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.

Effects of different concentrations of enterotoxigenic and verotoxigenic E. coli on boar sperm quality.

The presence of bacteria in boar semen causes economic losses in artificial insemination (AI) centers, as a consequence of alterations on boar sperm quality. For this reason, the effects of different concentrations of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) on boar sperm quality were determined in this study, by conducting two experiments. The first one consisted of assessing these effects on boar sperm quality after incubating the inoculated doses at 37°C for a 96-h period, whereas the second inoculated doses were stored at 15°C during 11 days. In both experiments, the infective concentrations ranged from 10(8)cfu mL(-1) to 10(2)cfu mL(-1); the negative control being a non-inoculated dose. Twenty-four hours after inoculation, we checked by PCR for the presence of bacteria in all tubes. Sperm quality (sperm motility, sperm viability and sperm morphology) was assessed at 24h, 48h, 72h and 96h after inoculations in the first experiment (37°C), and after 3, 5, 7, 9 and 11 days in the second (15°C). Whereas no changes were observed in sperm morphology in both experiments, the percentages of progressive motile spermatozoa dramatically diminished after 24h of incubation at 37°C, the effect being more detrimental at the highest infective concentration of microbes. Moreover, a significant decrease in the percentage of viable spermatozoa in the tube inoculated with the highest concentration (10(8)cfu mL(-1)) was detected after 24h of incubating contaminated doses at 37°C. After 48h of incubation, the presence of infective concentrations of ETEC and VTEC from 10(8)cfu mL(-1) to 10(3)cfu mL(-1) resulted in a significant diminution in the percentage of viable spermatozoa. These results suggest that ETEC and VTEC PCR analyses should be done in doses destined for AI to minimize the use of doses with diminished sperm quality due to the presence of bacteria and to avoid the potential spread of infective diseases.

Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriae.

Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1 A resolution and displayed the symmetry of space group P2(1). CfaBB exhibited a crystal diffraction limit of 2.3 A resolution and had the symmetry of space group P2(1)2(1)2. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3 A resolution. These structures were determined using the molecular-replacement method.

Enterotoxigenic Escherichia coli EtpA mediates adhesion between flagella and host cells.

Adhesion to epithelial cells and flagella-mediated motility are critical virulence traits for many Gram-negative pathogens, including enterotoxigenic Escherichia coli (ETEC), a major cause of diarrhoea in travellers and children in developing countries. Many flagellated pathogens export putative adhesins belonging to the two-partner secretion (TPS) family. However, the actual function of these adhesins remains largely undefined. Here we demonstrate that EtpA, a TPS exoprotein adhesin of enterotoxigenic E. coli, mimics and interacts with highly conserved regions of flagellin, the major subunit of flagella, and that these interactions are critical for adherence and intestinal colonization. Although conserved regions of flagellin are mostly buried in the flagellar shaft, our results suggest that they are at least transiently exposed at the tips of flagella where they capture EtpA adhesin molecules for presentation to eukaryotic receptors. Similarity of EtpA to molecules encoded by other motile pathogens suggests a potential common pattern for bacterial adhesion, whereas participation of conserved regions of flagellin in adherence has implications for development of vaccines for Gram-negative pathogens.