Cytolethal distending toxin B inoculation leads to distinct gut microtypes and IBS-D-like microRNA-mediated gene expression changes in a rodent model

ABSTRACT

Diarrhea-predominant irritable bowel syndrome (IBS-D), associated with increased intestinal permeability, inflammation, and small intestinal bacterial overgrowth, can be triggered by acute gastroenteritis. Cytolethal distending toxin B (CdtB) is produced by gastroenteritis-causing pathogens and may underlie IBS-D development, through molecular mimicry with vinculin. Here, we examine the effects of exposure to CdtB alone on gut microbiome composition, host intestinal gene expression, and IBS-D-like phenotypes in a rat model. CdtB-inoculated rats exhibited increased anti-CdtB levels, which correlated with increased stool wet weights, pro-inflammatory cytokines (TNFα, IL2) and predicted microbial metabolic pathways including inflammatory responses, TNF responses, and diarrhea. Three distinct ileal microbiome profiles (microtypes) were identified in CdtB-inoculated rats. The first microtype (most like controls) had altered relative abundance (RA) of genera Bifidobacterium, Lactococcus, and Rothia. The second had lower microbial diversity, higher Escherichia-Shigella RA, higher absolute E. coli abundance, and altered host ileal tissue expression of immune-response and TNF-response genes compared to controls. The third microtype had higher microbial diversity, higher RA of hydrogen sulfide (H₂S)-producer Desulfovibrio, and increased expression of H₂S-associated pain/serotonin response genes. All CdtB-inoculated rats exhibited decreased ileal expression of cell junction component mRNAs, including vinculin-associated proteins. Significantly, cluster-specific microRNA-mRNA interactions controlling intestinal permeability, visceral hypersensitivity/pain, and gastrointestinal motility genes, including several previously associated with IBS were seen. These findings demonstrate that exposure to CdtB toxin alone results in IBS-like phenotypes including inflammation and diarrhea-like stool, decreased expression of intestinal barrier components, and altered ileal microtypes that influenced changes in microRNA-modulated gene expression and predicted metabolic pathways consistent with specific IBS-D symptoms.

GRAPHICAL ABSTRACT

KEYWORDS:

• Irritable bowel syndrome
• cytolethal distending toxin
• gut microbiome
• host gene expression
• microRNAs

Acknowledgments

The authors would also like to thank the following donors for their support: The Gottesdiener foundation, Frank Lee, Joel Levine, the Monica Lester Charitable Trust, the Elias, Genevieve, and Georgianna Atol Charitable Trust, the Tull Family Foundation, Thomas Wurster and David Allen, and the John and Geraldine Cusenza Family Foundation.

Author contributions

Conceptualization: MP, RM; Formal analysis: GL, AR, MP; Funding acquisition: MP, GMB; Investigation: GL, WM, JFG, GP, MLP, GMB, MJVM, MS, SA, SW, AR, MP; Methodology: GL, JFG, WM, SW, MP; Project administration: RM, MP; Supervision: WM, SW, RM, MP; Visualization: GL; Writing – original draft: GL, GMB, JFG, WM, MP; Writing – review & editing: GL, GMB, JFG, WM, AR, RM, MP.

Disclosure statement

MP and AR are consultants for, and have received grant support from, Bausch Health Inc. The remaining authors have no relevant conflicts of interest to disclose.

Data availability statement

The datasets generated during this study are available in the National Center for Biotechnology Information (NCBI) BioProject Repository https://www.ncbi.nlm.nih.gov/bioproject under BioProject PRJNA858800.

Statistical analyses for non-high-throughput data

Paired and group analyses were performed using GraphPad Prism 9.1.0 (GraphPad Software) and IBM SPSS Statistics Version 24. Normally distributed data were analyzed with t-test or paired t-test, and data that were not normally distributed were analyzed with Mann-Whitney test or Wilcoxon paired test. Correlations were performed using Spearman’s rank associations.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2023.2293170

Additional information

Funding

This study was funded in part by funds from Bausch Health Companies Inc and the National Philanthropic Trust to MP. These sponsors played no role in the study design or in the collection, analysis, and interpretation of data.