F4 Fimbriae Research Articles

Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro

The role of flagella in the pathogenesis of F4ac+ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference C83902 ETEC strain (O8:H19:F4ac+ LT+ STa+ STb+), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac+ ETEC adhesion in vitro.

Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin–toxoid fusion (1FaeG-FedF-LTA2:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection

Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin–toxoid fusion (1FaeG-FedF-LTA2:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection

Development and Evaluation of Porous Chitosan Nanoparticles for Treatment of Enterotoxigenic <I>Escherichia</I> <I>Coli</I> Infection

Enterotoxigenic Escherichia coil (ETEC) infections result in large mortality rate and usually a frequent cause of diarrhea in infants and a major cause of economic losses in the swine industry. To prevent enterotoxigenic Escherichia coli infections animal needs an active mucosal immunity at the moment of weaning. In the present study, F4 loaded porous chitosan nanoparticles were prepared by spray drying method for oral vaccination. In order to prevent the release the antigen in upper GI tract and to release it at target site nanoparticles were coated with Eudragit L100 which protect the antigen against the detrimental effects in the gastro-intestinal tract. Average size of prepared nanoparticles varied between 548 +/- 2.3 to 98 +/- 1.1 nm with a polydispersity index ranging from 0.767 +/- 0.023 to 0.209 +/- 0.021. Zeta potential for prepared nanoparticles was found to be in range from +18.3 +/- 2.5 to +29.5 +/- 2.8 mV. SEM studies completely revealed that the drug loaded nanoparticles were found to be distinct, spherical in shape with pores formed. Practicability of NPs was compared to vaccination with F4 fimbriae in solution. Mucosal immune response study revealed that, immune response were elicited in solution was well as in NPs group but colonization of the small intestine by F4+ ETEC upon oral solution challenge could not be prevented. However animals vaccinated with porous NPs group reveal a significant reduction in excretion of F4+ E. coli. Studies indicate that a solid vaccine formulation will be more efficient as compared to oral solutions. These systems can contribute to the development of oral vaccines in veterinary as well as in human medicines.

Oral Immunization with F4 Fimbriae and CpG Formulated with Carboxymethyl Starch Enhances F4-Specific Mucosal Immune Response and Modulates Th1 and Th2 Cytokines in Weaned Pigs

F4 fimbriae are a potential candidate for an oral subunit vaccine for prevention of post-weaning diarrhea in swine due to infection with F4-positive enterotoxigenic Escherichia coli. However, large quantities of F4 fimbriae are required to induce a specific antibody response. The aim of the present study was to evaluate the effect of supplementation of F4 fimbriae with Cytosine-phosphate-Guanosine-oligodeoxynucleotide (CpG-A D19) or with complete cholera toxin (CT) as adjuvants on the F4-specific antibody response and cytokine production in weaned pigs following oral administration of F4 fimbrial antigen formulated with Carboxymethyl Starch (CMS). Oral dosage forms of F4 fimbriae alone or supplemented with CpG-A D19 or with CT were formulated with CMS as monolithic tablets, obtained by direct compression, and administered to weaned pigs. Blood and faecal samples were collected to determine the systemic and mucosal immune status of animals at various times until necropsy. During necropsy, contents of the jejunum and ileum were collected for determination of mucosal F4 specific antibodies. Segments of jejunum and ileum were also used to measure mRNA cytokine production. The presence of CpG in the formulation of the fimbriae significantly increased F4-specific immunoglobulin (Ig) IgM and IgG levels in intestinal secretions, and enhanced Th1 (Interferon-gamma / IFN-γ, Tumour Necrosis Factor-alpha / TNF-α, Interleukin-12p40 / IL-12p40, IL-1β) and Th2 (IL-4, IL-6) cytokine production in intestinal tissues. Supplementation with CT did not result in induction of F4-specific antibodies in secretions, although a significant Th1 response (IFN-α, IFN-γ, IL-18) was detected in tissues. Neither F4-specific systemic antibodies, nor intestinally secreted IgA were detected throughout the immunization trial for all groups. CpG-A D19 appeared to be a promising adjuvant for an oral F4 subunit vaccine formulated with CMS excipient as monolithic tablets. This matrix afforded gastro-protection and delivered the F4 fimbriae at their intestinal sites.

One-step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells

One-step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells

Flagella from F18+Escherichia coli play a role in adhesion to pig epithelial cell lines

Flagella from F18+Escherichia coli play a role in adhesion to pig epithelial cell lines

Differentiation of F4 receptor profiles in pigs based on their mucin 4 polymorphism, responsiveness to oral F4 immunization and in vitro binding of F4 to villi

Differentiation of F4 receptor profiles in pigs based on their mucin 4 polymorphism, responsiveness to oral F4 immunization and in vitro binding of F4 to villi

ETEC vaccination in pigs

ETEC vaccination in pigs

The effect of enterotoxigenic Escherichia coli F4ab,ac on early-weaned piglets: A gene expression study

The effect of enterotoxigenic Escherichia coli F4ab,ac on early-weaned piglets: A gene expression study

Development of A Loop-Mediated Isothermal Amplification (LAMP) for the Detection of F5 Fimbriae Gene in Enterotoxigenic Escherichia coli (ETEC)

The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. A set of four primers were designed based on the conservative sequence of coding F5 fimbriae. Temperature and time condition, specificity test, and sensitivity test were performed with the DNA of Escherichia coli (F5+). The results showed that the optimal reaction condition for LAMP was achieved at 61 °C for 45 min in a water bath. Ladder-like products were produced with those F5-positive samples by LAMP, while no product was generated with other negative samples. The assay of LAMP had a detection limit equivalent to 72 cfu/tube, which was more sensitive than PCR (7.2 × 102 cfu/tube). The agreement rate between LAMP and PCR was 100 % in detecting simulation samples. Thus, the LAMP assay may be a new method for rapid detection of F5 fimbriae gene of ETEC.

Structural insight in histo‐blood group binding by the F18 fimbrial adhesin FedF

F18-positive enterotoxigenic and Shiga toxin-producing Escherichia coli are responsible for post-weaning diarrhoea and oedema disease in pigs and lead to severe production losses in the farming industry. F18 fimbriae attach to the small intestine of young piglets by latching onto glycosphingolipids with A/H blood group determinants on type 1 core. We demonstrate the N-terminal domain of the F18 fimbrial subunit FedF to be responsible for ABH-mediated attachment and present its X-ray structure in ligand-free form and bound to A and B type 1 hexaoses. The FedF lectin domain comprises a 10-stranded immunoglobulin-like β-sandwich. Three linear motives, Q(47) -N(50), H(88) -S(90) and R(117) -T(119), form a shallow glycan binding pocket near the tip of the domain that is selective for type 1 core glycans in extended conformation. In addition to the glycan binding pocket, a polybasic loop on the membrane proximal surface of FedF lectin domain is shown to be required for binding to piglet enterocytes. Although dispensable for ABH glycan recognition, the polybasic surface adds binding affinity in the context of the host cell membrane, a mechanism that is proposed to direct ABH-glycan binding to cell-bound glycosphingolipids and could allow bacteria to avoid clearance by secreted glycoproteins.

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, rFaeGntd/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 rFaeGntd/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 rFaeGntd/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 rFaeGntd/dsc in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeGntd/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.

Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains

BackgroundEnterotoxigenic Escherichia coli (ETEC) is one of the most important pathogenic bacteria causing severe diarrhoea in human and pigs. In ETEC strains, the fimbrial types F4 and F18 are commonly found differently colonized within the small intestine and cause huge economic losses in the swine industry annually worldwide. To address the underlying mechanism, we performed a transcriptome study of porcine intestinal epithelial cells (IPEC-J2) with and without infection of three representative ETEC strains.ResultsA total 2443, 3493 and 867 differentially expressed genes were found in IPEC-J2 cells infected with F4ab ETEC (CF4ab), with F4ac ETEC (CF4ac) and with F18ac ETEC (CF18ac) compared to the cells without infection (control), respectively. The number of differentially expressed genes between CF4ab and CF4ac, CF4ab and CF18ac, and CF4ac and CF18ac were 77, 1446 and 1629, respectively. The gene ontology and pathway analysis showed that the differentially expressed genes in CF4abvs control are significantly involved in cell-cycle progress and amino acid metabolism, while the clustered terms of the differentially expressed genes in CF4acvs control comprise immune, inflammation and wounding response and apoptosis as well as cell cycle progress and proteolysis. Differentially expressed genes between CF18acvs control are mainly involved in cell-cycle progression and immune response. Furthermore, fundamental differences were observed in expression levels of immune-related genes among the three ETEC treatments, especially for the important pro-inflammatory molecules, including IL-6, IL-8, TNF-α, CCL20, CXCL2 etc.ConclusionsThe discovery in this study provides insights into the interaction of porcine intestinal epithelial cells with F4 ETECs and F18 ETEC, respectively. The genes induced by ETECs with F4 versus F18 fimbriae suggest why ETEC with F4 may be more virulent compared to F18 which seems to elicit milder effects.

Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response.

Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response.

Supplementary tryptophan downregulates the expression of genes induced by the gut microbiota in healthy weaned pigs susceptible to enterotoxigenic Escherichia coli F4

Supplementary tryptophan downregulates the expression of genes induced by the gut microbiota in healthy weaned pigs susceptible to enterotoxigenic Escherichia coli F4

Colibacillosis in lambs is associated to type I heat-stable enterotoxin in a farm in São Paulo State, Brazil

Twenty seven (48.2%) culture supernatants of 56 Escherichia coli isolated from diarrheic lamb feces (7 to 10 days old) in São Paulo State, Brazil, presented positive results to suckling mice assay (fluid accumulation) but none caused cytopathic effects on Vero and CHO cells, indicating that these strains did not produced LT or VT toxins. PCR assays showed that these 27 E. coli strains harbored estA, that codifies for STa, but not for stx1, stx2 or cnf genes. The positive STa strains were checked for genes that codify for F41, F17 and K99 fimbriae, wich are considered colonization factors in ETEC. Only F17 was detect in two samples (7.4%). Twelve of 27 STa positive carried hlyA gene and presented hemolytic activity in blood Agar. Presence of rotavirus was not detected among the diarrheic feces. These data suggests that STa must be an important diarrheagenic factor to small ruminants in São Paulo State.

A Genome-Wide Association Study Identifies Two Novel Promising Candidate Genes Affecting Escherichia coli F4ab/F4ac Susceptibility in Swine

Enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbria is the major pathogenic bacteria causing diarrhoea in neonatal and post-weaning piglets. Previous studies have revealed that the susceptibility to ETEC F4ab/F4ac is an autosomal Mendelian dominant trait and the loci controlling the F4ab/F4ac receptor are located on SSC13q41, between markers SW207 and S0283. To pinpoint these loci and further validate previous findings, we performed a genome-wide association study (GWAS) using a two generation family-based population, consisting of 301 piglets with phenotypes of susceptibility to ETEC F4ab/F4ac by the vitro adhesion test. The DNA of all piglets and their parents was genotyped using the Illumina PorcineSNP60 BeadChip, and 50,972 and 50,483 SNPs were available for F4ab and F4ac susceptibility, respectively, in the association analysis after quality control. In summary, 28 and 18 significant SNPs (p<0.05) were detected associated with F4ab and F4ac susceptibility respectively at genome-wide significance level. From these significant findings, two novel candidate genes, HEG1 and ITGB5, were firstly identified as the most promising genes underlying F4ab/F4ac susceptibility in swine according to their functions and positions. Our findings herein provide a novel evidence for unravelling genetic mechanism of diarrhoea risk in piglets.

Efficacy of thiolated eudragit microspheres as an oral vaccine delivery system to induce mucosal immunity against enterotoxigenic Escherichia coli in mice

Efficacy of thiolated eudragit microspheres as an oral vaccine delivery system to induce mucosal immunity against enterotoxigenic Escherichia coli in mice

Age-dependent competition of porcine enterotoxigenic E. coli (ETEC) with different fimbria genes — Short communication

To investigate the association of pathogenic Escherichia coli fimbrial adhesins with the development of diarrhoea in piglets of different age groups and to test their relative competitiveness, piglets were orally inoculated with a mixture of E. coli strains harbouring F4, F5, F6, F18 and F41 fimbrial genes. A total of 537 E. coli strains with haemolytic activity were isolated from 36 diarrhoeic piglets. The F4 fimbrial gene was observed in 98.5%, 97.6% and 80.6% strains carrying fimbrial genes isolated from diarrhoeic piglets that were infected at 1, 3 and 5 weeks of age, respectively. These data demonstrate that F4 fimbriae are highly associated with diarrhoea in piglets of all age groups. Interestingly, the F18 fimbrial gene was observed in 2.4% and 25.4% strains carrying fimbrial genes isolated from the 3- and 5-week-old groups, respectively, which confirms that F18 fimbriae are associated with diarrhoea in piglets from late stages of suckling to post-weaning, and are more related to diarrhoea in weaned than in unweaned piglets.

Susceptibility of piglets to enterotoxigenic Escherichia coli is not related to the expression of MUC13 and MUC20

Enterotoxigenic Escherichia coli (ETEC) is one of the most frequently isolated enteropathogens in production animals, especially pigs and calves. Economically, the swine industry is by far the most affected by infections with ETEC because of mortality, morbidity and decreased growth rate of newborn and early-weaned piglets. After ingestion by the animal, these bacteria attach themselves to specific receptors on the small intestinal epithelium by means of proteinaceous surface appendages, the fimbriae. The F4 fimbriae, which attach to the F4 receptor, are the most studied. The aim of our study was to investigate gene expression in the small intestine of piglets of MUC13 and MUC20 in relation to animals with a different treatment towards or a different reaction on ETEC-F4ac by means of quantitative reverse transcription chain reaction (qRT/PCR). MUC13 and MUC20 are positional candidate genes for this F4ac receptor and are located in the region on SSC13q41 that segregates with the susceptibility to ETEC-F4ac. The condition of the small intestine is crucial when examining expression differences between different samples. Therefore, the expression of two genes, fatty-acid binding protein 2, intestinal (FABP2) and pancreatitis-associated protein (PAP), now known as regenerating islet-derived 3 alpha (REG3A) in the small intestine was simultaneously checked. FABP2, a standard for epithelial content, reflects the state of damage, whereas REG3A is a measure for inflammation in the small intestine. The four different substudies presented here suggest that expression of MUC13 and MUC20 is not related to the susceptibility of piglets to ETEC-F4ac.