Article Figures & Data
Figures
- FIG 1
DHMA is a chemoattractant for EHEC 86-24. The chemotaxis response of EHEC was investigated using the plug-in-pond assay. EHEC cells expressing GFP were suspended in CB and incubated with an agarose plug containing either CB alone (control) or CB plus a signal molecule. The accumulation of cells at the plug edge was observed after 30 min with an inverted fluorescence microscope. The increase in the intensity of fluorescence at the plug boundary serves as a qualitative measure of the attractant response to the chemical in the plug. (A) Plug with CB plus 500 μM DHMA; (B) plug with CB only (negative control); (C) plug with CB plus 100 μM l-serine (positive control). The images shown are from one representative experiment of three independent experiments.
- FIG 2
EHEC 86-24 responds to DHMA in a dose-dependent manner. The dose-response of EHEC was measured using a microflow-based MMC assay. EHEC 86-24 cells expressing GFP were suspended in CB and introduced into the center of a microflow channel containing buffer or a fixed concentration of DHMA. One hundred images were collected over a period of 6 min and were overlaid to prepare representative snapshots. (A) Representative snapshot of cells in buffer. (B) Representative snapshot of cells in 50 μM DHMA. (C) The dose-dependent chemotaxis response of EHEC 86-24 to DHMA. The migration of cells from the center of the channel was determined by image analysis and used to calculate the MMC. The data represent the means and standard errors of the means from three independent experiments. *, **, and #, statistical significance for each response compared to that for the buffer control using Student's t test at significance levels of P < 0.01, 0.005, and 0.0005, respectively.
- FIG 3
DHMA induces expression of virulence genes in EHEC 86-24 in a QseC-dependent manner. The increase in the expression of LEE and non-LEE virulence genes upon exposure to DHMA (left) or NE (right) was determined by qRT-PCR. Untreated cultures were used as controls, and the housekeeping gene rpoA was used to normalize the data. The means and the standard errors of the means are shown for three biological replicates with two experimental replicates each. *, **, and #, statistical significance of the fold change in expression by the wild type (WT) relative to that by the mutant using Student's t test at significance levels of P < 0.1, 0.05, and 0.01, respectively.
- FIG 4
DHMA increases attachment of EHEC 86-24 to HeLa cells. The effect of DHMA and NE on attachment of EHEC 86-24 to HeLa cells was determined. The increase in attachment relative to that for the control (i.e., the ratio of the number of EHEC cells attached when the cells were grown with DHMA or NE relative to the number of EHEC cells attached when the cells were grown only with solvent) was calculated. The means and standard errors of the means are shown for six biological replicates with three experimental replicates each, using the EHEC 86-24 wild-type strain or the isogenic ΔqseC derivative. *, statistical significance of P < 0.001 by Student's t test.
- FIG 5
Presence of DHMA in fecal samples. Metabolites were extracted from fecal samples of SPF (n = 9; 4 to 12 weeks old) and GF (n = 6; 3 to 8 weeks old) mice. DHMA was detected by LC-MS and quantified using a standard curve. DHMA (exact mass, 184.037) was detected in the negative-ion mode as an ion of m/z 183.032 with a confirmatory fragment ion of m/z 137.025. (Top) Extracted ion current (XIC) chromatogram indicating the elution of pure DHMA using a Chromolith RP-18 column. (Inset) Standard curve of DHMA determined over a concentration range of 1 nM to 1 × 103 nM. (Middle) Representative XIC chromatogram showing the elution of DHMA in a fecal extract from an SPF mouse and a GF mouse. The peak area of DHMA in the fecal extract from the SPF mouse corresponds to a DHMA concentration of 1.9 ± 0.3 μM. (Bottom) Quantitation of DHMA in SPF and GF mice. Note that no DHMA was detected in extracts from GF mice.
- FIG 6
Proposed model for the effect of DHMA on EHEC chemotaxis and virulence. (Left) (A) Mesenteric nerves from lymphoid follicles release norepinephrine (NE; •) into the gut lumen. (Right) Enlarged image of the boxed area in the left panel. (B) NE is sensed by QseC in commensal bacteria (green), resulting in expression of tynA and feaB, whose products convert NE to DHMA (▲). (C) A gradient of DHMA is formed from NE. (D) DHMA attracts pathogens, such as EHEC (red), through Tsr-mediated chemotaxis and induces pathogenesis through QseC.
Additional Files
Supplemental material
- Supplemental file 1 -
Table S1. Primers used for qRT-PCR. Fig. S1. Dose-dependent chemotaxis response of E. coli RP437 to DHMA. Fig. S2. Attachment of EHEC 86-24 cells to the plastic of the well plates in the absence of HeLa cells. Fig. S3. Representative total ion current (TIC) chromatograms of fecal extracts from normal and germfree mice.
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- Supplemental file 1 -
