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.     

Histone H1 proteins act as receptors for the 987P fimbriae of enterotoxigenic Escherichia coli

The tip adhesin FasG of the 987P fimbriae of enterotoxigenic Escherichia coli mediates two distinct adhesive interactions with brush border molecules of the intestinal epithelial cells of neonatal piglets. First, FasG attaches strongly to sulfatide with hydroxylated fatty acyl chains. This interaction involves lysine 117 and other lysine residues of FasG. Second, FasG recognizes specific intestinal brush border proteins that migrate on a sodium-dodecyl sulfate-polyacrylamide gel like a distinct set of 32-35-kDa proteins, as shown by ligand blotting assays. The protein sequence of high performance liquid chromatography-purified tryptic fragments of the major protein band matched sequences of human and murine histone H1 proteins. Porcine histone H1 proteins isolated from piglet intestinal epithelial cells demonstrated the same SDS-PAGE migration pattern and 987P binding properties as the 987P-specific protein receptors from porcine intestinal brush borders. Binding was dose-dependent and shown to be specific in adhesion inhibition and gel migration shift assays. Moreover, mapping of the histone H1 binding domain suggested that it is located in their lysine-rich C-terminal domains. Histone H1 molecules were visualized on the microvilli of intestinal epithelial cells by immunohistochemistry and electron microscopy. Taken together these results indicated that the intestinal protein receptors for 987P are histone H1 proteins. It is suggested that histones are released into the intestinal lumen by the high turnover of the intestinal epithelium. Their strong cationic properties can explain their association with the negatively charged brush border surfaces. There, the histone H1 molecules stabilize the sulfatide-fimbriae interaction by simultaneously binding to the membrane and to 987P.     

Genetic analysis of 987P adhesion and fimbriation of Escherichia coli: the fas genes link both phenotypes.

The 987P fimbrial gene cluster has recently been shown to contain eight genes (fasA to fasH) clustered on large plasmids of enterotoxigenic Escherichia coli and adjacent to a Tn1681-like transposon encoding the heat-stable enterotoxin STIa. Different genetic approaches were used to study the relationship between 987P fimbriation and adhesion. TnphoA mutagenesis, complementation assays, and T7 RNA polymerase-promoted gene expression indicated that all of the fas genes were involved in fimbrial expression and adhesion. In contrast to other fimbrial systems, the lack of expression of any single fas gene never resulted in the dissociation of fimbriation and adhesion, indicating that the adhesin is required for fimbrial expression and suggesting that FasA, the fimbrial structural subunit itself, is the adhesin. In addition, fimbrial length was shown to be modulated by the levels of expression of different fas genes.     

Immunocytochemical analysis of P-fimbrial structure: localization of minor subunits and the influence of the minor subunit FsoE on the biogenesis of the adhesin

Antibodies recognizing the non-adhesive minor P-fimbral subunit protein E and the P-fimbrial adhesin were used in an immunocytochemical analysis of P-fimbrial structure. It was demonstrated that P-fimbriae of the serotypes F71, F72 and F11 carry their adhesin in a complex with protein E. These complexes are commonly found at the tip of the fimbrial structure. In P-fimbriae of serotype F9, expressed by the uropathogenic Escherichia coli strain 21086, adhesin-protein E complexes are localized at the tips as well as along the shafts of the fimbriae. Protein E of F71 fimbriae (FsoE) plays a catalysing role in the biogenesis of the adhesin, but has no effect on the eventual localization of the adhesin.     

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.     

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.     

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.     

Role of fimbrial adhesins in the pathogenesis of Escherichia coli infections.

Escherichia coli strains are able to cause intestinal (enteritis, diarrhoeal diseases) and extraintestinal (urinary tract infections, sepsis, meningitis) infections. Most pathogenic E. coli strains produce specific fimbrial adhesins, which represent essential colonization factors: intestinal E. coli strains very often carry transferable plasmids with gene clusters specific for fimbrial adhesins, like K88 and K99, or colonization factor antigens (CFA) I and II. In contrast, the fimbrial gene clusters of extraintestinal E. coli strains, such as P, S, or F1C fimbriae, are located on the chromosomes. The fimbrial adhesin complexes consist of major and minor subunit proteins. Their binding specificity can generally be assayed in hemagglutination tests. In the case of fimbrial adhesins of intestinal E. coli strains, the major subunit proteins preferentially represent the hemagglutinating adhesins, whereas minor subunit proteins are the hemagglutinins of extraintestinal E. coli strains. Recently "alternative" adhesin proteins were identified, which have the capacity to bind to eukaryotic structures different from the receptors of the erythrocytes. Fimbrial adhesins are not constitutively expressed but are stringently regulated on the molecular level. Extraintestinal E. coli wild-type strains normally carry three or more fimbrial adhesin determinants, which have the capacity to influence the expression of one another (cross talk). Furthermore the fimbrial gene clusters undergo phase variation, which seems to be important for their contribution to pathogenesis of E. coli.     

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.     

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.     

Organization and expression of genes involved in the biosynthesis of 987P fimbriae

Summary A chromosomal DNA segment encoding the biosynthesis of 987P fimbriae was isolated by cosmid-cloning and subsequent subcloning into pBR322. The 12 kb DNA segment expressed five polypeptides with apparent molecular weights of 81,000, 39,000, 28,500, 20,500, and 16,500, respectively. The location of the corresponding genes was determined by insertional mutagenesis using Tn5. The 20.5 K polypeptide was identified as the 987P fimbrial subunit by its reaction with specific anti-987P antibodies. The 81, 39, and 28.5 K polypeptides appeared to be accessory proteins involved in 987P production.     

Permissive linker insertion sites in the outer membrane protein of 987P fimbriae of Escherichia coli.

The FasD protein is essential for the biogenesis of 987P fimbriae of Escherichia coli. In this study, subcellular fractionation was used to demonstrate that FasD is an outer membrane protein. In addition, the accessibility of FasD to proteases established the presence of surface-exposed FasD domains on both sides of the outer membrane. The fasD gene was sequenced, and the deduced amino acid sequence was shown to share homologous domains with a family of outer membrane proteins from various fimbrial systems. Similar to porins, fimbrial outer membrane proteins are relatively polar, lack typical hydrophobic membrane-spanning domains, and posses secondary structures predicted to be rich in turns and amphipathic beta-sheets. On the basis of the experimental data and structural predictions, FasD is postulated to consist essentially of surface-exposed turns and loops and membrane-spanning interacting amphipathic beta-strands. In an attempt to test this prediction, the fasD gene was submitted to random in-frame linker insertion mutagenesis. Preliminary experiments demonstrated that it was possible to produce fasD mutants, whose products remain functional for fimbrial export and assembly. Subsequently, 11 fasD alleles, containing linker inserts encoding beta-turn-inducing residues, were shown to express functional proteins. The insertion sites were designated permissive sites. The inserts used are expected to be least detrimental to the function of FasD when they are inserted into surface-exposed domains not directly involved in fimbrial export. In contrast, FasD is not expected to accommodate such residues in its amphipathic beta-strands without being destabilized in the membrane and losing function. All permissive sites were sequenced and shown to be located in or one residue away from predicted turns. In contrast, 5 of 10 sequenced nonpermissive sites were mapped to predicted amphipathic beta-strands. These results are consistent with the structural predictions for FasD.     

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.     

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.     

Evaluation of Proteus mirabilis structural fimbrial proteins as antigens against urinary tract infections

Proteus mirabilis is a common cause of urinary tract infection (UTI) and produce several types of different fimbriae, including mannose-resistant/Proteus-like fimbriae, uroepithelial cell adhesin (UCA), and P. mirabilis fimbriae (PMF). Different authors have related these fimbriae with different aspects of P. mirabilis pathogenesis, although the precise role of fimbriae in UTI has not yet been elucidated. In this work we expressed and purified recombinant structural fimbrial proteins of these fimbriae (MrpA, UcaA, and PmfA) and assessed their role as protective antigens using an ascending and a haematogenous model of UTI in the mouse. MrpA protected subcutaneously immunised mice in both models, suggesting that it could be taken into account as a promising vaccine candidate against P. mirabilis UTI. UcaA could also be an interesting subunit to be studied although it only protected mice that were challenged intravenously. All subunits elicited a strong specific serum IgG response but there was no significant correlation between antibody levels and protection. Only PmfA-immunised mice elicited a significant urinary antibody response but this protein was unable to confer protection against P. mirabilis experimental challenges. These results may contribute to the development of vaccines against P. mirabilis, an important cause of complicated UTI.     

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.     

Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli.

Antigen 43 has been identified as a unique protein complex in the outer membrane of Escherichia coli. The complex contains two different polypeptides, alpha (Mr, 60,000) and beta (Mr, 53,000), in equal stoichiometry (P. Owen, P. Caffrey, and L.-G. Josefsson, J. Bacteriol. 169:3770-3777, 1987). The alpha subunit was released in a water-soluble form upon heating of outer membranes to 60 degrees C and was purified to apparent homogeneity by gel filtration and ion-exchange chromatography. The purified protein was acidic (pI 4.6) and had a polarity of 49.2%. The N-terminal sequence showed homology with the N termini of certain enterobacterial fimbrial subunits. In addition, antigen 43 underwent a reversible phase variation similar to that of type 1 fimbriae. By use of subunit-specific antisera, it was shown that the purified alpha subunit was capable of reassociating with the beta polypeptide. However, electron microscopic examination indicated that antigen 43 does not form a recognizable surface structure. The available evidence supports the view that antigen 43 is a complex consisting of a peripheral membrane protein (alpha) anchored to a subunit (beta) that is integral to the outer membrane.