Real-time HRMAS 13C NMR of obligately anaerobic cells identifies new metabolic targets in the pathogen Clostridioides difficile

Anaerobic microbial metabolism drives critical aspects of host-microbiome interactions and supports many economically important industrial applications. Yet, the metabolic pathways of anaerobic bacteria and their associated constraints for maintaining energy and redox balance are often poorly described. We employ High-Resolution Magic Angle Spinning Carbon-13 (13C) Nuclear Magnetic Resonance spectroscopy with dynamic flux balance analysis to resolve real-time metabolism in living cells of the obligately anaerobic pathogen Clostridioides difficile . Using 13C-labeled carbon sources, we elaborate the time-dependent progression of reductive and oxidative anaerobic fermentation pathways. Analyses identified new integration points for redox and nitrogen coupling between carbohydrate and amino acid metabolism, particularly in the production of 13C-alanine from 13C-glucose to provide an ammonia sink from co-occurring amino acid fermentation. Analyses conducted in the presence or absence of selenium, a required co-factor for the proline Stickland reductase, demonstrate further capacity to modulate cellular metabolism and resulting metabolites. Findings informed a genome-scale metabolic model of C. difficile , identifying alanine and associated electron carrier pools as critical metabolic integration points in energy flow and biomass expansion. We illustrate use of HRMAS NMR as a new analytical platform to resolve complex interactions in anaerobic metabolism and inform new metabolic targets to counter C. difficile infections. ### Competing Interest Statement LB is an inventor on patents for C. difficile therapeutics.