Identification of a fibronectin-binding domain within the Campylobacter jejuni CadF protein

The binding of Campylobacter jejuni to fibronectin (Fn), a component of the extracellular matrix, is mediated by a 37 kDa outer membrane protein termed CadF for Campylobacter adhesion to Fn. Previous studies have indicated that C. jejuni binds to Fn on the basolateral surface of T84 human colonic cells. To further characterize the interaction of the CadF protein with Fn, enzyme-linked immunosorbent assays were performed to identify the Fn-binding domain (Fn-BD). Using overlapping 30-mer and 16-mer peptides derived from translated cadF nucleotide sequence, maximal Fn-binding activity was localized to four amino acids (AA 134-137) consisting of the residues phenylalanine-arginine-leucine-serine (FRLS). A mouse alpha-CadF peptide polyclonal antibody (M alpha-CadF peptide pAb) was generated using FRLS containing peptides and found to react with viable C. jejuni as judged by indirect fluorescent microscopy, suggesting that the FRLS residues are surface-exposed. Binding of CadF to purified Fn and INT 407 human epithelial cells was significantly inhibited with peptides containing the Fn-BD. Moreover, a CadF recombinant variant protein, in which the Phe-Arg-Leu residues (CadF AA 134-136) were altered to Ala-Ala-Gly, exhibited a 91% decrease in Fn-binding activity as compared with the wild-type CadF protein. Collectively, these data indicate that the FRLS residues (CadF AA 134-137) of the C. jejuni CadF protein possess Fn-binding activity.     

Identification and characterization of a cell surface protein of Prevotella intermedia 17 with broad-spectrum binding activity for extracellular matrix proteins

Prevotella intermedia binds and invades a variety of host cells. This binding is most probably mediated through cell surface proteins termed adhesins. To identify proteins binding to the host extracellular matrix (ECM) component, fibronectin, and study the molecular mechanism underlying bacterial colonization, we applied proteomic approaches to perform a global investigation of P. intermedia strain 17 outer membrane proteins. 2-DE followed by Far Western Blot analysis using fibronectin as a probe revealed a 29-kDa fibronectin-binding protein, designated here AdpB. The molecular identity of the protein was determined using PMF followed by a search of the P. intermedia 17 protein database. Database searches revealed the similarity of AdpB to multiple bacterial outer membrane proteins including the fibronectin-binding protein from Campylobacter jejuni. A recombinant AdpB protein bound fibronectin as well as other host ECM components, including fibrinogen and laminin, in a saturable, dose-dependent manner. Binding of AdpB to immobilized fibronectin was also inhibited by soluble fibronectin, laminin, and fibrinogen, indicating the binding was specific. Finally, immunoelectron microscopy with anti-AdpB demonstrated the cell surface location of the protein. This is the first cell surface protein with a broad-spectrum ECM-binding abilities identified and characterized in P. intermedia 17.     

Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii , we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria–fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.     

Identification of a 47 kDa fibronectin-binding protein expressed by Borrelia burgdorferi isolate B31

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS–PAGE, transferred to nitrocellulose and probed with alkaline phosphatase-labelled fibronectin (fibronectin-AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin-binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin-binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys-C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli , and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.     

The outer membrane protein OmpA of Mannheimia haemolytica A1 is involved in the binding of fibronectin

An enzyme-linked immunosorbent assay using bovine fibronectin as the substrate was used to demonstrate that Mannheimia haemolytica A1 binds to fibronectin. This binding to fibronectin was specific as no binding was observed with bovine fibrinogen. The binding to fibronectin was not observed if the M. haemolytica A1 cells were pretreated with trypsin or proteinase K, suggesting that it involved a protein molecule on the cell surface. Interestingly, the fibronectin-binding activity was found to be higher in an acapsular mutant compared with its parent strain. The fibronectin-binding protein was shown to be present in the outer membrane fraction of M. haemolytica A1. A 45 kDa outer membrane protein that binds to fibronectin was identified by Far-Western immunoblot analysis. This protein was purified and subjected to MS matrix-assisted laser desorption ionization time-of-flight analysis. The results identified it to be outer membrane OmpA based on comparison with the M. haemolytica A1 genomic sequence.     

Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni

The outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections.     

Purification, characterization, and localization of a major membrane protein antigen from Porphyromonas (bacteroides) gingivalis.

Humans and rats infected with P. gingivalis develop a strong immune response to a 75 kDa major membrane component of P. gingivalis and hence knowledge of the nature of this molecule may aid in understanding the host response to P. gingivalis during infection. Purification of the 75 kDa protein was achieved by repeated precipitation from a crude sonicate of P. gingivalis 2561 at pH 5.0. Homogeneity of the purified 75 kDa protein was confirmed by SDS-PAGE and Western immunoblot analysis using monoclonal and polyclonal antibodies. The purified protein revealed an apparent molecular mass of 300 kDa in native form. Although most of the strains of P. gingivalis tested showed a major membrane protein band in the range of 61 to 78 kDa, on Western immunoblot analysis only the strains which have proteins in the range of 75 to 78 kDa were reactive with anti-75 kDa protein polyclonal antibodies. Affinity purified polyclonal antibodies were used to localized 75 kDa protein on the cell surface of P. gingivalis 2561 by immunogold electron microscopy. Immunolabeling of the 75 kDa protein demonstrated specific localization of the protein along the outer cell membrane, but not on the fimbriae. Furthermore, immunogold labeling of the 75 kDa protein on the thin sections showed that the 75 kDa component was present on not only the outer membrane, but also on the cell membrane, and on membrane bound organelles. Localization of this protein suggests that the 75 kDa component is a membrane-associated protein.     

JlpA, a novel surface-exposed lipoprotein specific to Campylobacter jejuni, mediates adherence to host epithelial cells

A 1116 bp open reading frame (ORF), designated jlpA, encoding a novel species-specific lipoprotein of Campylobacter jejuni TGH9011, was identified from recombinant plasmid pHIP-O. The jlpA gene encodes a polypeptide (JlpA) of 372 amino acid residues with a molecular mass of 42.3 kDa. JlpA contains a typical signal peptide and lipoprotein processing site at the N-terminus. The presence of a lipid moiety on the JlpA molecule was confirmed by the incorporation of [3H]-palmitic acid. Immunoblotting analysis of cell surface extracts prepared using glycine-acid buffer (pH 2.2) and proteinase K digestion of whole cells indicated that JlpA is a surface-exposed lipoprotein in C. jejuni. JlpA is loosely associated with the cell surface, as it is easily extracted from the C. jejuni outer membrane by detergents, such as sarcosyl and Triton X-100. JlpA is released to the culture medium, and its concentration increases in a time-dependent fashion. The adherence of both insertion and deletion mutants of jlpA to HEp-2 epithelial cells was reduced compared with that of parental C. jejuni TGH9011. Adherence of C. jejuni to HEp-2 cells was inhibited in a dose-dependent manner when the bacterium was preincubated with anti-GST-JlpA antibodies or when HEp-2 cells were preincubated with JlpA protein. A ligand-binding immunoblotting assay showed that JlpA binds to HEp-2 cells, which suggests that JlpA is C. jejuni adhesin.     

Brucella group 3 outer membrane proteins contain a heat-modifiable protein

Abstract Brucella melitensis and B. ovis outer membrane blebs contained a protein displaying a temperature-dependent molecular mass upshift from 25 kDa to 30 kDa. A fraction of the protein tightly bound to LPS did not show the molecular mass upshift which was also blocked by exposure of the protein to Zwittergent 314. The B. melitensis heat-modifiable protein and Escherichia coli OmpA shared antigenic determinants. These data indicate that the Brucella group 3 outer membrane proteins belonged to the OmpA family of proteins.     

PorA protein of Campylobacter jejuni is not a cytotoxin mediating inflammatory diarrhoea

Campylobacter jejuni is a major food-borne pathogen and a leading cause of diarrhoea. A cytotoxin is most likely involved in the pathogenesis of inflammatory diarrhoea due to C. jejuni. A 45-kDa outer membrane protein encoded by the porA gene was reported to exhibit cytotoxic activity for cultured mammalian cells in vitro. We cloned and expressed the porA gene in Escherichia coli BL21 codon plus RIL strain using the fusion vector pGEX-4T-1. The fusion protein solubilised in urea in denatured form or solubilised in Empigen BB in native form or their thrombin-cleaved products did not exhibit cytotoxic activity for Chinese hamster ovary (CHO) cells. The urea-solubilised fusion protein did not induce fluid accumulation in the rabbit ileal loop assay. All 76 clinical isolates of Campylobacter spp. tested were positive for porA by PCR, but only 13 isolates were positive for cytotoxin on CHO cells. Both cytotoxin-positive as well as cytotoxin-negative strains expressed PorA as determined by immunoblot analysis. These findings show that the porA gene product of C. jejuni is not a cytotoxin mediating inflammatory diarrhoea.     

Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post‐translational processing that removes immunogenicity while retaining fibronectin binding

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn‐binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2‐DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF₂₄) and 22 kDa (CadF₂₂) mass variants. CadF variants were fully characterized by MALDI‐TOF MS and MALDI‐MS/MS. These data confirmed that CadF forms re‐folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post‐translationally. CadF₂₂ and CadF₂₄, however, were characterized as N‐terminal, membrane‐associated polypeptides resulting from cleavage between serine¹⁹⁵ and leucine¹⁹⁶, and glycine²⁰¹ and phenylalanine²⁰², respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full‐length CadF (34 kDa), CadF₂₄, and the deleted C‐terminal OmpA domain (14 kDa; CadF₁₄) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full‐length CadF retained reactivity. Binding assays showed that CadF₂₄ retained Fn‐binding capability, while CadF₁₄ did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn‐binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.     

Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes

Campylobacter jejuni is one of the major causes of human diarrhea throughout the world. Attachment to host cells and extracellular matrix proteins is considered to be an essential primary event in the pathogenesis of enteritis. Outer membrane proteins of three C. jejuni strains, one of which was aflagellate, were investigated for their contribution to the process of adhesion to INT 407 cell membranes and the extracellular matrix protein fibronectin. Using a ligand-binding immunoblotting assay the flagellin, the major outer membrane protein and a 59-kDa protein were detected to be involved in adhesion to both substrates. The MOMP was able to inhibit the attachment of the bacteria to INT 407 cell membranes partly, when the protein was isolated under native conditions. However, it was totally lost when the protein was isolated in the presence of SDS. The 59-kDa protein of one strain was identified by N-terminal sequencing, and regarding the first 14 amino acids it was found to be identical to the 37-kDa CadF protein just recently described as fibronectin-binding protein of C. jejuni. Especially for the aflagellate strain this protein may be of special importance for adhesion of the bacteria to different substrates.     

A major outer membrane protein of Serratia marcescens which was easily solubilized and had a capacity to bind to calcium

Easily solubilized major outer membrane protein was found in Serratia marcescens. The protein was originally obtained as a membrane-associated, insoluble protein in the outer membrane when the cells were slightly disrupted. However, the amount of this protein in the outer membrane gradually decreased with the time of sonication. The decrease was not due to decomposition of the protein but to solubilization into a soluble fraction after a long period of disruption. The molecular weight of this protein was 47 kDa and it bound calcium. Another 40 kDa calcium binding protein was also found in the outer membrane of S. marcescens.     

The human cytomegalovirus receptor on fibroblasts is a 30-kilodalton membrane protein.

Previous studies have demonstrated that human cytomegalovirus (HCMV) binding to human foreskin fibroblasts (HFF) is mediated by a single type of molecule, likely a glycoprotein, which serves as a specific receptor for the virus. In the present experiments, HCMV was found to bind to an HFF membrane protein with an approximate molecular mass of 30 kilodaltons (kDa); weak binding to 28- and 92-kDa membrane components was also observed. Binding was specific, as it was inhibited by excess unlabeled HCMV. Radiolabeled HCMV also bound selectively to Raji and Daudi lymphoblastoid cell membrane proteins of the same molecular masses. The 30-kDa radiolabeled HFF membrane protein bound to HCMV in solution; this binding was also specific, as it was blocked by an excess of HCMV. These data suggest that a membrane protein with a molecular mass of approximately 30 kDa mediates HCMV binding to several cell types.     

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28 degrees C and 37 degrees C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Several proteins with molecular weights ranging from 34 kDa to 71 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could also be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discussed.     

Isolation of a type IV collagen binding protein from human platelets.

In order to study the molecular basis of platelet interaction with collagen IV of the basement membrane separating the arterial endothelium from the underlying subendothelial connective tissue, the possibility of presence of platelet membrane protein with affinity to type IV collagen was examined by subjecting the platelet membrane extract to affinity chromatography on collagen IV-sepharose. Urea (4 M) eluate was found to contain a protein with an apparent mol. wt of 68 kDa. The radioiodinated protein was isolated and used to test its specificity. By dot blot assay on nitrocellulose disks and solid-phase assays, the 68 kDa protein was found to bind with high affinity to collagen IV. Lack of significant binding to fibronectin and laminin when compared to albumin control indicated its high specificity for collagen. The radioiodinated protein was inserted into egg yolk lecithin liposomes. While these liposomes attached to microtitre plates coated with collagen IV, there was no significant binding to fibronectin or laminin coated wells, suggesting the membrane associated character of the protein as well as its specificity for collagen. These results indicate that presence of a 68 kDa protein in platelet membrane which interacts with very high specificity to collagen IV.     

Kinetic Properties of Purified Pseudomonas aeruginosa Phosphorylcholine Phosphatase Indicated That This Enzyme May Be Utilized by the Bacteria to Colonize in Different Environments

A protein oligomer with an approximate molecular weight of its 37-kDa monomer form was purified from the cell envelope fraction of Vibrio damsela cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with phosphatidyl choline. The functional properties for the 37-kDa protein suggest that it is a nonspecific or general porin, with an apparent pore size of 1.6 nm. This porin allows penetration of a variety of hydrophilic solutes according to their molecular mass. After electroelution, the oligomer was partially dissociated into monomers, whereas treatment with EDTA did not affect its dissociation. The monomers of the 37-kDa protein were not active in the reconstitution assay. The effect of culture media on the composition of the outer membrane protein of V. damsela was examined. Only one outer membrane protein with an apparent molecular weight of 37 kDa (37-kDa protein) was formed in cells grown in 3% NaCl–BHI broth and in 3% NaCl–nutrient broth with the addition of 2% glucose. Three outer membrane proteins, with apparent molecular weights of 37 kDa, 40 kDa, and 46 kDa, were produced in cells grown in 3% NaCl–nutrient broth. An additional outer membrane protein with an apparent molecular weight of 44 kDa (44-kDa protein) was found in cells grown in 3% NaCl–nutrient broth with the addition of 2% maltose. This protein was found to exhibit specificity to maltose derivatives. The results obtained in this study confirm the porin-like character of discussed proteins and give a basis for advanced study of those proteins. Received: 16 June 1998 / Accepted: 18 July 1998     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

A TonB-dependent outer membrane protein as a Bacteroides fragilis fibronectin-binding molecule

The binding of Bacteroides fragilis to plasmatic fibronectin was investigated using strains isolated from healthy subjects and from patients with bacteremia. They were cultivated in a synthetic media in which variations in cysteine concentrations determined alterations in the oxidation–reduction potential (Eh). All the strains assayed were capable of adhering to plasmatic fibronectin when cultivated under oxidizing and reducing conditions. Bacteroides fragilis 1405 showed the greatest difference when the results under these conditions were compared and it was selected for further investigations. Chemical treatments suggested the involvement of a protein in the interaction between B. fragilis and plasmatic fibronectin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of outer membrane proteins (OMPs) revealed differences between the extracts obtained from cultures grown under the two conditions. Protein bands of c . 102, 100, 77, 73, 50 and 40 kDa were more highly expressed under oxidizing than reducing conditions. Dot blot analysis showed a stronger recognition of plasmatic fibronectin by OMPs obtained from cultures grown under higher Eh, and Western blot assays confirmed a band of c . 102 kDa as fibronectin-binding protein. This protein was sequenced and revealed to be a putative TonB-dependent OMPs. PCR analysis confirmed the presence of this gene in all the studied strains.