Differential effects of epinephrine, norepinephrine, and indole on Escherichia coli O157:H7 chemotaxis, colonization, and gene expression

Abstract

During infection in the gastrointestinal tract, enterohemorrhagic Escherichia coli (EHEC) O157:H7 is exposed to a wide range of signaling molecules, including the eukaryotic hormones epinephrine and norepinephrine, and bacterial signal molecules such as indole. Since these signaling molecules have been shown to be involved in the regulation of phenotypes such as motility and virulence that are crucial for EHEC infections, we hypothesized that these molecules also govern the initial recognition of the large intestine environment and attachment to the host cell surface. Here, we report that, compared to indole, epinephrine and norepinephrine exert divergent effects on EHEC chemotaxis, motility, biofilm formation, gene expression, and colonization of HeLa cells. Using a novel two-fluorophore chemotaxis assay, it was found that EHEC is attracted to epinephrine and norepinephrine while it is repelled by indole. In addition, epinephrine and norepinephrine also increased EHEC motility and biofilm formation while indole attenuated these phenotypes. DNA microarray analysis of surface-associated EHEC indicated that epinephrine/norepinephrine up-regulated the expression of genes involved in surface colonization and virulence while exposure to indole decreased their expression. The gene expression data also suggested that autoinducer 2 uptake was repressed upon exposure to epinephrine/norepinephrine but not indole. In vitro adherence experiments confirmed that epinephrine and norepinephrine increased attachment to epithelial cells while indole decreased adherence. Taken together, these results suggest that epinephrine and norepinephrine increase EHEC infection while indole attenuates the process.

FIG. 1.

Agarose plug chemotaxis. Attraction or repulsion of E. coli O157:H7 to different chemical agents was determined using a modified agarose plug assay. Fluorescence images from bacteria exposed to 500 μM EPI (A), 500 μM NE (B), 2% Casamino Acids (C), 5,000 μM indole (D), 1 × 10⁻⁴ M glycerol (E), and 1× M9 salts solution (F) are shown. Green cells are E. coli O157:H7 expressing GFP, and red cells are kanamycin-killed E. coli TG1 expressing RFP. Fluorescence images were obtained on a Zeiss Axiovert 200 microscope after 30 min using a 10× objective. Data shown are representative images from three independent experiments. Arrows indicate chemoattractant ring (A), clearance zone (C), and chemorepellant band (D).

FIG. 2.

EPI, NE, and indole affect E. coli O157:H7 motility and biofilm formation. The relative change in EHEC motility and biofilm upon exposure to EPI (50 μM), NE (50 μM), and indole (500 μM) was determined. Motility data are shown as means ± 1 standard deviation from 18 motility agar plates from three independent experiments. Biofilm data are means ± 1 standard deviation from 36 wells and three independent experiments. The asterisk indicates statistical significance determined using a Student t test (P < 0.01).

FIG. 3.

Gene expression in E. coli O157:H7 biofilms upon exposure to EPI, NE, or indole. The effects of EPI (50 μM), NE (50 μM), and indole (500 μM) on gene expression in EHEC biofilms on glass wool were determined. The number of differentially expressed genes for each molecule, as well as the number of genes common to other molecules, is indicated in the Venn diagram. Genes common to all three molecules are not included in any of the other categories to facilitate interpretation. Annotated genes common to EPI, NE, and indole treatments (i.e., divergently expressed between EPI/NE and indole) are shown at right along with arrows indicating an increase or decrease in expression. E, epinephrine; IND, indole.

FIG. 4.

EPI, NE, and indole affect E. coli O157:H7 attachment to HeLa cells. The relative change in EHEC attachment to HeLa cells after 3 h of exposure to EPI (50 μM), NE (50 μM), or indole (500 μM) was determined. Cell counts (means ± 1 standard deviation) are from duplicate LB agar plates and were generated from five HeLa cell culture wells. Control values are based on EHEC cell counts obtained without the addition of any molecule. Statistical significance was determined using a Student t test. *, P < 0.01; **, P < 0.005.

FIG. 5.

Hypothetical model for E. coli O157:H7 colonization in the GI tract. Gradients of EPI and NE influence the chemotactic migration of E. coli O157:H7 to epithelial cell surfaces. Cells that do not encounter high concentrations of EPI and NE will continue to move parallel to, and not toward, the epithelial cell surface. (A) In the absence of indole-secreting commensal E. coli, colonization of the nonpathogenic biofilm occurs throughout the epithelial cell surface. (B) In the presence of commensal E. coli, the pathogen is exposed to gradients of indole which repel it from the host cells. In this scenario, colonization occurs only in regions where nonpathogenic, non-E. coli bacteria are present. The direction of migration is indicated by arrows.