Article Figures & Data
Figures
- 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.
Tables
- TABLE 1.
Primers used for quantitative RT-PCR
Gene ASAP identifier Forward primer Reverse primer lsrA ABH-0025965 5′-AACATCCTGTTTGGGCTGGCAA-3′ 5′-AAACAAGCGTTCGGTTTCCGCA-3′ lsrB ABH-0025962 5′-AGCATCCTGGCTGGGAAATTGT-3′ 5′-AAATTCTTTCACCGTGCCGCGT-3′ lsrC ABH-0025964 5′-ACAGCGTTTGGACGCAGTTT-3′ 5′-ACGCAGGCTGCAATTGCTTT-3′ rpoA ABH-0028324 5′-CGCGGTCGTGGTTATGTG-3′ 5′-GCGCTCATCTTCTTCCGAAT-3′ - TABLE 2.
Summary of changes in E. coli O157:H7 biofilm gene expression
Gene type or function and name Relative change in expression (n-fold) after exposure to:a Description EPI NE IND AI-2 group c1993 1.4 1.4 1.6 Putative integral membrane transport protein lsrA −9.8 −5.3 1.0 Fused AI-2 transporter subunits of ABC superfamily; ATP-binding components lsrB −7.0 −5.3 −1.4 Putative LACI-type transcriptional regulator lsrC −4.3 −2.3 1.2 AI-2 transporter lsrD −3.2 −1.4 1.5 AI-2 transporter lsrG −3.2 −3.0 −1.1 AI-2 modifying protein LsrG lsrK −2.8 −2.3 1.2 AI-2 kinase lsrR −3.7 −3.7 1.1 lsr operon transcriptional repressor Phosphate related phnD −2.1 1.2 2.1 Phosphonates-binding periplasmic protein precursor phnG −1.1 1.5 2.3 PhnG protein phnP −1.2 1.3 2.1 PhnP protein phoB 2.1 3.0 −2.6 Phosphate regulon transcriptional regulatory protein phoB phoH −4.9 −4.6 −1.9 PhoH protein phoR 2.6 3.5 −1.6 Phosphate regulon sensor protein phoR phoU 1.9 2.1 −3.7 Phosphate transport system protein phoU pstA 2.5 2.6 −3.2 Phosphate transport system permease protein pstA pstB 1.3 1.5 −3.7 Phosphate transport ATP-binding protein pstB pstC 2.6 3.0 −2.3 Phosphate transport system permease protein pstC pstS 1.1 1.1 −4.6 Phosphate-binding periplasmic protein precursor purE 3.0 3.7 1.4 Phosphoribosylaminoimidazole carboxylase catalytic subunit purK 1.2 2.6 1.6 Phosphoribosylaminoimidazole carboxylase ATPase subunit purT −1.4 2.5 2.1 Phosphoribosylglycinamide formyltransferase 2 Hydrognease hyaA −3.0 −3.2 1.1 Hydrogenase-1 large chain hyaB −4.0 −3.5 1.2 Hydrogenase-1 operon protein hyaE hyaC −3.7 −3.0 1.1 Hydrogenase-1 operon protein hyaF hyaD −3.2 −2.8 1.2 Hydrogenase-1 small chain precursor hyaE −3.5 −2.5 1.3 Hydrogenase-1 large chain hyaF −3.0 −2.6 1.1 Hydrogenase-1 operon protein hyaE PTS mtlA −13.9 −11.3 1.0 PTS system; mannitol-specific IIABC component manX −4.6 −3.5 1.1 PTS system; mannose-specific IIAB component manY −2.8 −2.3 1.1 PTS system; mannose-specific IIC component manZ −2.8 −2.1 1.2 PTS system; mannose-specific IID component fruB −1.7 −2.1 1.2 PTS system; fructose-specific IIA/FPr component Glycerol related glgC −2.5 −3.0 −1.3 Glucose-1-phosphate adenylyltransferase glpA −4.9 −1.7 1.7 Anaerobic glycerol-3-phosphate dehydrogenase subunit A glpD −9.2 −18.4 1.3 sn-Glycerol-3-phosphate dehydrogenase (aerobic) glpF −5.7 −5.7 1.3 Glycerol uptake facilitator protein glpK −7.5 −6.1 1.1 Glycerol kinase glpQ −2.1 −2.5 −1.1 Glycerophosphoryl diester phosphodiesterase; periplasmic precursor glpR −1.6 −2.1 −1.1 Glycerol-3-phosphate regulon repressor ugpB −7.0 −6.1 −1.4 Glycerol-3-phosphate-binding periplasmic protein precursor Histidine biosynthesis hisA −2.1 −2.8 −1.6 1-(5-Phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase hisB −2.3 −2.6 −2.1 Bifunctional: histidinol-phosphatase (N terminus) and imidazoleglycerol-phosphate dehydratase (C terminus) hisC −4.0 −4.6 −2.8 Histidinol-phosphate aminotransferase hisD −4.3 −4.3 −2.6 Histidine biosynthesis hisF −3.0 −3.0 −2.0 Imidazole glycerol phosphate synthase subunit hisF hisG 1.1 −1.3 −2.8 ATP phosphoribosyltransferase hisH −1.6 −2.1 −2.0 Imidazole glycerol phosphate synthase subunit hisH hisI −2.1 −2.0 −1.9 Histidine biosynthesis hisL 3.0 1.1 −3.7 His operon leader peptide hisLEDL 1.7 −1.2 −3.5 Histidine biosynthesis; hisL gene from strain EDL933 hisP −2.5 −1.4 −1.1 Nucleotide binding Two-component signal transduction dos 1.3 2.1 1.4 Putative phosphodiesterase, oxygen-sensing protein evgS −3.2 −1.5 −1.2 Two-component signal transduction system (phosphorelay) fimZ 1.1 2.1 3.0 Fimbiral protein Z; putative transcriptional regulator of fimbrial expression (LuxR/UhpA family) glnL 3.2 2.5 −1.9 Two-component signal transduction system (phosphorelay) narL −2.6 −2.0 −1.4 Nitrate/nitrite response regulator protein narL prpR −3.5 −3.7 1.1 Regulator for prp operon rstA 2.5 1.9 −1.3 Transcriptional Regulatory protein rstA yedV 1.4 2.6 2.0 Putative two-component sensor protein ygeV −2.1 −1.4 −1.5 Two-component signal transduction system (phosphorelay) Sulfate cysD −3.0 −3.0 −1.6 Sulfate adenylyltransferase (ATP) activity cysI −3.5 −3.0 −1.2 Sulfite reductase [NADPH] hemoprotein beta-component cysJ −4.3 −2.8 1.0 Sulfite reductase [NADPH] flavoprotein alpha-component sseA −2.1 −1.9 1.4 3-Mercaptopyruvate sulfurtransferase Motility/chemotaxis acs −8.0 −4.3 1.1 Acetyl-coenzyme A synthetase c2129 3.0 2.5 1.5 Cell division activator cedA c4004 1.3 2.1 1.1 Hypothetical protein csgA −4.3 −2.8 1.5 Cell adhesion csgB −5.7 −4.6 −1.1 Minor curlin subunit precursor csgF −3.7 −2.5 −1.4 Curli production assembly/transport component csgF precursor fimZ 1.1 2.1 3.0 Fimbiral protein Z; putative transcriptional regulator of fimbrial expression (LuxR/UhpA family) fliD 2.0 2.1 1.4 Cell motility fliR −1.1 1.9 2.3 Flagellar biosynthetic protein fliR gidB 1.4 2.1 1.5 Methyltransferase gidB motB −2.3 −1.2 1.3 Chemotaxis motB protein mreC 1.5 2.1 1.2 Rod shape-determining protein mreC sfmA −1.2 1.5 2.1 Putative fimbrial-like protein sfmF 1.0 1.5 2.3 Putative fimbrial-like protein sfmH −2.0 1.0 2.5 Fimbrial assembly protein yhhP 1.7 2.5 1.5 SirA protein Z0020 1.5 2.5 2.1 Cell adhesion Z4971 −1.9 3.7 1.9 Cell adhesion Cytochrome appB −3.0 −2.3 1.2 Cytochrome bd II oxidase subunit II appC −2.5 −2.1 1.1 Cytochrome bd II oxidase subunit I cyoC −2.1 −3.0 1.0 Cytochrome o ubiquinol oxidase subunit III nrfA −3.2 −1.5 1.5 Cytochrome c-552 precursor nrfB −2.8 1.0 1.5 Cytochrome c-type protein nrfB precursor Cold shock protein cspA 1.5 1.5 −3.2 Cold shock protein cspA cspE 2.3 2.5 −1.1 Cold shock-like protein cspE cspG 8.0 6.1 −4.0 Cold shock-like protein cspG cspH 22.6 21.1 −2.5 Cold shock-like protein cspH deaD 2.1 2.6 −1.2 Cold shock DEAD-box protein A Antisense RNA dsrA 3.5 4.9 1.3 Antisense RNA; silencer of rcsA gene, interacts with rpoS translation rdlD 4.0 4.0 −2.5 Antisense RNA; trans-acting regulator of ldrD translation sokB 9.2 7.0 −1.1 Antisense RNA blocking mokB and hokB translation sokC 2.0 2.6 −1.6 Antisense RNA blocking mokC (orf69) and hokC (gef) translation Glutamine/glutamate c0018 24.3 24.3 5.7 Putative glutamate dehydrogenase glnA 1.6 1.3 −2.3 Glutamine synthetase glnH −1.2 1.0 −2.1 Glutamine-binding periplasmic protein precursor glnP 2.5 3.0 −1.9 Glutamine transport system permease protein glnP gltJ 3.7 4.3 −1.6 Glutamate/aspartate transport system permease protein gltJ gltK 2.6 2.6 −1.6 Glutamate/aspartate transport system permease protein gltK gltL 1.4 1.7 −1.9 Glutamate/aspartate transport ATP-binding protein gltL ybeJ −1.3 −1.1 −2.5 Glutamate/aspartate periplasmic binding protein precursor Iron related c3773 2.0 2.1 1.6 Putative iron compound permease protein of ABC transporter family cydA 2.0 2.8 −1.1 Cytochrome d ubiquinol oxidase subunit I cydB 1.9 2.8 −1.2 Cytochrome d ubiquinol oxidase subunit II feoA 2.5 2.6 −1.3 Ferrous iron transport protein A feoB 2.0 3.2 1.0 Ferrous iron transport protein B fhuB 1.3 2.5 1.1 Ferrichrome transport system permease protein fhuB fhuC 1.6 2.5 1.0 Ferrichrome transport ATP-binding protein fhuC fhuD 2.5 4.6 1.1 Ferrichrome-binding periplasmic protein precursor yodB 2.5 2.6 −1.3 Cytochrome b561 homolog 1 NADH dehydrogenase nuoA −1.3 −2.5 1.1 NADH dehydrogenase I chain A nuoB −2.0 −3.5 1.0 NADH dehydrogenase I chain B nuoC −1.7 −4.0 −1.1 NADH dehydrogenase I chain C/D nuoE −2.0 −4.3 −1.1 NADH dehydrogenase I chain E nuoI −1.1 −2.5 1.1 NADH dehydrogenase I chain I nuoJ −1.3 −2.5 1.3 NADH dehydrogenase I chain J nuoK −1.3 −2.3 1.4 NADH dehydrogenase I chain K nuoL −1.2 −2.3 1.4 NADH dehydrogenase I chain L Tricarboxylic acid cycle sdhD −13.0 −21.1 1.1 Succinate dehydrogenase hydrophobic membrane anchor protein sdhC −12.1 −18.4 1.1 Succinate dehydrogenase cytochrome b-556 subunit sdhA −11.3 −16.0 1.3 Succinate dehydrogenase, catalytic and NAD/flavoprotein subunit sdhB −10.6 −12.1 1.0 Succinate dehydrogenase iron-sulfur protein sucA −7.5 −11.3 1.1 2-Oxoglutarate dehydrogenase E1 component sucB −7.0 −9.2 −1.1 Dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase complex sucD −6.5 −8.6 1.0 Succinyl-coenzyme A synthetase alpha chain sucC −6.5 −8.6 1.0 Succinyl-coenzyme A synthetase beta chain acnB −2.0 −4.3 1.0 Aconitate hydratase activity icdA −1.9 −2.6 1.1 Isocitrate dehydrogenase [NADP] yojH −1.2 −2.3 −1.5 Malate:quinone oxidoreductase mdh −2.0 −2.1 −1.1 Malate dehydrogenase ↵ a Important changes are shown in boldface. IND, indole.
- TABLE 3.
Changes in the expression of AI-2 uptake genes in E. coli O157:H7 biofilms after exposure to EPI and NE
Gene Biological process description Relative change in expression (n-fold) determined by the indicated methoda Microarray RT-PCR (EPI) EPI NE lsrB AI-2 transporter −7.0 −5.3 −2.5 lsrG Antibiotic biosynthesis −3.2 −3.0 lsrD Transport −3.2 −1.4 lsrA Transport −9.8 −5.3 −4.8 lsrC Transport −4.3 −2.3 −2.4 lsrK Carbohydrate metabolism −2.8 −2.3 lsrR Regulation of transcription; DNA dependent −3.7 −3.7 ↵ a Values are based on an exposure of 7 h. Significant changes are in boldface.
