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.     

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.     

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.     

Characterization of a Bordetella pertussis fimbrial gene cluster which is located directly downstream of the filamentous haemagglutinin gene

The biosynthesis of fimbriae is a complex process requiring multiple genes which are generally found clustered on the chromosome. In Bordetella pertussis, only major fimbrial subunit genes have been identified, and no evidence has yet been found that they are located in a fimbrial gene cluster. To locate additional genes involved in the biosynthesis of B. pertussis fimbriae, we used TnphoA mutagenesis. A PhoA+ mutant (designated B176) was isolated which was affected in the production of both serotype 2 and 3 fimbriae. Cloning and sequencing of the DNA region harbouring the transposon insertion revealed the presence of at least three additional fimbrial genes, designated fimB, fimC and fimD. The transposon was found to be located in fimD. Analysis of PhoA activity indicated that the fimbrial gene cluster was positively regulated by the bvg locus. A potential binding site for BvgA was observed upstream of fimB. FimB showed homology with the so-called chaperone-like fimbrial proteins, while FimC was homologous with a class of fimbrial proteins located in the outer membrane and presumed to be involved in transport and anchorage of fimbrial subunits. An insertion mutation in fimB abolished the expression of fimbrial subunits, implicating this gene in the biosynthesis of both serotype 2 and 3 fimbriae. Upstream of fimB a pseudogene (fimA) was observed which showed homology with the three major fimbrial subunit genes, fim2, fim3 and fimX. The construction of a phylogenetic tree suggested that fimA may be the primordial major fimbrial subunit gene from which the other three were derived by gene duplication. Interestingly, the fimbrial gene cluster was found to be located directly downstream from the gene coding for the filamentous haemagglutinin, an important B. pertussis adhesin, possibly suggesting co-operation between the two loci in the pathogenesis of pertussis.     

Identification and characterization of precursors in the biosynthesis of the K88ab fimbria of Escherichia coli

Four genes encoding for polypeptides with apparent molecular weights of 17,000, 26,000 (the fimbrial subunit), 27,000, and 81,000 have been implicated in the biosynthesis of the K88ab fimbria (Mooi et al., J. Bacteriol. 150:512-521, 1982). Escherichia coli mutants with defects in these genes were examined for the presence of fimbrial precursors. An analysis of these mutants revealed that fimbrial subunits accumulated transiently in the periplasmic space before being translocated across the outer membrane. The 81,000-dalton (d) polypeptide is probably involved in translocating fimbrial subunits across the outer membrane, because in the absence of this polypeptide the fimbrial subunits remained in the periplasmic space, where they were found to be associated with the 17,000- and 27,000-d polypeptides. In mutants with a deletion in the gene for the 27,000-d polypeptide, fimbrial precursors were not detected, because the fimbrial subunits were degraded. The 27,000-d polypeptide might be involved in stabilizing a conformation of the fimbrial subunit required to translocate it across the outer membrane. In the absence of the 17,000-d polypeptide, most fimbrial subunits were found in the periplasmic space associated with the 27,000-d polypeptide. However, small amounts of subunits were also translocated across the outer membrane. These extracellular subunits did not adhere to brushborders, suggesting that fimbrial subunits must be modified by the 17,000-d polypeptide to be assembled into functional fimbriae. A model for the biosynthesis of the K88ab fimbria is proposed.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

Characterization of two genes encoding antigenically distinct type-1 fimbriae of Klebsiella pneumoniae

A uropathogenic isolate of Klebsiella pneumoniae was shown to exhibit a mannose-sensitive hemagglutinating phenotype and to produce type-1 fimbriae consisting of subunits with a different electrophoretic mobility than those previously investigated. The gene cluster encoding expression of fimbriae was cloned and the genetic organization of the encoded polypeptides was determined. The gene encoding the major fimbrial subunit was localized and further examined by nucleotide sequence analysis. Comparison of two K. pneumoniae fimbrial genes revealed a nucleotide sequence agreement of 73%, and amino acid sequence agreement of 84% for the mature fimbrial subunits. Predictions of putative antigenic sites were correlated with regions demonstrating amino acid variability. In agreement with these predictions, no serological cross-reactivity between both fimbrial proteins could be demonstrated using an enzyme-linked immunosorbent assay (ELISA).     

Nucleotide sequences of the genes encoding type 1 fimbrial subunits of Klebsiella pneumoniae and Salmonella typhimurium.

The nucleotide sequences of the genes encoding the subunits of Klebsiella pneumoniae and Salmonella typhimurium type 1 fimbriae were determined. Comparison of the predicted amino acid sequences of the two subunits revealed domains in which the sequences were highly conserved. Both gene products possessed signal peptides, a fact consistent with the transport of the fimbrial subunit across the membrane, but these regions showed no amino acid homology between the two proteins. The predicted N-terminal amino acid sequences of the processed fimbrial subunits were in good agreement with those obtained by purification of the fimbrial subunits.     

Subcellular localization of K99 fimbrial subunits and effect of temperature on subunit synthesis and assembly.

Analysis of the biogenesis of K99 fimbriae indicated that the fimbrial subunits were synthesized at the cytoplasmic membrane and accumulated in the periplasm before they appeared at the cell surface. Cells grown at 18 degrees C contained a small pool of fimbrial subunits. After a shift to 37 degrees C, the cells required about 1 h before they were able to synthesize fimbriae, concomitant with a dramatic increase in K99 concentration.     

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.     

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.     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clonal analysis reveals high rate of structural mutations in fimbrial adhesins of extraintestinal pathogenic Escherichia coli

Type 1 fimbriae of Escherichia coli mediate mannose-specific adhesion to host epithelial surfaces and consist of a major, antigenically variable pilin subunit, FimA, and a minor, structurally conserved adhesive subunit, FimH, located on the fimbrial tip. We have analysed the variability of fimA and fimH in strains of vaginal and other origin that belong to one of the most prominent clonal groups of extraintestinal pathogenic E. coli, comprised of O1:K1-, O2:K1- and O18:K1-based serotypes. Multiple locus sequence typing (MLST) of this group revealed that the strains have identical (at all but one nucleotide position) eight housekeeping loci around the genome and belong to the ST95 complex defined by the publicly available E. coli MLST database. Multiple highly diverse fimA alleles have been introduced into the ST95 clonal complex via horizontal transfer, at a frequency comparable to that of genes defining the major O- and H-antigens. However, no further significant FimA diversification has occurred via point mutation after the transfers. In contrast, while fimH alleles also move horizontally (along with the fimA loci), they acquire point amino acid replacements at a higher rate than either housekeeping genes or fimA. These FimH mutations enhance binding to monomannose receptors and bacterial tropism for human vaginal epithelium. A similar pattern of rapid within-clonal structural evolution of the adhesive, but not pilin, subunit is also seen, respectively, in papG and papA alleles of the di-galactose-specific P-fimbriae. Thus, while structurally diverse pilin subunits of E. coli fimbriae are under selective pressure for frequent horizontal transfer between clones, the adhesive subunits of extraintestinal E. coli are under strong positive selection (Dn/Ds > 1 for fimH and papG) for functionally adaptive amino acid replacements.     

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.     

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.     

Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae

Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.     

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.     

The distinct binding specificities exhibited by enterobacterial type 1 fimbriae are determined by their fimbrial shafts

Type 1 fimbriae of enterobacteria are heteropolymeric organelles of adhesion composed of FimH, a mannose-binding lectin, and a shaft composed primarily of FimA. We compared the binding activities of recombinant clones expressing type 1 fimbriae from Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium for gut and uroepithelial cells and for various soluble mannosylated proteins. Each fimbria was characterized by its capacity to bind particular epithelial cells and to aggregate mannoproteins. However, when each respective FimH subunit was cloned and expressed in the absence of its shaft as a fusion protein with MalE, each FimH bound a wide range of mannose-containing compounds. In addition, we found that expression of FimH on a heterologous fimbrial shaft, e.g. K. pneumoniae FimH on the E. coli fimbrial shaft or vice versa, altered the binding specificity of FimH such that it closely resembled that of the native heterologous type 1 fimbriae. Furthermore, attachment to and invasion of bladder epithelial cells, which were mediated much better by native E. coli type 1 fimbriae compared with native K. pneumoniae type 1 fimbriae, were found to be dependent on the background of the fimbrial shaft (E. coli versus K. pneumoniae) rather than the background of the FimH expressed. Thus, the distinct binding specificities of different enterobacterial type 1 fimbriae cannot be ascribed solely to the primary structure of their respective FimH subunits, but are also modulated by the fimbrial shaft on which each FimH subunit is presented, possibly through conformational constraints imposed on FimH by the fimbrial shaft. The capacity of type 1 fimbrial shafts to modulate the tissue tropism of different enterobacterial species represents a novel function for these highly organized structures.     

Isolation and characterization of Bacteroides nodosus fimbriae: structural subunit and basal protein antigens

We examined the isolation of fimbriae from Bacteroides nodosus. It was found that the best preparations were obtained from the supernatant of washed cells cultured on solid medium, from which fimbriae could be recovered in high yield and purity by a simple one-step procedure. Analysis of such preparations by sodium dodecyl sulfate gel electrophoresis showed that greater than 98% of the protein consisted of fimbrial structural subunits whose molecular weight was ca. 17,000. These preparations also usually exhibited minor contamination with a polypeptide of ca. 80,000 molecular weight, as well as trace amounts of lipopolysaccharide. Attempts to release additional fimbriae by the traditional means of subjecting the bacterial cells to physical stress, such as shearing or heating, resulted primarily in an increase in the level of contamination, without significant gain in the yield of fimbriae. Removal of the 80,000-dalton component could not be achieved by any of a variety of techniques normally used in fimbriae purification, including isoelectric precipitation, MgCl2 precipitation, and CsCl gradient ultracentrifugation, implying a direct physical association with the fimbrial strand. Electron micrographs of fractions containing this protein show cap-shaped structures attached to the ends of what appeared to be fimbrial stubs. These observations suggest that the 80,000-dalton polypeptide may actually constitute the basal attachment site which anchors the fimbria to the outer membrane, analogous to a similar protein recently described in enterotoxigenic strains of Escherichia coli. In B. nodosus, this 80,000-dalton protein is a major surface antigen, and like the fimbrial subunit, exhibited variation in electrophoretic mobility between serotypically different isolates.