected gut.
The team, headed by Eric Skaar, PhD, the Ernest W. Goodpasture Professor of Pathology and director of the Vanderbilt Institute for Infection, Immunology, and Inflammation, discovered how C. difficile converts the poisonous compound 4-thiouracil, which could come from foods such as broccoli, into a usable nutrient. The findings from their in vitro experiments and work in mice increase understanding of the molecular drivers of C. difficile infection and could point to novel therapeutic strategies.
Senior author Skaar, together with first author Matthew Munneke, and colleagues, reported on their findings in Cell Host & Microbe, in a paper titled, “A thiouracil desulfurase protects Clostridioides difficile RNA from 4-thiouracil incorporation, providing a competitive advantage in the vertebrate gut.” In their paper, the team noted, “Collectively, these results reveal a molecular mechanism for C. difficile to utilize a poisonous pyrimidine analog in the vertebrate gut to outcompete commensal microbes.”
C. difficile causes about half a million infections in the United States each year, according to Centers for Disease Control and Prevention figures cited by the Vanderbilt University Medical Center team. Factors that increase the risk of C. difficile infection include antibiotic use, age over 65 years, and recent stays in hospitals and other healthcare facilities. “The gram-positive human gastrointestinal pathogen Clostridioides difficile (C. difficile) thrives in the antibiotic-perturbed gut and causes nearly half a million infections and 29,000 deaths annually,” the authors stated.
C. difficile—and other pathogens—must acquire nutrients to survive and grow. “We’re interested in trying to understand the nutrients that C. difficile needs during infection, and how what you eat influences what C. difficile eats in your gut,” said Munneke, a graduate student working with Skaar.
Matthew Munneke, left, and Eric Skaar, PhD, MPH, use anaerobic chambers to study bacteria like C. difficile that die in the presence of oxygen. [Vanderbilt University Medical Center]
Matthew Munneke, left, and Eric Skaar, PhD, use anaerobic chambers to study bacteria like C. difficile that die in the presence of oxygen. [Vanderbilt University Medical Center]
For their reported study the group focused on nucleotides—the building blocks of DNA and RNA—which are a class of nutrients that hasn’t been well studied for C. difficile. “Nucleotides are essential building blocks for major cellular macromolecules and are critical for life,” the team wrote in their paper. “Nucleotide acquisition contributes to the virulence of several human bacterial and parasitic pathogens in the host environment.”
Bacterial pathogens must acquire or synthesize nucleotides during infection. Through a series of studies, the researchers found that C. difficile must acquire a certain type of nucleotides (pyrimidines) to cause infection, and they discovered an enzyme thiouracil desulfurase (TudS) that C. difficile uses to salvage the pyrimidine nucleotide uracil from a related compound: 4-thiouracil.
The team’s experiments showed that 4-thiouracil gets incorporated into RNA and is toxic to resident gut microbes that do not have the TudS enzyme. In C. difficile, however, TudS modifies and detoxifies 4-thiouracil, making it available as a nutrient. The researchers demonstrated that TudS contributes to C. difficile “fitness” in mice fed 4-thiouracil and in a novel MiniBioreactor model of C. difficile infection (CDI) that contains a community of bacteria isolated from human feces with added 4-thiouracil. “… we reveal the importance of pyrimidine nucleotide acquisition for C. difficile infection (CDI) and describe a molecular mechanism by which C. difficile salvages a modified pyrimidine nucleobase, 4-thiouracil (4-TU), present in human stool,” they wrote.
“We think that 4-thiouracil metabolism is beneficial to C. difficile because it acts as a nutrient to fuel the bacteria, and it also may inhibit neighboring bacteria, which would give C. difficile a further competitive advantage within the gut environment,” Munneke said. “Taken together, these findings suggest that 4-TU benefits C. difficile in the gut by inhibiting competing microbes and serving as a pyrimidine source for growth,” the authors stated in their report.
The TudS enzyme may represent a novel therapeutic target for treating C. difficile infections. It is not present in many resident gut microbes (or in human cells), so an antimicrobial targeting it to kill C. difficile might help preserve the healthy gut microbiota, Munneke noted. “Since TudS is not conserved in multicellular eukaryotes, we propose that TudS-mediated 4-TU acquisition has the potential to serve as a therapeutic target to treat CDI,” the authors added.
The researchers also showed that adding C. difficile TudS to a probiotic strain of E. coli blunted C. difficile fitness advantage in an in vitro model. “It might be possible to use a probiotic with this enzyme to diminish C. difficile’s ability to thrive in the gut and push it out,” Munneke said. The authors further noted, “These data demonstrate that TudS is required for C. difficile to outcompete commensal microbes in the presence of 4-TU, and expression of tudS in a commensal strain is sufficient to diminish the competitive advantage conferred by 4-TU to C. difficile.”
Although the researchers showed that 4-thiouracil is present in the human gut, the source of this compound is unclear. Livestock that consume a diet rich in cruciferous vegetable family members (such as kale and other leafy greens, broccoli, and cauliflower) have elevated levels of 4-thiouracil, and it is present in broccoli, both suggestive that a dietary source may contribute to the presence of 4-thiouracil in the human gut. “More research is needed to understand the source of 4-thiouracil, but if it comes from the diet, that could inform dietary interventions for C. difficile infection,” added Munneke.
It’s not time to give up eating cruciferous vegetables though. In the healthy gut, some resident microbes contain a TudS-related enzyme and can likely convert 4-thiouracil into nutrients. These microbes may be missing in the C. difficile-infected gut, Munneke said.