Newly Identified Metabolic Targets May Improve Treatment of C. difficile

The gut microbiome is mainly composed of obligately anaerobic bacteria (those that don't replicate in the presence of oxygen). The metabolic pathways and nutrient requirements of anaerobes differ in many respects from those of oxygen-tolerant species such as Escherichia coli, and many haven't yet been characterized.

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Leo L. Cheng, PhD, an associate biophysicist in the Department of Pathology and Department of Radiology at Massachusetts General Hospital and associate professor of Radiology at Harvard Medical School, Lynn Bry, MD, PhD, director of Massachusetts Host-Microbiome Center and associate medical director in the Department of Pathology at Brigham and Women's Hospital and associate professor of Pathology at Harvard Medical School, and colleagues have devised a versatile approach to studying metabolism in obligate anaerobes. They describe the technique in Nature Chemical Biology using Clostridioides difficile as an example.

Background

The team used high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy, which permits the real-time study of metabolism in very small amounts of living cells. It's particularly well suited to the study of anaerobes because the rotor chamber is sealed.

"Magic angle" refers to the rotation of the samples at 54.74° relative to the magnetic field during NMR spectrum measurement. This enhances signal detection in colloidal or semisolid samples, so very few input cells are necessary (tens of microliters, or about 100,000 cells). When coupled with carbon-13–labeled substrates, HRMAS NMR can track carbon flow through complex metabolic pathways.

C. difficile is known to colonize the gut by fermenting diverse carbon sources, including carbohydrates and amino acids. It releases toxins to obtain nutrients from damaged mucosa as its growth exceeds the carrying capacity of gut environments. This study aimed to identify what metabolic strategies C. difficile uses to support its rapid colonization and expansion.

Results

The team applied dynamic flux balance analysis, which simulates the time-dependent recruitment of metabolic pathways in an organism. HRMAS NMR spectra were acquired from living C. difficile cells as they fermented ¹³C-labeled substrates.

To mimic the conditions in the gut after a person takes a course of antibiotics, the researchers presented C. difficile with abundant fermentable amino acids and carbohydrates. The bacterium rapidly recruited amino acid fermentation pathways, then engaged pathways to ferment simple sugars such as glucose.

Amino acid and glycolytic metabolism converged to produce the amino acid alanine, supporting efficient energy generation, nitrogen handling, and biomass generation.

Why It Matters

These results identify new targets for small-molecule drugs that might counter C. difficile colonization and infection in the gut. HRMAS NMR might also prove useful for understanding broader aspects of microbial metabolism, including antibiotic responses, and for developing industrially important chemicals.

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