Porcine aminopeptidase N binds to F4+ enterotoxigenic Escherichia coli fimbriae

Abstract

F4(+) enterotoxigenic Escherichia coli (ETEC) strains cause diarrheal disease in neonatal and post-weaned piglets. Several different host receptors for F4 fimbriae have been described, with porcine aminopeptidase N (APN) reported most recently. The FaeG subunit is essential for the binding of the three F4 variants to host cells. Here we show in both yeast two-hybrid and pulldown assays that APN binds directly to FaeG, the major subunit of F4 fimbriae, from three serotypes of F4(+) ETEC. Modulating APN gene expression in IPEC-J2 cells affected ETEC adherence. Antibodies raised against APN or F4 fimbriae both reduced ETEC adherence. Thus, APN mediates the attachment of F4(+) E. coli to intestinal epithelial cells.

Figure 1

Yeast two-hybrid (Y2H) and pulldown assays. A Y2H. pGADT7-FaeG and pGBKT7-APN were co-expressed in yeast and positive clones were tested for β-galactosidase activity. Samples 1–3: F4ab FaeG-APN; Samples 4–6: F4ac FaeG-APN; Samples 7–9: F4ad FaeG-APN; N: negative control; P: positive control. B GST-pulldown assays. The binding between the recombinant FaeG and APN proteins was studied using the Pierce™ GST Protein Interaction Pull-Down Kit. Western blotting with anti-F4 monoclonal antiserum and anti-APN polyclonal antiserum was used for detection. C Metaperiodate treatment. PVDF membranes were treated with either 0, 10, or 20 mM NaIO₄ before using the membranes in Western blots as described in B. Each experiment was repeated three times and representative results are shown.

Figure 2

apn knockdown and overexpression in IPEC-J2 cells. A RT-PCR. apn mRNA levels in pEC129-APN-IPEC-J2 cells, pcDNA™6.2-GW/miR-APN-IPEC-J2 cells, and IPEC-J2 cells were quantified using RT-PCR and normalized to gapdh expression. The asterisk indicates a statistically significant difference in expression when compared to the original cells (p < 0.05). B Western blotting. Proteins from IPEC-J2, pEC129-APN-IPEC-J2, and pcDNA™6.2-GW/miR-APN-IPEC-J2 cells were harvested in RIPA buffer. Blots were incubated overnight with polyclonal antibodies against APN and stained with enhanced chemiluminescence (ECL) (Pierce) reagents. Each experiment was repeated three times and representative results are shown.

Figure 3

Modulating APN expression affects ETEC adherence. A Adhesion of F4 E. coli strains to IPEC-J2 cells. Bacterial adherence to the original IPEC-J2 cell line was normalized to 100%. B In vitro inhibition assay. Adherence of F4⁺ ETEC to IPEC-J2 cells after pre-incubation with anti-F4 fimbriae monoclonal antiserum (1:100 dilution) or with anti-APN polyclonal antiserum (1:1, 1:10, 1:100 dilutions). The adhesion of the untreated samples was normalized 100%. The experiments were repeated three times and data are expressed as mean ± standard deviations (p < 0.05).