Species-specific enhancement of enterohemorrhagic E. coli pathogenesis mediated by microbiome metabolites

Abstract

Background: Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia coli (EHEC) infection, whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities.

Results: We utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites-4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid-that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity.

Conclusion: Organ-on-chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection.

Fig. 1

Microbiome metabolites recapitulate species-specific tolerance in Colon Chips. a A schematic representation of the experimental design illustrating how human or mouse intestinal microbiome metabolites were added to the intestinal channel (red) of optically clear, human Colon Chips that are lined by primary human colon epithelial cells (Epi) and directly opposed to a second parallel vascular microchannel (blue) in which HIMVECs (Endo.) are cultured; the two channels are separated by a thin, porous, ECM-coated membrane; F-actin filaments in epithelial and endothelial cells were stained with Phalloidin (magenta) and nuclei with DAPI (white). b–d Analysis of EHEC-induced epithelial injury on-chip. b Representative differential interference contrast (DIC) images of the colonic epithelium in the presence of Hmm or Mmm in the presence or absence of EHEC (bar, 100 μm). c Pseudo-colored images of the entire colon epithelium within the upper channel of the Colon Chip (yellow) cultured in the presence of Hmm or Mmm with or without EHEC (dark regions indicate lesion areas). d Quantification of epithelial lesion areas under the experimental conditions described in b, c. Epithelial lesion defined as regions in which cells normally contained within a continuous intact epithelium have fully detached from the ECM-coated membrane and their neighboring cells, thus, leaving exposed regions of the membrane below. e Changes in levels of various indicated cytokines released into the vascular channel of the Colon Chips by cells cultured under the conditions described in b, c. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

Human microbiome metabolites stimulate bacterial motility. a–d Changes in the EHEC transcriptome induced by exposure to human (Hmm) versus mouse (Mmm) gut microbiome metabolites. a Heatmap of differentially expressed genes (red indicates higher levels of expression). b Gene enrichment analysis. c Heatmap of chemotaxis and flagellar assembly pathways showing expression levels for relevant motility-related genes in EHEC cultured in the presence of Hmm versus Mmm. d Schematic of key genes critical in regulating chemotaxis and flagellar assembly in EHEC. e EHEC swimming motility tracking (lines: bacterial movement tracks; dots: starting points for all tracked bacteria; bar, 100 μm). f Quantification of the fraction (%) of moving EHEC. g Mean velocity of each tracked bacterium (red and black: velocity < or > 3 μm s⁻¹, respectively). h Distance traveled (μm) by the moving bacteria. i Fli-C-luciferase expression levels in medium supplemented with Hmm or Mmm [determined by quantifying area under the curve (AUC), and normalizing for the medium control]. *p < 0.05; ****p < 0.0001

Fig. 3

Identification of specific metabolites that mediate EHEC motility. a–c Results of metabolomics analysis of human versus mouse gut microbiome metabolites. a Venn-diagram illustrating metabolomics analysis workflow and total numbers of compounds identified in the Hmm and Mmm samples compared to the pre-fermentation medium (Pre-ferm.; label p_25: human pre-fermentation medium; label p_26 murine pre-fermentation medium). b Heatmap of 426 compounds produced by commensal bacteria that were differentially abundant in human (Hmm) versus mouse (Mmm) microbiome metabolites. c Relative abundance of 30 microbiome metabolites that were tested (blue and red: higher levels in Mmm or Hmm, respectively). d Results of FliC- luciferase (FliC-lux) screening for the 30 selected metabolites (FliC-lux levels are presented based on quantification of the AUC; grape seed oligomeric proanthocyanidins (PAC) was used as a negative control; the 4 active metabolites that induced higher FliC levels are highlighted in red; all values were normalized against the DMSO control)

Fig. 4

The identified active metabolites mediate increased pathogenicity. a–c Effect of 3,4-dimethylbenzoic acid, 4-methylbenzoic acid, hexanoic acid, and heptanoic acid (4 metab.) on epithelial injury in the Colon Chip in the presence or absence of EHEC, with or without Mmm, compared to the effects of Hmm with EHEC. a Representative DIC images of the colon epithelium under the various experimental conditions (bar, 100 μm). b Pseudocolored view of the entire epithelial layer in the Colon Chip (yellow) under the same conditions. c Quantification of epithelial lesion area size under conditions shown in b. Epithelial lesion defined as regions in which cells normally contained within a continuous intact epithelium have fully detached from the ECM-coated membrane and their neighboring cells, thus, leaving exposed regions of the membrane below. *p < 0.05; **p < 0.01