Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures

Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole-cell extracts. To engineer plasmids capable of directing the expression in E. coli of high levels of this product, both as a pre-protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda P(L) and P(R) promoters and the E. coli atpE translational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in several E. coli strains including DH5. However, they did in E. coli CAG629, which is lon protease and heat shock protein deficient. Both pre-protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface of E. coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionylated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with whole B. pertussis serotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild-type form of the fimbrial subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence homology between the subunits of two immunologically and functionally distinct types of fimbriae of Actinomyces spp.

Nucleotide sequencing of the type 1 fimbrial subunit gene of Actinomyces viscosus T14V revealed a consensus ribosome-binding site followed by an open reading frame of 1,599 nucleotides. The encoded protein of 533 amino acids (Mr = 56,899) was predominantly hydrophilic except for an amino-terminal signal peptide and a carboxy-terminal region identified as a potential membrane-spanning segment. Edman degradation of the cloned protein expressed in Escherichia coli and the type 1 fimbriae of A. viscosus T14V showed that both began with alanine at position 31 of the deduced amino acid sequence. The amino acid compositions of the cloned protein and fimbriae also were comparable and in close agreement with the composition of the deduced protein. The amino acid sequence of the A. viscosus T14V type 1 fimbrial subunit showed no significant global homology with various other proteins, including the pilins of gram-negative bacteria. However, 34% amino acid sequence identity was noted between the type 1 fimbrial subunit of strain T14V and the type 2 fimbrial subunit of Actinomyces naeslundii WVU45 (M. K. Yeung and J. O. Cisar, J. Bacteriol. 170:3803-3809, 1988). This homology included several different conserved sequences of up to eight identical amino acids that were distributed in both the amino- and carboxy-terminal thirds of each Actinomyces fimbrial subunit. These findings indicate that the different types of fimbriae on these gram-positive bacteria share a common ancestry.     

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.     

Structure-function analysis of the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli

Several in-frame linker insertions have been made in various positions in the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The effects of the linker insertions have been investigated with regard to the ability of the mutated fimbrial subunits to be exported to the surface of the bacterial cell and assembled into a fimbrial structure. The structure/function relationship of the subunit protein is discussed in the light of the phenotypes of the mutations constructed, secondary structure predictions, and hydrophilicity plots.     

Cloning and nucleotide sequence analysis of the serotype 2 fimbrial subunit gene of Bordetella pertussis

An oligonucleotide probe complementary to the beginning of the gene encoding the serotype 2(ST2) fimbrial subunit of Bordetella pertussis was synthesized and a cloned DNA fragment hybridizing with the probe identified and sequenced. Several lines of evidence indicate that an open reading frame with coding information for a polypeptide of 207 amino acids, including a 26-amino-acid signal sequence, is the ST2 gene. The protein deduced from the nucleotide sequence shows good agreement with the NH2-terminal amino acid sequence, amino acid composition and molecular weight of the purified fimbrial subunit. In addition, the proposed ST2 subunit is shown to have homology with other fimbrial subunits.     

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.     

Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.     

Ordered translocation of 987P fimbrial subunits through the outer membrane of Escherichia coli.

The 987P fimbria of enterotoxigenic Escherichia coli is a heteropolymeric structure which consists essentially of a major FasA subunit and a minor subunit, the FasG adhesin. The latter harbors the binding moiety for receptor molecules on piglet intestinal epithelial cells. In this study, anti-FasF antibody probes were developed and used to demonstrate that the FasF protein represents a new minor fimbrial component. FasF was identified in highly purified fimbriae, and its sequence demonstrated significant levels of similarity with that of FasA. Immune electron microscopy localized both the FasG and FasF proteins at the fimbrial tip as well as at broken ends and at various intervals along the fimbrial length. The presence of these minor proteins in purified 987P fimbriae was corroborated by enzyme-linked immunosorbent assay inhibitions. Finally, the use of nonfimbriated fasG, fasF, and fasA mutants indicated that subunit translocation through the outer membrane follows a specific order, FasG being the first, FasF being the second, and FasA being the third type of exported subunit. Since fimbriae are thought to grow from the base, FasG is proposed to be a tip adhesin and FasF is proposed to be a linker molecule between the adhesin and the fimbrial shaft. Moreover, export of FasG (or FasF) in the absence of FasF (or FasA) indicates that during the process of fimbrial biogenesis in the outer membrane, translocating events precede the initiation of subunit heteropolymerization.     

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.     

Dependence of secretion and assembly of type 1 fimbrial subunits of Escherichia coli on normal protein export.

The export of fimbrial subunits was found to be diminished at the restrictive temperature in a strain bearing a secA(Ts) mutation. Likewise, export was inhibited in a strain harboring a malE-lacZ protein fusion upon induction of hybrid protein synthesis. Both conditions resulted in the accumulation of a precursor protein ca. 2,000 daltons larger than the mature fimbrial subunit.     

Characterization and molecular cloning of the PCF8775 CS5 antigen from an enterotoxigenic Escherichia coli 0115:H40 isolated in Central Australia

The genes determining the biosynthesis of the colonization factor CS5 have been cloned from Escherichia coli 0115:H40:PCF8775 isolated during an outbreak of diarrhoea among aboriginal children in Central Australia. Electron microscopy has shown purified CS5 to be of semi-rigid fimbrial type. NH2-terminal analysis has shown the CS5 determinant to be distinct from other fimbriae, although there is some conservation of certain residues. Expression in minicells of the cloned fimbrial genes encoded on pPM1312 has shown that proteins of 70 and 46.5 kD which co-purity with the 23 kD major fimbrial subunit protein are also co-expressed along with proteins of 45, 31, 17 and 14 kD. The major CS5 subunit is synthesized in precursor form (approximately 26 kD). A synthetic oligonucleotide to the NH2-terminal amino acid coding sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1312 encoding the structural gene for the major pilin subunit.     

Analysis of genes coding for the sialic acid-binding adhesin and two other minor fimbrial subunits of the S-fimbrial adhesin determinant of Escherichia coli

The S fimbrial adhesin (Sfa) enables Escherichia coli to attach to sialic acid-containing receptor molecules of eukaryotic cells. As previously reported, the genetic determinant coding for the Sfa of an E. coli O6 strain was cloned, the gene coding for the major fimbrial subunit was identified and sequenced and the S specific adhesin was detected. Here we present evidence that in addition to the major subunit protein SfaA three other minor subunit proteins, SfaG (17 kD), SfaS (14 kD) and SfaH (31 kD) can be isolated from the S-specific fimbrial adhesin complex. The genes coding for these minor subunits were identified, mutagenized separately and sequenced. Using haemagglutination tests, electron-microscopy and quantitative ELISA assays with monoclonal anti-SfaA and anti-SfaS antibodies the functions of the minor subunits were determined. It was determined that SfaS is identical to the S-specific adhesin, which also plays a role in determination of the degree of fimbriation of the cell. The minor subunit SfaH also had some influence on the level of fimbriation of the cell, while SfaG is necessary for full expression of S-specific binding. It was further shown that the amino-terminal protein sequence of the isolated SfaS protein was identical to the protein sequence calculated from the DNA sequence of the sfaS gene locus.     

Functional analysis of the fsoC gene product of the F71 (fso) fimbrial gene cluster

Contrary to what would be expected from data in the literature, mutations in the fsoC gene of the F7(1) (fso) P-fimbrial gene cluster do not completely block fimbrial biogenesis. fsoC mutants still express small amounts of fimbriae of normal length, which carry the non-adhesive minor subunit protein, FsoE, but lack the adhesin, FsoG. The FsoC protein operates at the same stage in fimbrial biogenesis as the FsoF and FsoG proteins. The data suggest that FsoC, FsoF and FsoG interact to form an initiation complex for fimbrial biogenesis.     

The fimA gene encoding the type-1 fimbrial subunit of Escherichia coli. Nucleotide sequence and primary structure of the protein

The nucleotide sequence was determined of a region of 1450 base pairs encompassing the fimA gene for the subunit of type 1 fimbriae of Escherichia coli as well as flanking regions containing potential regulator sequences. The 'translated' protein contains a 23-residue signal peptide; the processed fimbrial subunit consists of 158 amino acid residues yielding a relative molecular mass of 15706. The elucidated sequence shows significant homology with those of other E. coli fimbrial proteins.     

Immunogenicity of a Cholera Toxin B Subunit <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> Fimbrial Antigen Fusion Protein Expressed in <Emphasis Type="Italic">E. coli</Emphasis>

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.     

Molecular cloning of the fimbrial subunit gene from a benign type B isolate of Bacteroides nodosus

Abstract The fimbrial subunit gene from the benign type B Bacteroides nodosus isolate AC/6 was cloned into the Sph I site of the multicopy vector plasmid pUC19. Five Escherichia coli recombinants that were positive in a colony immunoassay were shown, by Western transfer analysis, to produce an immunologically cross-reacting protein of identical molecular size to fimbrial subunits prepared from B. nodosus AC/6. Restriction endonuclease analysis showed that 4 of the recombinant plasmids carried a 6.7 kb Sph I fragment. Recloning experiments showed that the fimbrial subunit gene was located within a 2.5 kb Eco RI- Sph I fragment and that there was a Pst I site located within the structural gene or its regulatory region. These recombinant clones will prove useful for the construction of a multivalent recombinant vaccine for the control of ovine footrot.     

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.     

Sortase-catalyzed assembly of distinct heteromeric fimbriae in Actinomyces naeslundii

Two types of adhesive fimbriae are expressed by Actinomyces; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two fimbrial gene clusters present in the genome of Actinomyces naeslundii strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the fimQ-fimP-srtC1-fimR gene cluster encodes a fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the fimB-fimA-srtC2 gene cluster encodes a distinct fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 fimbriae. Thus, SrtC2 is a fimbria-specific sortase that is essential for assembly of the type 2 fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the fimbrial assembly pathways in Actinomyces and their function in the pathogenesis of different biofilm-related oral diseases.     

Expression of foreign epitopes in P-fimbriae of Escherichia coli.

Summary Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes. Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively. Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors. Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids. The inserted peptides appeared to be exposed in the fimbrial filament. One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice.