Bicarbonate ion stimulates the expression of locus of enterocyte effacement-encoded genes in enterohemorrhagic Escherichia coli O157:H7

Abstract

Enterohemorrhagic Escherichia coli (EHEC) strains adhere to the intestinal mucosa and produce an attaching and effacing (A/E) lesion. Most of the genes required to produce A/E lesions are thought to be encoded by the 36-kb pathogenicity island termed the locus for enterocyte effacement (LEE). Although the mechanisms underlying the bacterial adherence, including the genes involved, are still poorly understood, the preferential adherence phenotype of EHEC is thought to depend on the nature of the genes and/or the response of these genes to changes in environmental conditions. To explore the environmental factors affecting EHEC adherence, we used an O157:H7 strain and investigated the optimal growth conditions for its adherence to Caco-2 cells. We observed that EHEC grown in Dulbecco's modified Eagle's medium (DMEM) adhered more efficiently to Caco-2 cells than EHEC grown in Luria-Bertani (LB) broth. Among the components of DMEM, only NaHCO(3) was found to remarkably stimulate bacterial adherence. When bacteria were grown in LB broth containing NaHCO(3), the production of intimin, Tir, EspA, and EspB was greatly enhanced compared with the production in LB broth. Indeed, the transcription of ler required for LEE-encoded gene expression was promoted in response to the concentration of NaHCO(3) in LB broth. Since the concentration of NaHCO(3) in the lower intestinal tract has been shown to be relatively high compared with that in the upper small intestine, our results may imply that NaHCO(3) is an important signaling factor for promoting colonization of EHEC in the lower intestinal tract in humans.

FIG. 1.

Effect of preincubation conditions on EHEC adherence to Caco-2 cells. (A) EHEC grown in DMEM shows a greater capacity to adhere to Caco-2 cells than EHEC grown in LB broth. After 30, 60, 90, 120, and 150 min, bacterial cultures were removed to measure optical density at 600 nm, and the bacteria were allowed to adhere to Caco-2 cells. The adherence assay was performed in triplicate, and values are expressed relative to the value for EHEC preincubated in DMEM for 120 min. The data are the means and standard errors of the means for microscopic fields. (B) Caco-2 cells were infected with EHEC strain O157Sakai preincubated in LB broth or DMEM for 120 min. Microcolony formation was examined by Giemsa staining and phase-contrast microscopy (40).

FIG. 2.

Effect of DMEM components on EHEC adherence to Caco-2 cells. Bacteria were preincubated in test tubes containing LB broth supplemented with 1.8 mM CaCl₂, with 5.3 mM KCl, with 0.8 mM MgSO₄, with 44 mM NaHCO₃, with 1 mM phosphate buffer (pH 7.4), with 25 mM HEPES-Tris (pH 7.4), with 1 mM sodium pyruvate, or with 0.1% Casamino Acids. (A) EHEC grown in LB broth supplemented with NaHCO₃ shows a greater capacity to adhere to Caco-2 cells than EHEC grown in LB broth and DMEM. After 120 min, bacterial cultures were allowed to adhere to Caco-2 cells. The adherence assay was performed in triplicate, and values are expressed relative to the value for EHEC preincubated in DMEM for 120 min. The data are the means and standard errors of the means for microscopic fields. (B) Caco-2 cells were infected with EHEC O157Sakai preincubated in LB broth supplemented with NaHCO₃ or in DMEM for 120 min. Microcolony formation was examined by Giemsa staining and phase-contrast microscopy.

FIG. 3.

Effect of NaHCO₃ on the expression of LEE-encoded genes. Whole-cell extracts of EHEC strain O157Sakai grown in LB broth supplemented with 0, 2, 5, 11, 22, and 44 mM NaHCO₃ (lanes 1, 2, 3, 4, 5, and 6, respectively) and in DMEM supplemented with 2, 5, 11, 22, and 44 mM NaHCO₃ (lanes 7, 8, 9, 10, and 11, respectively) were analyzed. The expression levels of intimin, Tir, EspB, and EspA were determined by Western blotting with protein-specific antiserum (40).

FIG.4.

Effect of the HCO₃⁻ ion on the expression of LEE-encoded genes. EHEC strain O157Sakai was grown in LB broth overnight and then diluted 1:20 with fresh medium. Bacteria were grown for 120 min at 37°C, and 1 ml of the culture was removed to measure optical density and to prepare whole-cell extracts. (A) The pH of LB broth did not affect the expression of LEE-encoded genes. Whole-cell extracts of EHEC strain O157Sakai grown in LB broth supplemented with 0 and 44 mM NaHCO₃ (lanes 1 and 2) and in DMEM (lane 3) were analyzed by using the experimental procedure. Whole-cell extracts of EHEC strain O157Sakai grown in MOPS-LB broth at pH 6.0, 6.5, 6.7, 7.0, 7.2, 7.5, 7.7, and 8.0 (lanes 4, 5, 6, 7, 8, 9, 10, and 11, respectively) and at pH 7.5 supplemented with 44 mM NaHCO₃ (lane 13) were analyzed. (B) KHCO₃ also stimulated the expression of intimin, Tir, EspB, and EspA. Whole-cell extracts of EHEC strain O157Sakai grown in LB broth supplemented with 0, 2, 5, 11, 22, 44, and 88 mM KHCO₃ (lanes 1, 3, 4, 5, 6, 7, and 8, respectively) and with 44 mM NaHCO₃ (lane 2) were analyzed by using the experimental procedure described above. (C) Whole-cell extracts of EHEC strain O157Sakai (lanes 1 to 3), EPEC strain E2348/69 (lanes 4 to 6), and EPEC strain B171-8 (lanes 7 to 9) grown in LB broth (lanes 1, 4, and 7), in LB broth supplemented with 44 mM NaHCO₃ (lanes 2, 5, and 8), and in DMEM (lanes 3, 6, and 9) were analyzed by using experimental procedure described above. The expression levels of EspB were determined by Western blotting with EspB-specific antiserum. (D) Whole-cell extracts of EHEC strains O157Sakai (lanes 1 and 2), O157Okayama01 (lanes 3 and 4), O157V425 (lanes 5 and 6), O157Oku133 (lanes 7 and 8), O157Oku702 (lanes 9 and 10), and O157Niimi14 (lanes 11 and 12) grown in LB broth (lanes 1, 3, 5, 7, 9, and 11) and in LB broth supplemented with 44 mM NaHCO₃ (lanes 2, 4, 6, 8, 10, and 12) were analyzed by using the experimental procedure described above.

FIG.5.

HCO₃⁻ ion stimulates the expression of LEE-encoded regulator. EHEC O157:H7 was grown in LB broth overnight and then diluted 1:20 with fresh medium. Bacteria were grown for 120 min at 37°C, and 10 ml of the culture was removed to measure optical density and to isolate total cellular RNAs. (A) NaHCO₃ stimulated transcription of ler. Total RNAs (10 μg) prepared from EHEC strain O157Sakai grown in LB broth not supplemented with NaHCO₃ (lane 1) or supplemented with 44 mM NaHCO₃ (lane 2) and in DMEM (lane 3) were subjected to Northern blot analysis. The transcription levels of ler mRNA were determined by using the labeled Sau3AI DNA fragment containing the ler gene as the probe (upper panel). The levels of ihf mRNA were determined as the control by using the labeled PCR fragment containing the ihfα gene as the probe (lower panel). (B) Cloning of the ler regulatory region into the pKK232-8 vector. The blunted 930-bp EcoRI PCR fragment encompassing the ler regulatory region was cloned into the SmaI site between the rrnB T1 terminator and the cat gene. (C) Activation of the ler promoter in response to addition of NaHCO₃ to LB broth. EHEC strain O157Sakai containing pKK232-8 (triangles) or plerCAT (squares) was grown in LB broth (open symbols) or in LB broth supplemented with 44 mM NaHCO₃ (solid symbols). The solid triangles are not visible due to overlap with the open triangles. CAT activity was determined at different times (38). The data are the means and standard errors of the means (SEM) obtained in triplicate. SEMs are not visible due to the very narrow range. OD₆₀₀, optical density at 600 nm; w.t, EHEC strain O157Sakai wild type. (D) Effect of ler overproduction on adherence of EHEC to Caco-2 cells. EHEC strain O157Sakai containing pBR322 or pBRler was grown in LB broth. After 120 min, bacterial cultures were allowed to adhere to Caco-2 cells. The adherence assay was performed in triplicate, and values are expressed relative to the value for EHEC preincubated in DMEM for 120 min. The data are the means and standard errors of the means for microscopic fields. (E) Caco-2 cells were infected with EHEC strain O157Sakai containing pBRler preincubated in LB broth for 120 min. Microcolony formation was examined by Giemsa staining and phase-contrast microscopy.

FIG.5.

HCO₃⁻ ion stimulates the expression of LEE-encoded regulator. EHEC O157:H7 was grown in LB broth overnight and then diluted 1:20 with fresh medium. Bacteria were grown for 120 min at 37°C, and 10 ml of the culture was removed to measure optical density and to isolate total cellular RNAs. (A) NaHCO₃ stimulated transcription of ler. Total RNAs (10 μg) prepared from EHEC strain O157Sakai grown in LB broth not supplemented with NaHCO₃ (lane 1) or supplemented with 44 mM NaHCO₃ (lane 2) and in DMEM (lane 3) were subjected to Northern blot analysis. The transcription levels of ler mRNA were determined by using the labeled Sau3AI DNA fragment containing the ler gene as the probe (upper panel). The levels of ihf mRNA were determined as the control by using the labeled PCR fragment containing the ihfα gene as the probe (lower panel). (B) Cloning of the ler regulatory region into the pKK232-8 vector. The blunted 930-bp EcoRI PCR fragment encompassing the ler regulatory region was cloned into the SmaI site between the rrnB T1 terminator and the cat gene. (C) Activation of the ler promoter in response to addition of NaHCO₃ to LB broth. EHEC strain O157Sakai containing pKK232-8 (triangles) or plerCAT (squares) was grown in LB broth (open symbols) or in LB broth supplemented with 44 mM NaHCO₃ (solid symbols). The solid triangles are not visible due to overlap with the open triangles. CAT activity was determined at different times (38). The data are the means and standard errors of the means (SEM) obtained in triplicate. SEMs are not visible due to the very narrow range. OD₆₀₀, optical density at 600 nm; w.t, EHEC strain O157Sakai wild type. (D) Effect of ler overproduction on adherence of EHEC to Caco-2 cells. EHEC strain O157Sakai containing pBR322 or pBRler was grown in LB broth. After 120 min, bacterial cultures were allowed to adhere to Caco-2 cells. The adherence assay was performed in triplicate, and values are expressed relative to the value for EHEC preincubated in DMEM for 120 min. The data are the means and standard errors of the means for microscopic fields. (E) Caco-2 cells were infected with EHEC strain O157Sakai containing pBRler preincubated in LB broth for 120 min. Microcolony formation was examined by Giemsa staining and phase-contrast microscopy.

FIG.5.

HCO₃⁻ ion stimulates the expression of LEE-encoded regulator. EHEC O157:H7 was grown in LB broth overnight and then diluted 1:20 with fresh medium. Bacteria were grown for 120 min at 37°C, and 10 ml of the culture was removed to measure optical density and to isolate total cellular RNAs. (A) NaHCO₃ stimulated transcription of ler. Total RNAs (10 μg) prepared from EHEC strain O157Sakai grown in LB broth not supplemented with NaHCO₃ (lane 1) or supplemented with 44 mM NaHCO₃ (lane 2) and in DMEM (lane 3) were subjected to Northern blot analysis. The transcription levels of ler mRNA were determined by using the labeled Sau3AI DNA fragment containing the ler gene as the probe (upper panel). The levels of ihf mRNA were determined as the control by using the labeled PCR fragment containing the ihfα gene as the probe (lower panel). (B) Cloning of the ler regulatory region into the pKK232-8 vector. The blunted 930-bp EcoRI PCR fragment encompassing the ler regulatory region was cloned into the SmaI site between the rrnB T1 terminator and the cat gene. (C) Activation of the ler promoter in response to addition of NaHCO₃ to LB broth. EHEC strain O157Sakai containing pKK232-8 (triangles) or plerCAT (squares) was grown in LB broth (open symbols) or in LB broth supplemented with 44 mM NaHCO₃ (solid symbols). The solid triangles are not visible due to overlap with the open triangles. CAT activity was determined at different times (38). The data are the means and standard errors of the means (SEM) obtained in triplicate. SEMs are not visible due to the very narrow range. OD₆₀₀, optical density at 600 nm; w.t, EHEC strain O157Sakai wild type. (D) Effect of ler overproduction on adherence of EHEC to Caco-2 cells. EHEC strain O157Sakai containing pBR322 or pBRler was grown in LB broth. After 120 min, bacterial cultures were allowed to adhere to Caco-2 cells. The adherence assay was performed in triplicate, and values are expressed relative to the value for EHEC preincubated in DMEM for 120 min. The data are the means and standard errors of the means for microscopic fields. (E) Caco-2 cells were infected with EHEC strain O157Sakai containing pBRler preincubated in LB broth for 120 min. Microcolony formation was examined by Giemsa staining and phase-contrast microscopy.

FIG.5.

HCO₃⁻ ion stimulates the expression of LEE-encoded regulator. EHEC O157:H7 was grown in LB broth overnight and then diluted 1:20 with fresh medium. Bacteria were grown for 120 min at 37°C, and 10 ml of the culture was removed to measure optical density and to isolate total cellular RNAs. (A) NaHCO₃ stimulated transcription of ler. Total RNAs (10 μg) prepared from EHEC strain O157Sakai grown in LB broth not supplemented with NaHCO₃ (lane 1) or supplemented with 44 mM NaHCO₃ (lane 2) and in DMEM (lane 3) were subjected to Northern blot analysis. The transcription levels of ler mRNA were determined by using the labeled Sau3AI DNA fragment containing the ler gene as the probe (upper panel). The levels of ihf mRNA were determined as the control by using the labeled PCR fragment containing the ihfα gene as the probe (lower panel). (B) Cloning of the ler regulatory region into the pKK232-8 vector. The blunted 930-bp EcoRI PCR fragment encompassing the ler regulatory region was cloned into the SmaI site between the rrnB T1 terminator and the cat gene. (C) Activation of the ler promoter in response to addition of NaHCO₃ to LB broth. EHEC strain O157Sakai containing pKK232-8 (triangles) or plerCAT (squares) was grown in LB broth (open symbols) or in LB broth supplemented with 44 mM NaHCO₃ (solid symbols). The solid triangles are not visible due to overlap with the open triangles. CAT activity was determined at different times (38). The data are the means and standard errors of the means (SEM) obtained in triplicate. SEMs are not visible due to the very narrow range. OD₆₀₀, optical density at 600 nm; w.t, EHEC strain O157Sakai wild type. (D) Effect of ler overproduction on adherence of EHEC to Caco-2 cells. EHEC strain O157Sakai containing pBR322 or pBRler was grown in LB broth. After 120 min, bacterial cultures were allowed to adhere to Caco-2 cells. The adherence assay was performed in triplicate, and values are expressed relative to the value for EHEC preincubated in DMEM for 120 min. The data are the means and standard errors of the means for microscopic fields. (E) Caco-2 cells were infected with EHEC strain O157Sakai containing pBRler preincubated in LB broth for 120 min. Microcolony formation was examined by Giemsa staining and phase-contrast microscopy.

FIG.5.

HCO₃⁻ ion stimulates the expression of LEE-encoded regulator. EHEC O157:H7 was grown in LB broth overnight and then diluted 1:20 with fresh medium. Bacteria were grown for 120 min at 37°C, and 10 ml of the culture was removed to measure optical density and to isolate total cellular RNAs. (A) NaHCO₃ stimulated transcription of ler. Total RNAs (10 μg) prepared from EHEC strain O157Sakai grown in LB broth not supplemented with NaHCO₃ (lane 1) or supplemented with 44 mM NaHCO₃ (lane 2) and in DMEM (lane 3) were subjected to Northern blot analysis. The transcription levels of ler mRNA were determined by using the labeled Sau3AI DNA fragment containing the ler gene as the probe (upper panel). The levels of ihf mRNA were determined as the control by using the labeled PCR fragment containing the ihfα gene as the probe (lower panel). (B) Cloning of the ler regulatory region into the pKK232-8 vector. The blunted 930-bp EcoRI PCR fragment encompassing the ler regulatory region was cloned into the SmaI site between the rrnB T1 terminator and the cat gene. (C) Activation of the ler promoter in response to addition of NaHCO₃ to LB broth. EHEC strain O157Sakai containing pKK232-8 (triangles) or plerCAT (squares) was grown in LB broth (open symbols) or in LB broth supplemented with 44 mM NaHCO₃ (solid symbols). The solid triangles are not visible due to overlap with the open triangles. CAT activity was determined at different times (38). The data are the means and standard errors of the means (SEM) obtained in triplicate. SEMs are not visible due to the very narrow range. OD₆₀₀, optical density at 600 nm; w.t, EHEC strain O157Sakai wild type. (D) Effect of ler overproduction on adherence of EHEC to Caco-2 cells. EHEC strain O157Sakai containing pBR322 or pBRler was grown in LB broth. After 120 min, bacterial cultures were allowed to adhere to Caco-2 cells. The adherence assay was performed in triplicate, and values are expressed relative to the value for EHEC preincubated in DMEM for 120 min. The data are the means and standard errors of the means for microscopic fields. (E) Caco-2 cells were infected with EHEC strain O157Sakai containing pBRler preincubated in LB broth for 120 min. Microcolony formation was examined by Giemsa staining and phase-contrast microscopy.