matB, a common fimbrillin gene of Escherichia coli, expressed in a genetically conserved, virulent clonal group.

A novel fimbrial type in Escherichia coli was identified and characterized. The expression of the fimbria was associated with the O18acK1H7 clonal group of E. coli, which cause newborn meningitis and septicemia when grown at low temperature; hence, it was named the Mat (meningitis associated and temperature regulated) fimbria. The fimbriae were purified from a fimA::cat sfaA::Gm fliC::St derivative of the O18K1H7 isolate E. coli IHE 3034. The purified Mat fimbrillin had an apparent molecular mass of 18 kDa and did not serologically cross-react with the type 1 or S fimbria of the same strain. The matB gene encoding the major fimbrillin was cloned from the genomic DNA of the fimA::cat sfaA::Gm fliC::St derivative of IHE 3034. The predicted MatB sequence was of 195 amino acids, contained a signal sequence of 22 residues, and did not show significant homology to any of the previously characterized fimbrial proteins. The DNA sequence of matB was 97.8% identical to a region from nucleotides 17882 to 18469 in the 6- to 8-min region of the E. coli K-12 chromosome, reported to encode a hypothetical protein. The 7-kb DNA fragment containing matB of IHE 3034 was found by restriction mapping and partial DNA sequencing to be highly similar to the corresponding region in the K-12 chromosome. Trans complementation of the matB::cat mutation in the IHE 3034 chromosome showed that matB in combination with matA or matC restored surface expression of the Mat fimbria. A total of 27 isolates representing K-12 strains and the major pathogroups of E. coli were analyzed for the presence of a matB homolog as well as for expression of the Mat fimbria. A conserved matB homolog was found in 25 isolates; however, expression of the Mat fimbriae was detected only in the O18acK1H7 isolates. Expression of the Mat fimbria was temperature regulated, with no or a very small amount of fimbriae or intracellular MatB fimbrillin being detected in cells cultivated at 37(o)C. Reverse transcriptase PCR and complementation assays with mat genes controlled by the inducible trc promoter indicated that regulation of Mat fimbria expression involved both transcriptional and posttranscriptional events.

Expression of pls, a gene closely associated with the mecA gene of methicillin-resistant Staphylococcus aureus, prevents bacterial adhesion in vitro.

The pls gene, coding for a large surface protein of methicillin-resistant Staphylococcus aureus, was cloned from a strain which adheres poorly to several mammalian proteins. The structure of pls revealed three distinct repeat regions, one of which was a serine-aspartate repeat characteristic of the Clf-Sdr family of surface proteins in staphylococci. The lengths of the repeat regions varied in different clinical strains and could be used as epidemiological markers. pls was found to be closely associated with the mecA gene by pulsed-field gel electrophoresis analysis of SmaI-digested DNA. A pls mutant constructed by allele replacement adhered well to immobilized fibronectin and immunoglobulin G, in contrast to the parental strain, suggesting that Pls could have a role in preventing adhesion at some stages during an infection.

The gaf fimbrial gene cluster of Escherichia coli expresses a full-size and a truncated soluble adhesin protein.

The GafD lectin of the G (F17) fimbriae of diarrhea-associated Escherichia coli was overexpressed and purified from the periplasm of E. coli by affinity chromatography on GlcNAc-agarose. The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named DeltaGafD. Expression of gafD from the cloned gaf gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of DeltaGafD. The peptide was also detected in the periplasm of the wild-type E. coli strain from which the gaf gene cluster originally was cloned. We expressed gafD fragments encoding C-terminally truncated peptides. Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of DeltaGafD. Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases. Pulse-chase assays using gafD indicated that DeltaGafD was processed from GafD and is not a primary translation product. The DeltaGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose. Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant E. coli strain and in the binding of [(14)C]DeltaGafD to GlcNAc-agarose. DeltaGafD bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when gafD is expressed alone or from the gaf gene cluster.

Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus.

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

Peptide display on bacterial flagella: principles and applications.

Expression of foreign peptides as fusions to bacterial cell surface proteins has gained increasing attention in basic, as well as applied research during the last decade. A wide range of heterologous peptides have been expressed, and the spectrum of available carrier proteins is also wide. The choice of carrier protein is frequently ruled by the application of the fusion protein constructed. This review is focused on flagella display, which is based on genetic fusion of foreign peptides into a surface-exposed, dispensable region of flagellin, the flagellar major subunit present in thousands of copies per filament. Expression of these constructs in flagellin-deficient host strains results in hybrid flagella carrying the heterologous peptides in thousands of intimately-associated copies. The first and still most frequent application of flagella display is the construction of novel recombinant vaccines. Flagella display has also been used in peptide display as an alternative to the phage-display technique. One application involves fusion into a disulfide loop of Escherichia coli thioredoxin that has been inserted into flagellin, this system facilitates expression of random peptides in a conformationally constrained manner readily accessible on the flagellar surface. The random peptide library has been applied in antibody epitope mapping and is suitable for biopanning procedures in the study of ligand-receptor interactions. Many bacterial adhesins are of complex nature and thereby difficult to analyse by conventional methods. Direct flagella display has proven to be applicable also in bacterial adhesion technology since large fragments, up to 302 amino acid residues in length, of bacterial adhesins can be functionally expressed as fusions to flagellin. Hybrid flagella are easily purified and can easily be analysed for binding to various targets, such as immobilized proteins, tissue sections, as well as cell cultures.

Construction of a multihybrid display system: flagellar filaments carrying two foreign adhesive peptides.

A multivalent, bifunctional flagellum carrying two different adhesive peptides in separate flagellin subunits within a filament was constructed in Escherichia coli. The inserted peptides were the fibronectin-binding 115-mer D repeat region of Staphylococcus aureus and the 302-mer collagen-binding region of YadA of Yersinia enterocolitica. Western blotting, immunoelectron microscopy, and adhesion tests with hybrid flagella from an in trans-complemented DeltafliC E. coli strain showed that individual filaments consisted of both recombinant flagellins.

Identification of two laminin-binding fimbriae, the type 1 fimbria of Salmonella enterica serovar typhimurium and the G fimbria of Escherichia coli, as plasminogen receptors.

Escherichia coli strains carrying recombinant plasmids encoding either the type 1 fimbria of Salmonella enterica serovar Typhimurium or the G fimbria of E. coli exhibited binding of human 125I-Glu-plasminogen and enhanced the tissue-type plasminogen activator-catalyzed conversion of plasminogen to plasmin. Purified type 1 or G fimbriae similarly bound plasminogen and enhanced its activation. The binding of plasminogen did not involve the characteristic carbohydrate-binding property of the fimbriae but was inhibited at low concentrations by the lysine analog epsilon-aminocaproic acid. Because these fimbrial types bind to laminin of basement membranes (M. Kukkonen et al., Mol. Microbiol. 7:229-237, 1993; S. Saarela et al., Infect. Immun. 64:2857-2860, 1996), the results demonstrate a structural unity in the creation and targeting of bacterium-bound proteolytic plasmin activity to basement membranes.

Immunohistological localization of the MrkD adhesin in the type 3 fimbriae of Klebsiella pneumoniae.

The adhesive minor protein MrkD of the type 3 fimbria of Klebsiella pneumoniae was expressed and purified from Escherichia coli as a fusion protein with an N-terminal polyhistidine tail. Polyclonal antibodies raised against MrkD specifically recognized the MrkD peptide in Western blots of fimbrial preparations. Immunoelectron microscopic analyses showed that the anti-MrkD immunoglobulins bound to the tip of the plasmid-encoded variant of the type 3 fimbria of K. pneumoniae, whereas no binding to the chromosomally encoded MrkD-deficient type 3 fimbrial variant of K. pneumoniae was detected. Immunoglobulins from an antiserum raised against purified type 3 fimbrial filaments bound laterally to both type 3 fimbrial variants. The anti-MrkD antibodies also bound to the tip of a papG deletion derivative of the E. coli P fimbria complemented with mrkD, indicating that MrkD structurally complements a PapG mutation in the P fimbria of E. coli.

Characterization of the fimA gene encoding bundle-forming fimbriae of the plant pathogen Xanthomonas campestris pv. vesicatoria.

The fimA gene of Xanthomonas campestris pv. vesicatoria was identified and characterized. A 20-mer degenerate oligonucleotide complementary to the N-terminal amino acid sequence of the purified 15.5-kDa fimbrillin was used to locate fimA on a 2.6-kb SalI fragment of the X. campestris pv. vesicatoria 3240 genome. The nucleotide sequence of a 1.4-kb fragment containing the fimA region revealed two open reading frames predicting highly homologous proteins FimA and FimB. FimA, which was composed of 136 amino acids and had a calculated molecular weight of 14,302, showed high sequence identity to the type IV fimbrillin precursors. fimB predicted a protein product of 135 amino acids and a molecular weight of 13,854. The open reading frame for fimB contained near the 5' end a palindromic sequence with a terminator loop potential, and the expression level of fimB in vitro and in Xanthomonas was considerably lower than that of fimA. We detected an efficiently transcribed fimA-specific mRNA of 600 bases as well as two weakly expressed, longer mRNA species that reacted with both fimA and fimB. A homolog of fimA but not of fimB was detected by Southern hybridization in strains of X. campestris pv. vesicatoria, campestris, begoniae, translucens, and graminis. A fimA::omega mutant of strain 3240 was not significantly reduced in virulence or adhesiveness to tomato leaves. However, the fimA mutant was dramatically reduced in cell aggregation in laboratory cultures and on infected tomato leaves. The fimA mutant strain also exhibited decreased tolerance to UV light.

Plasminogen receptors. Turning Salmonella and Escherichia coli into proteolytic organisms.

We evaluated in vitro the hypothesis that bacterial adhesiveness to the mammalian extracellular matrix and the activation of plasminogen on bacterial plasminogen receptors promote bacterial penetration through basement membranes. We used the strain SH401 of Salmonella enterica serovar Typhimurium, which adheres to the high-mannose chains of laminin, a major glycoprotein of basement membranes, and expresses plasminogen receptors. Bacterium-bound plasmin was able to degrade laminin and extracellular matrix preparations as well as to potentiate the penetration of bacteria through a reconstituted basement membrane. The results suggest that metastatic tumour cells and bacterial pathogens use similar mechanisms to penetrate through tissue barriers.

Functional expression of adhesive peptides as fusions to Escherichia coli flagellin.

An expression system for studying epitopes of adhesion proteins based on fusion of gene fragments into fliC(H7) of Escherichia coli is described. We constructed the system by an in-frame insertion of DNA fragments encoding one, two or three of the fibronectin-binding D repeats present in the fibronectin-binding protein A (FnBPA) of Staphylococcus aureus, into the fliC(H7) gene region encoding the variable domain of the H7 flagellin. The constructs were expressed by in trans complementation in the E. coli strain JT1 which harbours knock-out mutations for the expression of FliC as well as of the mannoside-binding fimbrial adhesin. The resulting chimeric flagella, which contained 39, 77 or 115 heterologous amino acid residues, efficiently bound soluble and immobilized human plasma and cellular fibronectin, and the binding was most efficient with the flagella containing the three D repeats of FnBPA. The chimeric flagella bound to frozen sections of human kidney and to cultured human cells. Antibodies raised against the chimeric flagella bound to Protein A-deficient S. aureus cells and inhibited the binding of staphylococci to immobilized fibronectin. We also expressed peptides, ranging in size between 48 and 302 amino acids, of the collagen-binding YadA adhesin of Yersinia enterocolitica. A fragment of 302 amino acids representing the middle region of YadA was needed for collagen binding. Chimeric flagellar filaments expressing hundreds of intimately associated adhesive epitopes offer versatile tools to analyze adhesin-receptor interactions and functional epitopes of adhesion proteins.

The GafD protein of the G (F17) fimbrial complex confers adhesiveness of Escherichia coli to laminin.

Escherichia coli IHE11088(pRR-5) expressing the G (F17) fimbria adhered to immobilized laminin as well as to reconstituted basement membranes. No adhesion was seen with the plasmidless strain IHE11088 or with the deletion derivative IHE11088(pHUB110), which expresses the G-fimbrial filament with a defective GafD lectin and lacks N-acetyl-D-glucosamine-specific binding. Adhesion of IHE11088(pRR-5) to laminin and to reconstituted basement membranes was specifically inhibited by N-acetyl-D-glucosamine, and adhesion was abolished after N-glycosidase F treatment of laminin. The results show that the GafD lectin binds to laminin carbohydrate and suggest a novel function for the F17 fimbria in binding to mammalian basement membranes.