Biology subjects

Cheng, L. L.

Publications and source records attributed to Cheng, L. L..

3 recordsLinked to original sources

bioRxiv2025

Deconvoluting Metabolomic Flux in Living Cells with Real-time 13C J-coupling-edited Proton High-Resolution Magic Angle Spinning (HRMAS) NMR

Carbon-13 (13C) NMR provides a powerful approach to non-invasively investigate complex cellular metabolism. However, limitations arise per 13C NMRs reduced sensitivity as compared to proton (1H) NMR, even when using 13C tracer molecules to enrich signal, and particularly when investigating metabolic processes in low volume samples. To overcome these limitations, we developed a 1H NMR method that harnesses 1H NMRs sensitivity with 13C NMRs specificity by capturing 13C NMR information in the 1H spectrum via 13C-1H J-couplings (1H[13C-Jed]). This approach improves 13C signal detection 13.9-fold and incorporates additional 1H NMR information to investigate complex metabolism on a cellular scale. Here, we demonstrate application of 1H[13C- Jed] NMR to deconvolute complex cellular metabolism in the anaerobic pathogen Clostridioides difficile. Analyses enabled precise tracking of branching metabolic reactions under anoxic and reducing conditions over time in a 30{micro}L volume, and improved predictions of the pathogens genome scale metabolism. 1H[13C-Jed] NMR provides methodological advancements to investigate dynamic cellular metabolism and its role in complex biologic processes.

biophysics↗

bioRxiv2023

HRMAS 13C NMR and genome-scale metabolic modeling identify threonine as a preferred dual redox substrate for Clostridioides difficile

Stickland-fermenting Clostridia preferentially ferment amino acids to generate energy and anabolic substrates for growth. In gut ecosystems, these species prefer dual redox substrates, particularly mucin-abundant leucine. Here, we establish how theronine, a more prevalent, mucin-abundant substrate, supports dual redox metabolism in the pathogen Clostridioides difficile. Real-time, High-Resolution Magic Angle Spinning NMR spectroscopy, with dynamic flux balance analyses, inferred dynamic recruitment of four distinct threonine fermentation pathways, including ones with intermediate accrual that supported changing cellular needs for energy, redox metabolism, nitrogen cycling, and growth. Model predictions with 13C isotopomer analyses of [U-13C]threonine metabolites inferred threonines reduction to butyrate through the reductive leucine pathway, a finding confirmed by deletion of the hadA 2-hydroxyisocaproate CoA transferase. In vivo metabolomic and metatranscriptomic analyses illustrate how threonine metabolism in C. difficile and the protective commensal Paraclostridium bifermentans impacts pathogen colonization and growth, expanding the range of dual-redox substrates that modulate host risks for disease.

microbiology↗

bioRxiv2021

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.

microbiology↗