The N-terminal -barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization

The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer. The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit-binding specificity were investigated by analysis of mutant chaperones. FaeE contains a C-terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones. A C-terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C-terminal amino acid residues. Expression and complementation experiments revealed that this C-terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis. Two hybrid chaperones were constructed. Each hybrid protein contained one -barrel domain of FaeE and the other -barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively). Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae. The Fan/FaeE hybrid chaperone was active in the biosynthesis of K99 fimbriae. The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG. Cross-linking experiments suggested that the C-terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild-type K88 chaperone. Altogether, the data suggested that the N-terminal -barrel domain of a fimbrial chaperone determines subunit specificity. In the case of the K88 periplasmic chaperone, this N-terminal domain also determines dimerization of the protein.     

Combining sites of bacterial fimbriae

The few known crystal structures of receptor-binding domains of fimbrial tip adhesins, FimH, PapGII, and F17G, tell us that each of these structures is unique and surprising. Despite little to no sequence identity, common to them all is their variable immunoglobulin (Ig)-fold. Nevertheless, their glycan-binding sites have evolved in different locations onto this similar scaffold, and with distinct, highly specific binding properties. Difficult to capture is the often dominant role played by the fimbrial shaft in host cell recognition and biofilm formation. The major pilin FaeG, building up the shaft of F4 fimbriae, also harbors the carbohydrate receptor-binding property and has thereto an enlarged Ig-domain, with the insertion of two beta-strands and two alpha-helices. Bordetella and CFA/I fimbriae combine a tip adhesin with major subunit adhesins. Still other fimbriae incorporate a specialized invasin at the very tip of polyadhesive fibers for uptake of bacteria in cells of the immune system and host epithelia. Finally, glycan recognition by fimbrial adhesins has often been found to coincide with the binding of cell-surface integrins and components of the extracellular matrix, such as collagen IV and laminin.     

Structural and Thermodynamic Characterization of Pre- and Postpolymerization States in the F4 Fimbrial Subunit FaeG

Enterotoxigenic Escherichia coli expressing F4 fimbriae are the major cause of porcine colibacillosis and are responsible for significant death and morbidity in neonatal and postweaned piglets. Via the chaperone-usher pathway, F4 fimbriae are assembled into thin, flexible polymers mainly composed of the single-domain adhesin FaeG. The F4 fimbrial system has been labeled eccentric because the F4 pilins show some features distinct from the features of pilins of other chaperone-usher-assembled structures. In particular, FaeG is much larger than other pilins (27  versus ∼ 17 kDa), grafting an additional carbohydrate binding domain on the common immunoglobulin-like core. Structural data of FaeG during different stages of the F4 fimbrial biogenesis process, combined with differential scanning calorimetry measurements, confirm the general principles of the donor strand complementation/exchange mechanisms taking place during pilus biogenesis via the chaperone-usher pathway.     

Fimbrial Subunit Protein FaeG Expressed in Transgenic Tobacco Inhibits the Binding of F4ac Enterotoxigenic Escherichia coli to Porcine Enterocytes

Plants offer a promising alternative for the production of foreign proteins for pharmaceutical purposes in tissues that are consumed as food and/or feed. Our long-term strategy is to develop edible vaccines against piglet diarrhoea caused by enterotoxigenic Escherichia coli (F4 ETEC) in feed plants. In this work, we isolated a gene, faeG, encoding for a major F4ac fimbrial subunit protein. Our goal was to test whether the FaeG protein, when isolated from its fimbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, that is, stability in gastrointestinal conditions, binding to intestinal receptors and inhibition of the F4 ETEC attachment. For this purpose, tobacco was first transformed with a faeG construct that included a transit peptide encoding sequence to target the FaeG protein to the chloroplast. The best transgenic lines produced FaeG protein in amounts of 1% total soluble protein. The stability of the plant-produced FaeG was tested in fluids simulating piglet gastric (SGF) and intestinal (SIF) conditions. Plant-produced FaeG proved to be stable up to 2 h under these conditions. The binding and inhibition properties were tested with isolated piglet villi. These results showed that the plant-produced FaeG could bind to the receptors on the villi and subsequently inhibit F4 ETEC binding in a dose-dependent manner. Thus, the first two prerequisites for the development of an oral vaccine have been met.     

Biosynthesis of K88 fimbriae in Escherichia coli: interaction of tip-subunit FaeC with the periplasmic chaperone FaeE and the outer membrane usher FaeD

K88 fimbriae are ordered polymeric protein structures at the surface of enterotoxigenic Escherichia coli cells. Their production and assembly requires a molecular chaperone located in the periplasm (FaeE) and a molecular usher located in the outer membrane (FaeD). FaeC is the tip component of the K88 fimbriae. We studied the expression of the subcloned faeC gene, the subcellular localization of FaeC and its interaction with the chaperone and the outer membrane usher. In the absence of the chaperone or the usher, FaeC could not be detected in E. coli cells harbouring the faeC gene and its ribosome binding site under contol of the IPTG inducible lpp/lac promoter/operator. The expression of FaeC was detectable in the presence of chaperone FaeE, but a direct interaction between the chaperone and FaeC was not found. The expression of FaeC was also detectable in cells co-expressing the outer membrane usher FaeD. Overexpression of FaeC after changing the faeC ribosome binding site appeared to induce lethality. Expression of subcloned FaeC in the absence of FaeE or FaeD could be detected when faeC was cloned under the tight control of the ara promoter/operator and when lethality induction was avoided. The direct interaction of FaeC with outer membranes containing the usher FaeD was studied by cell fractionation, isopycnic sucrose density gradient centrifugation, SDS-PAGE and immunoblotting. FaeC was found to bind to outer membranes containing FaeD or a FaeD-PhoA hybrid construct containing 215 amino-terminal residues of FaeD. This binding was not observed when control outer membranes without FaeD were used. No other K88 specific proteins were required for this interaction. The direct interaction between FaeC and FaeD in the outer membranes was shown by affinity blotting experiments. FaeE was not required for this interaction. Together these data indicate that the minor fimbrial subunit FaeC, unlike FaeG, H and F, does not have a strong interaction with the chaperone FaeE in the E. coli periplasm, but directly binds to the outer membrane molecular usher FaeD.     

Deletion and duplication of specific sequences in the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli.

Summary Small, defined in-frame deletions and in-frame duplications of specific sequences were made within the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The cellular localization and proteolytic stability of the different mutated fimbrial subunit proteins were determined, and compared with those of the wild-type protein. Based upon these results, we predict a functional role of specific structures in the K88ab fimbrial subunit protein in subunit-subunit interactions as well as in interactions between FaeG and the other proteins encoded by the K88ab operon. The results obtained were further compared with results obtained from operon deletions, linker insertion mutagenesis and the current model for biogenesis of K88 fimbriae. One of the mutated fimbrial subunit genes was used to construct a secreted in-frame fusion between FaeG and a characterized epitope (lacking cysteine) from the Hepatitis B pre-S2 protein. Such fusion proteins might be useful in the design of recombinant vaccines.     

Structural and Functional Insight into the Carbohydrate Receptor Binding of F4 Fimbriae-producing Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4_ab, F4_ac, and F4_ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeG_ad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D′-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe¹⁵⁰–Glu¹⁵² and Val¹⁶⁶–Glu¹⁷⁰ of FaeG_ad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4_ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D′-α1 loop that borders the FaeG_ad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes.     

Structure and function of peripiasmic chaperone-like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichia coli

The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced. Immunoblotting of subcellular fractions with an antiserum raised against purified FaeE confirmed that FaeE is located in the periplasm. Indications were obtained that FaeE functions as a chaperone-like protein. Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilized this polypeptide and prevents its degradation by the cell-envelope protease DegP. Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae. The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration. Indications about the domains in FaeG involved in the interaction with FaeE are discussed.     

Identification of minor fimbrial subunits involved in biosynthesis of K88 fimbriae.

The nucleotide sequences of the genes faeF, faeH, faeI, and faeJ encoding K88 minor fimbrial subunits were determined. Analysis of the primary structure of the gene products revealed that all four proteins are synthesized with an amino-terminal signal sequence. The molecular masses of the mature FaeF, FaeH, FaeI, and FaeJ proteins were calculated to be 15,161, 25,461, 24,804, and 25,093 Da, respectively. FaeH, FaeI, and FaeJ showed significant homology with FaeG, the major fimbrial subunit of K88 fimbriae. Mutations in the respective genes were constructed. Analysis of the mutants showed that the minor fimbrial subunits FaeF and FaeH play an essential role in the biogenesis but not in the adhesive properties of the K88 fimbriae. Mutations in faeI or faeJ had no significant effect on K88 production or adhesive capacity. Specific antisera against FaeF and FaeH were raised by immunization with hybrid Cro-LacZ-FaeF and Cro-LacZ-FaeH proteins. Immunoblotting and immunoelectron microscopy revealed that FaeF and FaeH are located in or along the K88 fimbrial structure.     

F4 fimbriae expressed by porcine enterotoxigenic Escherichia coli, an example of an eccentric fimbrial system?

An overwhelming number of infectious diseases in both humans and animals are initiated by bacterial adhesion to carbohydrate structures on a mucosal surface. Most bacterial pathogens mediate this adhesion by fimbriae or pili which contain an adhesive lectin subunit. The importance of fimbriae as virulence factors led to research elucidating the regulation of fimbrial expression and their molecular assembly process. This review provides an overview of the current knowledge of induction, expression and assembly of F4 (K88) fimbriae and discusses its unique as well as its identical characteristics compared to other intensively studied fimbriae or pili expressed by Escherichia coli.     

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.     

Glycosylated F4 (K88) Fimbrial Adhesin FaeG Expressed in Barley Endosperm Induces ETEC-neutralizing Antibodies in Mice

The F4-positive enterotoxigenic Escherichia coli (ETEC) strains are a frequent cause of porcine post-weaning diarrhea. Orally administered F4 fimbriae or FaeG, the major subunit and adhesin of F4, induce a protective mucosal immune response in F4 receptor-positive piglets. Feed plants carrying immunogenic subunit proteins can offer great advantages for oral vaccination of domestic animals. Here, we describe high-level endosperm-specific production (1% of total soluble proteins) of FaeG in the crop plant barley. The endoplasmic reticulum-targeted recombinant endospermic FaeG (erFaeG) was shown to be heterogeneously glycosylated. The erFaeG showed resistance at digestive conditions simulating piglet gastric fluid. Glycosylation did not abolish the immunogenic character of the FaeG protein, since erFaeG was able to induce F4 fimbria-specific antibodies in mice. Biological activity of these anti-F4 antibodies was demonstrated in vitro by blocking the attachment of the F4⁺ ETEC to the F4 receptors present on porcine intestinal enterocytes.     

Fimbrial adhesins: similarities and variations in structure and biogenesis

Abstract Fimbriae are wiry (2 to 4 nm diam.) or rod-shaped (6 to 8 nm diam.), fibre-like structures on the surfaces of bacteria which mediate attachment to host cells. Much has been learned in recent years about the biogenesis, structure and regulation of expression of these adhesive organelles in Gram-negative bacteria. Analyses of the genetic determinants encoding the biogenesis of fimbriae has revealed that the adhesive interaction of fimbriae can be mediated by major subunits (CFA/I and CS1 fimbriae) or minor subunits (P, S, and type 1 fimbriae), with the adhesin being located either at the tip of the fimbria or along the length of the fimbrial shaft. Minor subunits can also act as adapters, anchors, initiators or elongators. Post-translational glycosylation of the type 4 pilins of Neisseria gonorrhoeae, Neisseria meningitidis and Pseudomonas aeruginosa has been demonstrated. The structures of the PapD chaperone of Escherichia coli and of N. gonorrhoeae type 4 fimbrin have been resolved at 2.0–2.6 Å. Rod-shaped fimbriae should not be thought of as being rigid inflexible structures but rather as dynamic structures which can undergo transition from a helicoidal to a fibrillar conformation to provide a degree of elasticity and plasticity to the fimbriae so that they can resist shear forces, rather like a bungee cord. At least four mechanisms have been identified in the assembly of fimbriae from fimbrin subunits, namely the chaperone-usher pathway (e.g., P-fimbriae of uropathogenic E. coli ), the general secretion assembly pathway (e.g., type 4 fimbriae or N -methylphenylalanine fimbriae of P. aeruginosa , the extracellular nucleation-precipitation pathway (e.g., curli of E. coli ) and the CFA/I, CS1 and CS2 fimbrial pathway.     

Molecular and structural aspects of fimbriae biosynthesis and assembly in Escherichia coli

Abstract: Fimbriae are long filamentous polymeric protein structures located at the surface of bacterial cells. They enable the bacteria to bind to specific receptor structures and thereby to colonise specific surfaces. Fimbriae consist of so-called major and minor subunits, which form, in a specific order, the fimbrial structure. In this review emphasis is put on the genetic organisation, regulation and especially on the biosynthesis of fimbriae of enterotoxigenic Escherichia coli strains, and more in particular on K88 and related fimbriae, with ample reference to the well-studied P and type 1 fimbriae. The biosynthesis of these fimbriae requires two specific and unique proteins, a periplasmic chaperone and an outer membrane located molecular usher ('doorkeeper'). Molecular and structural aspects of the secretion of fimbrial subunits across the cytoplasmic membrane, the interaction of these subunits with the periplasmic molecular chaperone, their translocation to the inner site of the outer membrane and their interaction with the usher protein, as well as the (ordered) translocation of the subunits across the outer membrane and their assembly into a grwoing fimbrial structure will be described. A model for K88 fimbriae is presented.     

Chaperone-usher fimbriae in a diverse selection of Gallibacterium genomes

Background

Fimbriae are bacterial cell surface organelles involved in the pathogenesis of many bacterial species, including Gallibacterium anatis, in which a F17-like fimbriae of the chaperone-usher (CU) family was recently shown to be an important virulence factor and vaccine candidate. To reveal the distribution and variability of CU fimbriae 22 genomes of the avian host-restricted bacteria Gallibacterium spp. were investigated. Fimbrial clusters were classified using phylogeny-based and conserved domain (CD) distribution-based approaches. To characterize the fimbriae in depth evolutionary analysis and in vitro expression of the most prevalent fimbrial clusters was performed.

Results

Overall 48 CU fimbriae were identified in the genomes of the examined Gallibacterium isolates. All fimbriae were assigned to γ4 clade of the CU fimbriae of Gram-negative bacteria and were organized in four-gene clusters encoding a putative major fimbrial subunit, a chaperone, an usher and a fimbrial adhesin. Five fimbrial clusters (Flf-Flf4) and eight conserved domain groups were defined to accommodate the identified fimbriae. Although, the number of different fimbrial clusters in individual Gallibacterium genomes was low, there was substantial amino acid sequence variability in the major fimbrial subunit and the adhesin proteins. The distribution of CDs among fimbrial clusters, analysis of their flanking regions, and evolutionary comparison of the strains revealed that Gallibacterium fimbrial clusters likely underwent evolutionary divergence resulting in highly host adapted and antigenically variable fimbriae. In vitro, only the fimbrial subunit FlfA was expressed in most Gallibacterium strains encoding this protein. The absence or scarce expression of the two other common fimbrial subunits (Flf1A and Flf3A) indicates that their expression may require other in vitro or in vivo conditions.

Conclusions

This is the first approach establishing a systematic fimbria classification system within Gallibacterium spp., which indicates a species-wide distribution of γ₄ CU fimbriae among a diverse collection of Gallibacterium isolates. The expression of only one out of up to three fimbriae present in the individual genomes in vitro suggests that fimbriae expression in Gallibacterium is highly regulated. This information is important for future attempts to understand the role of Gallibacterium fimbriae in pathogenesis, and may prove useful for improved control of Gallibacterium infections in chickens.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1093) contains supplementary material, which is available to authorized users.     

Escherichia coli periplasmic chaperone FaeE is a homodimer and the chaperone-K88 subunit complex is a heterotrimer

The interaction of FaeE, a periplasmic chaperone involved in K8B biosynthesis, and the major fimbrial subunit FaeG was Investigated. The genes encoding the two proteins were subcloned together in the expression vector pINIIIA1, Cells expressing the sub-cloned genes accumulated in their periplasm a complex of FaeE and FaeG. This complex was purified by isoelectric focusing and anion-exchange fast-protein liquid chromatography. SOS-PAGE, native gel etectrophoresis, immunoblotting and determination of the N-terminal amino acid sequences and the molar ratio of the W-terminal amino acid residues revealed that the complex is a heterotrimer consisting of two molecules of FaeE and one molecule of FaeG. The periplasmic chaperone FaeE was purified from the periplasm of cells expressing only the subcloned faeE gene. Gel filtration, protein cross-linking analysis and a biophysical approach in which the rotation diffusion coefficient of the purified FaeE was determined led to the conclusion that the native FaeE chaperone is a homodimer.     

Identification of major and minor chaperone proteins involved in the export of 987P fimbriae.

The 987P fimbriae of Escherichia coli consist mainly of the major subunit, FasA, and two minor subunits, FasF and FasG. In addition to the previously characterized outer membrane or usher protein FasD, the FasB, FasC, and FasE proteins are required for fimbriation. To better understand the roles of these minor proteins, their genes were sequenced and the predicted polypeptides were shown to be most similar to periplasmic chaperone proteins of fimbrial systems. Western blot (immunoblot) analysis and immunoprecipitation of various fas mutants with specific antibody probes identified both the subcellular localizations and associations of these minor components. FasB was shown to be a periplasmic chaperone for the major fimbrial subunit, FasA. A novel periplasmic chaperone, FasC, which stabilizes and specifically interacts with the adhesin, FasG, was identified. FasE, a chaperone-like protein, is also located in the periplasm and is required for optimal export of FasG and possibly other subunits. The use of different chaperone proteins for various 987P subunits is a novel observation for fimbrial biogenesis in bacteria. Whether other fimbrial systems use a similar tactic remains to be discovered.     

More than one way to control hair growth: regulatory mechanisms in enterobacteria that affect fimbriae assembled by the chaperone/usher pathway

Many gram-negative enterobacteria produce surface-associated fimbriae that facilitate attachment and adherence to eucaryotic cells and tissues. These organelles are believed to play an important role during infection by enabling bacteria to colonize specific niches within their hosts. One class of these fimbriae is assembled using a periplasmic chaperone and membrane-associated scaffolding protein that has been referred to as an usher because of its function in fimbrial biogenesis. The presence of multiple types of fimbriae assembled by the chaperone/usher pathway can be found both within a single bacterial species and also among different genera. One way of controlling fimbrial assembly in these bacteria is at the genetic level by positively or negatively regulating fimbrial gene expression. This minireview considers the mechanisms that have been described to control fimbrial gene expression and uses specific examples to demonstrate both unique and shared properties of such regulatory mechanisms.     

F1C fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits.

The genetic organization of the foc gene cluster has been studied; six genes involved in the biogenesis of F1C fimbriae were identified. focA encodes the major fimbrial subunit, focC encodes a product that is indispensable for fimbria formation, focG and focH encode minor fimbrial subunits, and focI encodes a protein which shows similarities to the subunit protein FocA. Apart from the FocA major subunits, purified F1C fimbriae contain at least two minor subunits, FocG and FocH. Minor proteins of similar size were observed in purified S fimbriae. Remarkably, some mutations in the foc gene cluster result in an altered fimbrial morphology, i.e., rigid stubs or long, curly fimbriae.