ETEC colonisation factors disrupt the antigen presenting capacity of porcine intestinal dendritic cells

Enterotoxigenic E. coli (ETEC) are not only a major cause of diarrhoea in travellers to and children in developing countries, but also cause neonatal and postweaning diarrhoea in piglets, leading to a reduced feed conversion and a higher mortality rate. As a consequence ETEC infections result in severe economic losses in the swine production industry. This intestinal pathogen displays colonisation factors or fimbriae on its surface enabling the microorganism to adhere to the intestinal epithelium (Fig. 1). In pig, F4 and F18 fimbriae are the most frequently associated with ETEC-induced diarrhoea1. As opposed to F4 fimbriae, oral immunisation with F18 fimbriae doesn’t protect piglets from a subsequent challenge infection2. F18 fimbriae bind glycosphingolipids in the apical membrane of enterocytes, but no transcytosis occurs, resulting in lower sunepithelial antigen concentrations as compared to F4 fimbriae, which bind the transcytotic receptor aminopeptidase N3,4. However, M-cell mediated transport of F18 fimbriae should still occur. Hence, besides a lower antigen concentration, these fimbriae could affect the function of intestinal antigen presenting cells. Here, we investigated the influence of purified F18 fimbriae on the antigen presentation capacity of small intestinal lamina propria dendritic cells (LPDCs)

Enterotoxigenic Escherichia coli induce pro-inflammatory responses in porcine intestinal epithelial cells

F4+ enterotoxigenic Escherichia coli (ETEC) cause severe diarrhoea in both neonatal and weaning piglets, resulting in morbidity and mortality. F4 fimbriae are a key virulence factor involved in the attachment of F4+ ETEC to the intestinal epithelium. Intestinal epithelial cells (IEC) are recently being recognized as important regulators of the intestinal immune system through the secretion of cytokines, however, data on how F4+ ETEC affect this cytokine secretion are scarce. By using ETEC strains expressing either polymeric, monomeric or F4 fimbriae with a reduced polymeric stability, we demonstrated that polymeric fimbriae are essential for the adhesion of ETEC to porcine IEC as well as for the secretion of IL-6 and IL-8 by ETEC-stimulated intestinal epithelial cells. Remarkably, this cytokine secretion was not abrogated following stimulation with an F4-negative strain. As this ETEC strain expresses flagellin, TLR5 mediated signalling could be involved. Indeed, porcine IEC express TLR5 and purified flagellin induced IL-6 and IL-8 secretion, indicating that, as for other pathogens, flagellin seems to be the dominant virulence factor involved in the induction of proinflammatory responses in IEC upon ETEC infection. These results indicate a potential mucosal adjuvant capacity of ETEC-derived flagellin and may improve rational vaccine design against F4+ ETEC infections

ETEC colonization factors modulate the antigen presentation function of porcine intestinal mononuclear phagocytes

C

Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeG(ntd/dsc), engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeG(ntd/dsc) per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeG(ntd/dsc) protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeG(ntd/dsc) in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeG(ntd/dsc) protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD