# Proteomic antibacterial characterization of flavonoid xanthohumol and probiotic Clostridium butyricum on pathogenic Clostridioides difficile

## TL;DR

This study explores how xanthohumol and Clostridium butyricum reduce the harmful effects of Clostridioides difficile infection by altering bacterial metabolism.

## Contribution

The study identifies lysine acetylation as a novel mechanism by which xanthohumol and Clostridium butyricum inhibit C. difficile pathogenicity.

## Key findings

- Xanthohumol and Clostridium butyricum reduce C. difficile colonization and toxin production.
- Both treatments modulate lysine acetylation, affecting glycolysis and pathogen virulence.
- A specific lysine acetylation at fructose-1,6-bisphosphate aldolase is crucial for enzyme activity.

## Abstract

The management of dysbiotic gut microbiota in Clostridioides difficile infection has attracted increasing scholarly attention. The development of therapeutic agents with low toxicity, derived from both the flavonoid xanthohumol and the short-chain fatty acid-producing probiotic Clostridium butyricum, holds considerable promise for combating Clostridioides difficile infection. Despite their therapeutic potential, the molecular mechanisms underlying the anti-Clostridioides difficile effects remain inadequately characterized.

In this study, we established a dextran sulfate sodium-induced inflammatory model using Caco-2 intestinal epithelial cells. The protective effects of xanthohumol against Clostridioides difficile infection superimposed on colitis were evaluated through cell viability assays, analysis of inflammatory signaling pathways, and proteomic profiling. Subsequent in vitro assays and proteomic analyses were conducted to assess the influence of xanthohumol and Clostridium butyricum supernatant on Clostridioides difficile. Furthermore, tandem mass tag-based post-translational modification proteomics was employed to elucidate the underlying molecular mechanisms and key pathways. Finally, critical metabolic enzyme activity assays were performed to validate the regulatory roles of these pathways.

Xanthohumol significantly alleviated C. difficile-induced damage in Caco-2 cells, enhanced cell viability, and suppressed the activation of inflammatory signaling pathways. In vitro experiments demonstrated that both xanthohumol and C. butyricum supernatant reduced bacterial colonization, inhibited growth, and attenuated toxin production. Proteomic analyses revealed substantial alterations in the proteome of C. difficile in response to each treatment. Post-translational modification proteomics further indicated that both treatments modulate lysine acetylation levels, influencing glycolysis pathways and ultimately diminishing the pathogen’s virulence. Furthermore, mass spectrometry identified a specific lysine acetylation at the K280 site of fructose-1,6-bisphosphate aldolase, a key enzyme in glycolysis. Functional validation via site-directed mutagenesis confirmed the essential role of this acetylation in regulating the catalytic activity of fructose-1,6-bisphosphate aldolase.

Our study demonstrates that xanthohumol and Clostridium butyricum attenuate the pathogenicity of Clostridioides difficile through modulation of lysine acetylation and disruption of glycolysis metabolism. These findings highlight their potential as promising therapeutic strategies for treating Clostridioides difficile infection.

The online version contains supplementary material available at 10.1186/s13020-026-01343-x.