Wild-type C1845-induced gene expression

<p>Cells were infected for four hours with Afa/Dr DAEC wild-type strain C1845 and total RNA extracted. Selected gene expression was assayed by q-PCR. The oligonucleotides used in this study were designed by Applied Biosystems, the manufacturer's references are given in brackets. Abbreviations are as follows: VEGF: Vascular Endothelial Growth Factor, IL8: interleukin 8, PlGF: Placental growth factor, TGFβ R2: Transforming growth factor Receptor 2, EGF R1: Epithelial growth factor Receptor 1. Results (means±SD) are representative of independent experiments (where n represents the number of experiments). *, p<0.001 versus non infected cells.</p

Increase in VEGF mRNA by wild-type C1845 bacteria requires binding to brush border-associated DAF.

<p>In A, binding to DAF is necessary for the C1845-induced increase in VEGF mRNA expression. Confluent serum-starved T84 cells infected were with wild-type C1845 at 5×10⁷ CFU/ml, which represents a multiplicity of infection of 20 bacteria per epithelial cell, for two hours. When indicated, cells were treated for 30 minutes prior to infection with the anti-DAF blocking IH4 monoclonal antibody. VEGF mRNA expression was assayed by q-PCR. *, p = 0,03 (n = 7). In B, wild-type C1845-induced VEGF mRNA increase is blocked by transcription inhibitor. Confluent serum-starved T84 cells were pre-treated for 30 minutes with 25 µg/ml of the reversible transcription inhibitor, 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB). Cells were then infected with 5×10⁷ CFU/ml wild-type C1845 for four hours and total RNA prepared. The relative quantity of VEGF mRNA was measured by q-PCR. Quantification of the results from three independent experiments (means±SD) is shown. *, p<0,01.</p

C1845 bacteria increase VEGF expression in cultured intestinal epithelial cells.

<p>In A, increase in VEGF mRNA expression. Confluent serum-starved T84 cells (5×10⁶ cells/well) were infected with wild-type C1845 bacteria and total RNA was prepared as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001359#s4" target="_blank">Materials and Methods</a> section. Results for non-infected (NI) or cells infected with 5×10⁷ CFU/ml C1845 bacteria for the indicated time are shown on the left panel. The dose response effect is presented on the right. Cells were infected for four hours with the indicated number of bacteria. Results of Northern blots are presented in the upper part of the Figure and q-PCR shown in the lower part. These results are representative of three independent experiments. In B, confluent serum-starved LS174, Caco-2/TC7 or INT407 cells were infected with 5×10⁷ CFU/ml wild-type C1845 bacteria for four hours. VEGF mRNA expression was assayed by q-PCR. The signal corresponding to VEGF and 36B4 transcripts was quantified using a phosphoImager. Under each condition the signal was normalized to the 36B4 probe. Results are expressed as arbitrary units corresponding to the fold stimulation of treated versus non-treated conditions. In C, increase in the VEGF protein in the culture medium of wild-type C1845-infected T84 cells. Cells were infected with 5×10⁷ CFU/ml wild-type C1845 bacteria for four hours and the supernatant were collected as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001359#s4" target="_blank">Materials and Methods</a> section. The VEGF protein level was then quantified using an ELISA. Quantification of the results from two independent experiments (means±SD) is shown. *, p<0,01</p

Proliferative response, Erk activation and tube like structure formation in HUVEC in the presence of culture medium of wild-type C1845-infected T84 cells.

<p>³H-thymidine incorporation into DNA and Erk activation were essayed in HUVEC incubated with the following. In A, ³H-thymidine incorporation into DNA. Control (1), VEGF at 10 ng/ml (2), culture medium of non-infected T84 cells (3), culture medium of wild-type C1845-infected T84 cells (1×) (4), culture medium of wild-type C1845-infected T84 cells (0.5×) (5). In B, Western blot showing phospho-Erk1/2 and total Erk1/2 visualized with respectively anti-phospho-Erk and anti-Erk antibodies. Control (1), VEGF at 10 ng/ml (23), culture medium of wild-type C1845-infected T84 cells (1×) (3), culture medium of wild-type C1845-infected T84 cells (0.5×) (4). The results are representative of at least three independent experiments. *, p<0,01. In C, Endothelial cells were seeded on matrigel and incubated with growing medium (control) alone or supplemented with T84 conditionned medium (CM), C1845–condition medium (C1845) or VEGF. Images show that only C1845-conditionned medium (C1845) and VEGF allow the alignment of endothelial cells in tube like structure.</p

Wild-type C1845 bacteria induce Akt and Erk signaling pathways in T84 epithelial cells.

<p>Confluent serum-starved T84 cells were non-infected (NI) or infected with increasing concentrations of wild-type C1845 bacteria, for two hours. When indicated, cells were pre-treated with an anti-DAF blocking IH4 monoclonal antibody prior to or in the absence of infection. In A, phospho-Akt and total Akt were assayed by Western blotting using respectively anti-phospho-Akt and anti-Akt antibodies. In B, phospho-Erk1/2 and total Erk1/2 were visualized with respectively anti-phospho-Erk and anti-Erk antibodies. These results are representative of at least three independent experiments.</p

Wild-type C1845-induced VEGF expression is dependant on Src and Erk proteins.

<p>Confluent serum-starved T84 cells were pre-treated with 5 µM PP2, 10 µM U0126 or 15 µM LY294002 for 30 minutes. Cells were then infected with wild-type C1845 bacteria at 5×10⁷ CFU/ml for four hours. Total RNA was prepared and the relative quantity of VEGF mRNA was measured by q-PCR. Quantification of the results from four independent experiments (means±SD.) is shown. *, p<0.005 versus non infected cells.</p

A Src protein is involved in wild-type C1845-induced Akt and Erk activation.

<p>Confluent serum-starved T84 cells were pre-treated with 15 µM LY294002 (LY), 10 µM U0126 (U0), 5 µM GF109203X (GFX) or 5 µM PP2 for 30 minutes prior to infection or no infection with wild-type C1845 bacteria at 5×10⁷ CFU/ml for two hours. Cells were lyzed in SDS sample buffer and phospho-Akt and total Akt (in A) and phospho-Erk and total Erk1/2 (in B) assayed by Western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001359#pone-0001359-g005" target="_blank">Figure 5</a>. These results are representative of at least three independent experiments.</p

F1845 adhesin but not LPS, activates Erk and increases VEGF expression.

<p>In A, confluent serum-starved T84 cells were non infected (NI), infected with 5×10⁷ CFU/ml wild-type C1845 bacteria, or treated for 2 hours with indicated concentration of LPS purified from non-pathogenic AAEC185 or C1845 strains. Phospho-Erk1/2 and total Erk1 were visualized by Western Blotting with respectively anti-phospho-Erk and anti-Erk1 antibodies. This experiment is representative of two independent experiments. In B, confluent serum-starved T84 cells were non-infected (NI), infected with increasing concentrations of AAEC185 or recombinant AAEC185-F1845 strains for 2 h. Phospho-Erk and total Erk-1 were assayed by Western blotting using respectively anti-phospho-Erk and anti-Erk1 antibodies. In C, coomassie-stained polyacrylamide-SDS gel of fimbriae purified from C1845 or AAEC185 bacteria. 1 µg of bovine serum albumine (BSA) are showed on the left of the gel. In D, confluent serum-starved T84 cells were non treated (NT) or incubated 2 h in the presence of extracts of C1845 bacteria (containing 2 µg of purified F1845 adhesin) or AAEC185 bacteria. These result are representative of three independent experiments. In E, confluent serum-starved T84 cells (5×10⁶ cells/well) were infected with AAEC185 bacteria or recombinant AAEC185-F1845 bacteria expressing the pSSS1 operon encoding the F1845 adhesin (5×10⁷ CFU/ml). Total RNA were extracted and analysed by q-PCR. These results are representative of at least two independent experiments (means±SD).</p