Sheep β-Defensin 2 Regulates Escherichia coli F17 Resistance via NF-κB and MAPK Signaling Pathways in Ovine Intestinal Epithelial Cells

Abstract

Escherichia coli (E. coli) F17 is a member of enterotoxigenic Escherichia coli, which can cause massive diarrhea and high mortality in newborn lambs. β-defensin is mainly produced by the epithelial tissue of the gastrointestinal tract in response to microbial infection. However, the molecular mechanism of sheep β-defensin 2 (SBD-2) against E. coli F17 remains unclear. This study aims to reveal the antibacterial ability of SBD-2 against E. coli F17 infection in sheep. Firstly, we established the culture system of ovine intestinal epithelial cells (OIECs) in vitro, treated with different concentrations of E. coli F17 for an indicated time. Secondly, we performed RNA interference and overexpression to investigate the effect of SBD-2 expression on E. coli F17 adhesion to OIECs. Finally, inhibitors of NF-κB and MAPK pathways were pre-treated to explore the possible relationship involving in E. coli F17 infection regulating SBD-2 expression. The results showed that E. coli F17 markedly (p < 0.01) upregulated the expression levels of SBD-2 mRNA and protein in a concentration- and time-dependent manner. Overexpression of SBD-2 contributed to enhancing E. coli F17 resistance in OIECs, while silencing SBD-2 dramatically improved the adhesion of E. coli F17 to OIECs (p < 0.05 or p < 0.01). Furthermore, E. coli F17 stimulated SBD-2 expression was obviously decreased by pre-treatment with NF-κB inhibitor PDTC, p38 MAPK inhibitor SB202190 and ERK1/2 MAPK inhibitor PD98095 (p < 0.05 or p < 0.01). Interestingly, adhesion of E. coli F17 to OIECs were highly enhanced by pre-treated with PDTC, SB202190 and PD98095. Our data suggested that SBD-2 could inhibit E. coli F17 infection in OIECs, possibly through NF-κB and MAPK signaling pathways. Our results provide useful theoretical basis on developing anti-infective drug and breeding for E. coli diarrhea disease-resistant sheep.

Keywords: Escherichia coli F17; MAPK pathway; NF-κB pathway; SBD-2; inflammation; sheep.

Figure 1

E. coli F17 stimulates mRNA and protein expression levels of SBD-2. (A) SBD-2 mRNA expression levels detected by RT-PCR in OIECs treated with the indicated concentrations of E. coli F17 compared with non-treated controls. (B) SBD-2 mRNA expression levels detected by RT-PCR in OIECs treated with E. coli F17 (10⁷ CFU/mL) for various time intervals compared with non-treated controls. (C) Standard curve drawn by SBD-2 protein concentration value (y axis) and OD value at 450 nm (x axis). (D) SBD-2 protein expression levels detected by ELISA in OIECs treated with the indicated concentrations of E. coli F17 compared with non-treated controls. (E) SBD-2 protein expression levels detected by ELISA in OIECs treated with E. coli F17 (10⁷ CFU/mL) for various time intervals compared with non-treated controls. Mean values with different letters in the same row are significantly different (p < 0.05) according to Duncan’s multiple range test; data were shown as mean ± SD, n = 3 biological replicates.

Figure 2

The effect of SBD-2 expression on the adhesion of E. coli F17 to OIECs. (A) Overexpression efficiency of SBD-2 mRNA expression level detected by RT-PCR in pGH (Control) cells and pGH-SBD-2 cells. (B) Overexpression efficiency of SBD-2 protein expression level detected by ELISA in pGH (Control) cells and pGH-SBD-2 cells. (C) Adhesion of the F17 fimbria to OIECs transfected with pGH-SBD-2 compared with pGH (Control) analyzed by bacteria enumeration. (D) Interference efficiency of SBD-2 mRNA expression level detected by RT-PCR in OIECs transfected with siRNA-SBD-2 compared with negative control. (E) Interference efficiency of SBD-2 protein expression level detected by ELISA in OIECs transfected with siRNA-SBD-2 compared with negative control. (F) Adhesion of the F17 fimbria to OIECs transfected with siRNA-SBD-2 compared with negative control analyzed by bacteria enumeration. ** p < 0.01, extremely significant difference; NS = no difference. Data were shown as mean ± SD, n = 3 biological replicates.

Figure 3

The effect of E. coli F17 stimulation in OIECs on NF-κB and MAPK pathways. (A) p65, p50, p38, ERK1/2 and JNK expression level determined by RT-PCR in OIECs treated with E. coli F17 (10⁷ CFU/mL) for 6 h compared with non-treated control. (B) p50 expression level determined by RT-PCR in OIECs treated with NF-κB inhibitor PDTC, PDTC+E. coli F17 and E. coli F17 compared with non-treated control. (C) p38 expression level determined by RT-PCR in OIECs treated with p38 MAPK inhibitor SB202190, SB202190+E. coli F17 and E. coli F17 compared with non-treated control. (D) ERK1/2 expression level determined by RT-PCR in OIECs treated with ERK1/2 MAPK inhibitor PD98095, PD98095+E. coli F17 and E. coli F17 compared with non-treated control. * p < 0.05, significant difference; ** p < 0.01, extremely significant difference; NS = no difference. Data were shown as mean ± SD, n = 3 biological replicates.

Figure 4

The effect of NF-κB and MAPK pathways on SBD-2 expression. (A) SBD-2 relative expression determined by RT-PCR in OIECs treated with PDTC, PDTC+E. coli F17 and E. coli F17 compared with non-treated control. (B) SBD-2 protein expression determined by ELISA in OIECs treated with NF-κB inhibitor PDTC, PDTC+E. coli F17 and E. coli F17 compared with non-treated control. (C) SBD-2 relative expression determined by RT-PCR in OIECs treated with p38 MAPK inhibitor SB202190, SB202190+E. coli F17 and E. coli F17 compared with non-treated control. (D) SBD-2 relative expression determined by RT-PCR in OIECs treated with ERK1/2 MAPK inhibitor PD98095, PD98095+E. coli F17 and E. coli F17 compared with non-treated control. (E) SBD-2 protein expression determined by ELISA in OIECs treated with p38 MAPK inhibitor SB202190, SB202190+E. coli F17 and E. coli F17 compared with non-treated control. (F) SBD-2 protein expression determined by ELISA in OIECs treated with ERK1/2 MAPK inhibitor PD98095, PD98095+E. coli F17and E. coli F17 compared with non-treated control. * p < 0.05, significant difference; ** p < 0.01, extremely significant difference; NS = no difference. Data were shown as mean ± SD, n = 3 biological replicates.

Figure 5

The effect of NF-κB and MAPK pathways on the adhesion of E. coli F17 to OIECs. (A) Adhesion of the F17 fimbria to OIECs treated with NF-κB inhibitor PDTC compared with negative control analyzed by bacteria enumeration. (B) Adhesion of the F17 fimbria to OIECs treated with p38 MAPK inhibitor SB202190 and ERK1/2 MAPK inhibitor PD98095 compared with negative control analyzed by bacteria enumeration. ** p < 0.01, extremely significant difference; data were shown as mean ± SD, n = 3 biological replicates.