Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues

The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new insights into understanding the interplay between Brucella and host cells.     

The BtaF Trimeric Autotransporter of Brucella suis Is Involved in Attachment to Various Surfaces,Resistance to Serum and Virulence

The adhesion of bacterial pathogens to host cells is an event that determines infection, and ultimately invasion and intracellular multiplication. Several evidences have recently shown that this rule is also truth for the intracellular pathogen Brucella. Brucella suis displays the unipolar BmaC and BtaE adhesins, which belong to the monomeric and trimeric autotransporter (TA) families, respectively. It was previously shown that these adhesins are involved in bacterial adhesion to host cells and components of the extracellular matrix (ECM). In this work we describe the role of a new member of the TA family of B. suis (named BtaF) in the adhesive properties of the bacterial surface. BtaF conferred the bacteria that carried it a promiscuous adhesiveness to various ECM components and the ability to attach to an abiotic surface. Furthermore, BtaF was found to participate in bacterial adhesion to epithelial cells and was required for full virulence in mice. Similar to BmaC and BtaE, the BtaF adhesin was expressed in a small subpopulation of bacteria, and in all cases, it was detected at the new pole generated after cell division. Interestingly, BtaF was also implicated in the resistance of B. suis to porcine serum. Our findings emphasize the impact of TAs in the Brucella lifecycle.     

Genomic island 2 is an unstable genetic element contributing to Brucella lipopolysaccharide spontaneous smooth-to-rough dissociation

Brucella is a Gram-negative bacterium that causes a worldwide-distributed zoonosis. The genus includes smooth (S) and rough (R) species that differ in the presence or absence, respectively, of the O-polysaccharide of lipopolysaccharide. In S brucellae, the O-polysaccharide is a critical diagnostic antigen and a virulence determinant. However, S brucellae spontaneously dissociate into R forms, a problem in antigen and S vaccine production. Spontaneous R mutants of Brucella abortus, Brucella melitensis, and Brucella suis carried the chromosomal scar corresponding to genomic island 2 (GI-2) excision, an event causing the loss of the wboA and wboB O-polysaccharide genes, and the predicted excised circular intermediate was identified in B. abortus, B. melitensis, and B. suis cultures. Moreover, disruption of a putative phage integrase gene in B. abortus GI-2 caused a reduction in O-polysaccharide loss rates under conditions promoting S-R dissociation. However, spontaneous R mutants not carrying the GI-2 scar were also detected. These results demonstrate that the phage integrase-related GI-2 excision is a cause of S-R brucella dissociation and that other undescribed mechanisms must also be involved. In the R Brucella species, previous works have shown that Brucella ovis but not Brucella canis lacks GI-2, and a chromosomal scar identical to those in R mutants was observed. These results suggest that the phage integrase-promoted GI-2 excision played a role in B. ovis speciation and are consistent with other evidence, suggesting that this species and B. canis have emerged as two independent lineages.     

METABOLIC CHARACTERIZATION OF THE GENUS BRUCELLA IV. 3: Correlation of Oxidative Metabolic Patterns and Susceptibility to Brucella Bacteriophage, Type abortus, Strain.

Meyer, Margaret E. (University of California, Davis). Metabolic characterization of the genus Brucella. IV. Correlation of oxidative metabolic patterns and susceptibility to Brucella bacteriophage, type abortus, strain 3. J. Bacteriol. 82:950-953. 1961.-A total of 212 strains of brucellae that had been identified as Brucella melitensis, B. abortus, B. suis, or B. neotomae by their oxidative metabolism were tested for their susceptibility to Brucella bacteriophage, type abortus, strain 3. It was demonstrated that only those organisms that displayed the oxidative metabolic pattern that is singular for B. abortus were susceptible to this strain of phage, irrespective of their identity by the conventional methods usually employed for differentiating members of this genus. Strains of organisms that display the features of B. melitensis by the conventional determinative methods, but display the metabolic characteristics of B. abortus, are susceptible to lysis by this phage. These organisms are in fact B. abortus. Strains of organisms that display the features of B. melitensis by the classical methods, and display the metabolic pattern of B. melitensis, are not lysed by this phage. These organisms are B. melitensis. The conclusions then were drawn that B. abortus is the only species that can serve as host for this strain of phage, that oxidative metabolic patterns accurately identify the species in this genus, and that by the conventional methods of differentiation, many strains of B. abortus are misidentified as B. melitensis.     

Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain.

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.     

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.     

Brucella outer membrane protein Omp31 is a haemin-binding protein

The expression of haemin-binding proteins (HBPs) in the outer membrane is one of the strategies used by Gram-negative bacteria to obtain iron from the host. No HBP has been described in Brucella spp. We investigated whether Omp31, an outer membrane protein from Brucella with homology to HBPs from Bartonella quintana, is an HBP. Soluble recombinant Omp31 bound specifically to haemin-agarose, while an unrelated Brucella protein (SurA) did not. A similar experiment showed that native Omp31 found in the Brucella suis membrane fraction also binds to haemin-agarose. Recombinant Omp31 was electrophoresed by SDS-PAGE, transferred to nitrocellulose, and incubated with a haemin solution. Haemin bound to Omp31 and to albumin (positive control) but not to SurA. IPTG-induced recombinant Escherichia coli cells expressing Omp31 on their membrane bound significantly more haemin than uninduced cells or controls carrying a similar plasmid without the omp31 gene, showing that Omp31 also binds haemin in a bacterial membrane environment. Viable Brucella ovis cells bound haemin in solution, and this binding was markedly inhibited by preincubation of cells with antibodies to Omp31 and to an exposed prominent loop of the protein, thus showing that Omp31 functions as an HBP in brucellae. To test whether the expression of Omp31 is iron-regulated, B. suis was grown in trypticase-soy broth (TSB) and in iron-depleted TSB. The expression of Omp31, as assessed by Western blot, was significantly higher in bacteria grown under iron limitation. Overall, these results show that Omp31 from B. suis, B. melitensis and B. ovis is an HBP, whose expression seems to be induced by iron limitation.     

Triflavin, an Arg-Gly-Asp-containing peptide, inhibits human cervical carcinoma (HeLa) cell-substratum adhesion through an RGD-dependent mechanism

Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins, that have been isolated from the venoms of various vipers and shown to be potent inhibitors of platelet aggregation. The interaction of tumor cells with extracellular matrices such as fibronectin, vitronectin, and collagen has been shown to be mediated through a family of cell surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) sequence within each adhesive protein. In this study, we show that triflavin dose-dependently inhibited adhesion of human cervical carcinoma (HeLa) cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, and vitronectin). On the other hand, triflavin exerted a limited inhibitory effect on cell adhesion to laminin and collagen (type I and IV). On a molar basis, triflavin is approximately 800 times more potent than Gly-Arg-Gly-Asp-Ser (GRGDS) at inhibiting cell adhesion. When immobilized on plate, triflavin significantly promoted HeLa cell adhesion, and this attachment was inhibited by GRGDS. Furthermore, FITC-conjugated triflavin bound to cells in a saturable manner and its binding was inhibited by GRGDS. In addition, triflavin did not affect [³H]thymidine uptake of HeLa cells during a 3-day incubation. These results suggest that triflavin probably binds to integrin receptors expressed on HeLa cell surface via its RGD sequence within its molecule, thereby inhibiting the adhesion of extracellular matrices to HeLa cells.     

The pavA gene of Streptococcus pneumoniae encodes a fibronectin-binding protein that is essential for virulence

Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell-surface anchorage signals within the primary sequence. Full-length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid-phase fibronectin, in a heparin-sensitive interaction, and blocked binding of wild-type pneumococcal cells to fibronectin. However, a C-terminally truncated PavA' polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2-2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2-2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C-terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell-surface physico-chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104-fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin-binding protein plays a direct role in the pathogenesis of pneumococcal infections.     

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.     

The head of Bartonella adhesin A is crucial for host cell interaction of Bartonella henselae

Human pathogenic Bartonella henselae cause cat scratch disease and vasculoproliferative disorders (e.g. bacillary angiomatosis). Expression of Bartonella adhesin A (BadA) is crucial for bacterial autoagglutination, adhesion to host cells, binding to extracellular matrix proteins and proangiogenic reprogramming via activation of hypoxia inducible factor (HIF)-1. Like the prototypic Yersinia adhesin A, BadA belongs to the class of trimeric autotransporter adhesins and is constructed modularly consisting of a head, a long and repetitive neck-stalk module and a membrane anchor. Until now, the exact biological role of these domains is not known. Here, we analysed the function of the BadA head by truncating the repetitive neck-stalk module of BadA (B. henselae badA(-)/pHN23). Like B. henselae Marseille wild type, B. henselae badA(-)/pHN23 showed autoagglutination, adhesion to collagen and endothelial cells and activation of HIF-1 in host cells. Remarkably, B. henselae badA(-)/pHN23 did not bind to fibronectin (Fn) suggesting a crucial role of the deleted stalk domain in Fn binding. Additionally, the recombinantly expressed BadA head adhered to human umbilical vein endothelial cells and to a lesser degree to epithelial (HeLa 229) cells. Our data suggest that the head represents the major functional domain of BadA responsible for host adhesion and angiogenic reprogramming.     

Chinese hamster ovary cell motility to fibronectin is modulated by the second extracellular loop of CD9. Identification of a putative fibronectin binding site

CD9, a member of the tetraspanin family of proteins, is characterized by four transmembrane domains and two extracellular loops. Surface expression of CD9 on Chinese hamster ovary (CHO) cells dramatically enhances spreading and motility on fibronectin. To elucidate the mechanistic basis of CD9-fibronectin interaction, binding to fibronectin was investigated using purified and recombinant forms of CD9. The affinity of fibronectin for CD9 in enzyme-linked immunosorbent assay was 81 +/- 25 nm. The binding of fibronectin to immobilized CD9 was enhanced by Ca(2+) ions. Protein binding and peptide competition studies demonstrated that peptide 6 derived from CD9 extracellular loop 2 (amino acids 168-192) contained part of the fibronectin-binding domain. Additionally, enhanced adhesion of CD9-CHO-B2 cells to fibronectin was significantly reduced by peptide 6. CD9-CHO cells had a 5-fold increase in motility to fibronectin as compared with mock-transfected controls, an effect that correlated with CD9 cell surface density. Truncation of CD9 extracellular loop 2 and peptide 6 caused inhibition of CD9-CHO cell motility to fibronectin. Deletion of CD9 extracellular loop 1 had no significant effect on CHO cell motility. These findings demonstrate a critical role for CD9 extracellular loop 2 in cell motility to fibronectin and clarify the mechanism by which CD9-fibronectin interaction modulates cell adhesion and motility.     

Characterization of SP41, a surface protein of Brucella associated with adherence and invasion of host epithelial cells

Brucella is an invasive organism that multiplies and survives within eukaryotic cells. The brucellae are able to adhere to the surface of cultured epithelial cells, a mechanism that may facilitate penetration and dissemination to other host tissues. However, no adhesins that allow the bacteria to interact with the surface of epithelial cells before migration within polymorphonuclear leukocytes, monocytes and macrophages have been described. Here, we show that Brucella surface proteins (SPs) with apparent molecular masses of 14, 18 and 41 kDa bound selectively to HeLa cells. However, only antibodies directed against the 41 kDa surface protein (SP41) inhibited in dose-response manner, bacterial adherence and invasion of HeLa cells. HeLa cells treated with neuraminidase did not bind SP41, suggesting the involvement of cellular sialic acid residues in this interaction. Biochemical analysis of SP41 revealed that this protein is the predicted product of the ugpB locus, which showed significant homology to the glycerol-3-phosphate-binding ATP-binding cassette (ABC) transporter protein found in several bacterial species. SP41 appears to be exposed on the bacterial surface as determined by immunofluorescence and immunogold labelling with anti-SP41 antibody. An isogenic DeltaugpB mutant showed a significant inhibitory effect on Brucella adherence and invasion of human cultured epithelial cells and this effect could be reversed by restoration of the ugpB on a plasmid. Lastly, we also show that most of the sera from individuals with acute brucellosis, but not sera obtained from healthy donors or patients with chronic brucellosis, mount antibody reactivity against SP41, suggesting that this protein is produced in vivo and that it elicits an antibody immune response. These data are novel findings that offer new insights into understanding the interplay between this bacterium and host target cells, and identify a new target for vaccine development and prevention of brucellosis.     

A fibronectin-binding protein from Streptococcus equi binds collagen and modulates cell-mediated collagen gel contraction

The N-terminal fragment (FNZN) of the fibronectin-binding protein FNZ from Streptococcus equi subspecies zooepidemicus was investigated as to effects on murine cell interactions with extracellular matrix proteins. FNZN bound to immobilized fibronectin (FN) and native, but not denatured, collagen type I. FNZN had no effect on primary adhesion of cells from the murine myoblastic C2C12 cell line to immobilized fibronectin. C2C12 cells adhered to immobilized FNZN, a process that was not inhibited by anti-human FN IgG or by an inhibitor of integrin alphaVbeta3. C2C12 cells lack collagen-binding beta1 integrins and neither adhere to native collagen nor mediate contraction of three-dimensional collagen gels. FNZN stimulated collagen gel contraction by C2C12 cells but not adhesion of C2C12 cells to collagen. Experiments with an alphaVbeta3-inhibitor suggested that FNZN promoted contraction by a process requiring alphaVbeta3. Our data suggest that FNZN by binding to cells, collagen, and FN modulate complex adhesive processes mediated by the alphaVbeta3 integrin. Since alphaVbeta3-mediated contractile events function to counteract edema formation during inflammation, it is possible that FNZN and its secreted homologue FNE modulate edema responses in infected tissues.     

The N-terminal A domain of fibronectin-binding proteins A and B promotes adhesion of Staphylococcus aureus to elastin

The ability of Staphylococcus aureus to adhere to components of the extracellular matrix is an important mechanism for colonization of host tissues during infection. We have previously shown that S. aureus binds elastin, a major component of the extracellular matrix. The integral membrane protein, elastin-binding protein (EbpS), binds soluble elastin peptides and tropoelastin via its surface-exposed N-terminal domain. In this study, we demonstrate that some strains of S. aureus adhere strongly to immobilized human elastin and that this interaction is independent of EbpS but instead is mediated by the fibronectin-binding proteins, FnBPA and FnBPB. Our results show that EbpS mutant cells adhere to elastin-coated plates, whereas the cells negative for FnBPA and FnBPB do not adhere to the plates. Furthermore, only wild-type cells from the exponential phase of growth adhered when FnBPs were expressed maximally. We show that adherence to elastin promoted by FnBPA was not affected by soluble fibronectin, suggesting that the elastin binding domain is distinct from the fibronectin binding regions. Recombinant FnBPA(37-544) (rFnBPA(37-544)) protein corresponding to the A region of FnBPA and anti-FnBPA(37-544) antibodies inhibited FnBPA-mediated bacterial adherence to immobilized elastin. Finally, recombinant A domain proteins, rFnBPA(37-544) and rFnBPB(37-540), bound immobilized elastin dose-dependently and saturably. This interaction was inhibited by soluble elastin peptides, suggesting a specific receptor-ligand interaction.     

The adhesion of protein A-bearing Staphylococcus aureus organisms to soluble-staphylococcal-antigens-coated HeLa cells mediated by specific antibodies

The adhesion of staphylococcal protein A (SpA)-bearing Staphylococcus aureus Cowan I organisms to HeLa cells was enhanced by pretreatment of HeLa cells with staphylococcal extracellular antigens and antibodies to them. The adhesion of HLj, an SpA-poor mutant derived from Cowan I, to HeLa cells was not enhanced by the same pretreatment of HeLa cells. Furthermore, the enhanced staphylococcal adhesion was inhibited by soluble SpA. The antigen(s) responsible for the enhanced staphylococcal adhesion was(were) heat stable. Pretreatment of HeLa cells with the mixture of staphylococcal extracellular antigens and antibodies to them also enhanced the adhesion of Cowan I. Similarly the adhesion of Cowan I was enhanced by pretreatment of HeLa cells with extracellular antigens of Pseudomonas aeruginosa and antibodies to them. These results indicated that cell-bound SpA mediated the binding of S. aureus to immune complexes composed of extracellular bacterial products and antibodies to them bound to the surface of HeLa cells, and suggested another role of cell-bound SpA as a co-adhesin with other factors in infections due to S. aureus.     

Definition of the Native and Denatured Type II Collagen Binding Site for Fibronectin Using a Recombinant Collagen System

Interaction of collagen with fibronectin is important for extracellular matrix assembly and regulation of cellular processes. A fibronectin-binding region in collagen was identified using unfolded fragments, but it is not clear if the native protein binds fibronectin with the same primary sequence. A recombinant bacterial collagen is utilized to characterize the sequence requirement for fibronectin binding. Chimeric collagens were generated by inserting the putative fibronectin-binding region from human collagen into the bacterial collagen sequence. Insertion of a sufficient length of human sequence conferred fibronectin affinity. The minimum sequence requirement was identified as a 6-triplet sequence near the unique collagenase cleavage site and was the same in both triple-helix and denatured states. Denaturation of the chimeric collagen increased its affinity for fibronectin, as seen for mammalian collagens. The fibronectin binding recombinant collagen did not contain hydroxyproline, indicating hydroxyproline is not essential for binding. However, its absence may account, in part, for the higher affinity of the native chimeric protein and the lower affinity of the denatured protein compared with type II collagen. Megakaryocytes cultured on chimeric collagen with fibronectin affinity showed improved adhesion and differentiation, suggesting a strategy for generating bioactive materials in biomedical applications.     

Fibronectin binding to the Salmonella enterica serotype Typhimurium ShdA autotransporter protein is inhibited by a monoclonal antibody recognizing the A3 repeat

ShdA is a large outer membrane protein of the autotransporter family whose passenger domain binds the extracellular matrix proteins fibronectin and collagen I, possibly by mimicking the host ligand heparin. The ShdA passenger domain consists of approximately 1,500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence: an N-terminal nonrepeat region followed by a repeat region composed of two types of imperfect direct amino acid repeats, called type A and type B. The repeat region bound bovine fibronectin with an affinity similar to that for the complete ShdA passenger domain, while the nonrepeat region exhibited comparatively low fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger domain to fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3, and B9. Furthermore, a monoclonal antibody that specifically recognized an epitope in a recombinant protein containing the A3 repeat inhibited binding of ShdA to fibronectin.     

Identification of a fibronectin-binding protein of Treponema lecithinolyticum by two-dimensional gel electrophoresis and ligand binding assay

Treponema lecithinolyticum is associated with periodontitis and endodontic infections. As a critical early step in the infection process, fibronectin-binding protein (Fbp) is known to be involved in the adhesion of bacteria to cell surfaces for colonization and, hence, is considered to be a virulence factor. In this study, we identified an Fbp from the T. lecithinolyticum cell surface with a molecular mass of about 52 kDa by using 2-dimensional gel electrophoresis followed by a ligand binding assay. As T. lecithinolyticum is capable of binding to soluble and immobilized fibronectin, this Fbp may contribute to bacterial attachment to host cells.     

Fibronectin fibrillogenesis, a cell-mediated matrix assembly process.

The extracellular matrix provides a framework for cell adhesion, supports cell movement, and serves to compartmentalize tissues into functional units. Fibronectin is a core component of many extracellular matrices where it regulates a variety of cell activities through direct interactions with cell surface integrin receptors. Fibronectin is synthesized by many adherent cells which then assemble it into a fibrillar network. The assembly process is integrin-dependent and fibronectin-integrin interactions initiate a step-wise process involving conformational activation of fibronectin outside and organization of the actin cytoskeleton inside. During assembly, fibronectin undergoes conformational changes that expose fibronectin-binding sites and promote intermolecular interactions needed for fibril formation. In this review, the main steps of fibronectin assembly are described and recent studies on fibronectin conformational changes are discussed.