Staphylococcus saprophyticus hemagglutinin binds fibronectin.

Attachment of microorganisms to host tissue is regarded as an important step in the pathogenesis of infections. Staphylococcus saprophyticus adheres to various epithelial cells and hemagglutinates sheep erythrocytes. The hemagglutinin has been identified, but a human target for this surface protein is still not known. In our report, we show that hemagglutinating strains of S. saprophyticus bind to immobilized fibronectin, whereas nonhemagglutinating strains do not. Bacterial binding was inhibited by antibody to the hemagglutinin but not by antibody to Ssp, another surface protein of S. saprophyticus. The purified hemagglutinin but not other surface proteins bound biotin-labeled fibronectin. Binding was saturable and could be inhibited by unbound hemagglutinin, unlabeled fibronectin, and by antibody to the hemagglutinin. We thus conclude that the hemagglutinin of S. saprophyticus may act as a fibronectin receptor in the human host. Heparin, the D3 peptide, or Arg-Gly-Asp-Ser (RGDS) containing peptides did not inhibit binding of fibronectin to the hemagglutinin, indicating that the binding site is different from that of Staphylococcus aureus or Treponema pallidum.     

Virulent Treponema pallidum 47 kDa antigen regulates the expression of cell adhesion molecules and binding of T-lymphocytes to cultured human dermal microvascular endothelial cells

Perivasculitis and endothelial cell abnormalities are prominent histopathologic features of syphilis. Various cutaneous lesions are the main clinical features of syphilis. We examined whether Treponema pallidum 47 kDa antigen regulates the expression of cell adhesion molecules on human dermal microvascular endothelial cells (HDMEC) and the regulation of T-lymphocytes binding to HDMEC. Using immunofluorescence flow cytometry and enzyme-linked immunosorbent assay (ELISA), we demonstrated that T. pallidum upregulated the expression of adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. The 47 kDa antigen of T. pallidum also activated endothelium as measured by the upregulation of the expression of adhesion molecules on HDMEC, and it also promoted an increased adherence of T-lymphocytes to HDMEC. The expressions of ICAM-1 and VCAM-1 on HDMEC and the adherence of T-lymphocytes to HDMEC were inhibited by treatment with anti-TNF-alpha antibody or anti-IL-1alpha antibody. These results show that T. pallidum or T. pallidum-specific 47 kDa antigen are capable of stimulating HDMEC to increase the expression of ICAM-1, VCAM-1 and E-selectin and thereby, promote the adherence of T-lymphocytes. The whole process may play an important role in the immunopathogenesis of syphilis and it is likely that TNF-alpha and IL-1alpha are involved.     

Antibody-independent interactions of fibronectin, C1q, and human neutrophils with Treponema pallidum.

Although recent evidence suggests that fibronectin may be involved in the attachment of treponemes to mammalian cells, its possible role in promoting phagocytosis of Treponema pallidum has not been investigated. In the present study, we examined the antibody-independent interactions of fibronectin, C1q, and human polymorphonuclear leukocytes with T. pallidum. Binding of [125I]fibronectin was specific and saturable with an affinity constant of approximately 2 X 10(7) M-1. The number of binding sites per treponeme at 37 degrees C, irrespective of the mammalian source of fibronectin, was between 2,500 and 7,500, with a mean of approximately 4,700. Binding of [125I]C1q to T. pallidum, in the absence of antibodies to the organism, also was saturable and specific. Pretreatment of treponemes with C1q enhanced binding of soluble [125I]fibronectin two- to threefold and also increased attachment of 125I-surface-labeled treponemes to fibronectin-coated surfaces. Treatment of 125I-labeled T. pallidum with fibronectin alone, or together with C1q, however, did not enhance surface phagocytosis by neutrophils.     

Fibronectin degradation; an in-vitro model of neutrophil mediated endothelial cell damage

We have observed that fibronectin has a characteristic fibrillar morphology within the extracellular matrix surrounding endothelial cells. This morphology, which is easily recognizable by conventional immunoperoxidase techniques, is disrupted if neutrophils are induced to degranulate on endothelial monolayers. Loss of the fibrillar morphology (degraded fibronectin) is characterized by fragmentation and diffuse spreading of fibronectin over the surface of the endothelial cells. Loss of the normal fibronectin architecture following neutrophil degranulation is more rapid and extensive in endothelium pretreated with Interleukin-1 (IL-1). In addition, there is loss from the fibronectin molecule of a chymotryptic protease-sensitive epitope recognized by a cellular fibronectin specific antibody. Degraded fibronectin is stimulatory for neutrophils, and is likely to induce further fibronectin breakdown. This sequence has the potential to set up an amplification inflammatory loop with neutrophil mediated loss of vascular homeostasis. Alteration of fibronectin architecture is a useful marker of endothelial injury, and has important pathophysiological consequences.     

Putative Treponema pallidum cytadhesins share a common functional domain.

Three putative Treponema pallidum ligands (P1, P2, and P3) that bind host fibronectin were characterized by peptide mapping. Papain digestion of each protein yielded a comigrating peptide of approximately 12,000 molecular weight. An antibody to this protein fragment inhibited T. pallidum host cytadherence, indicating that this peptide may be the functional domain of these treponemal adhesins.     

A novel Treponema pallidum antigen, TP0136, is an outer membrane protein that binds human fibronectin

The antigenicity, structural location, and function of the predicted lipoprotein TP0136 of Treponema pallidum subsp. pallidum were investigated based on previous screening studies indicating that anti-TP0136 antibodies are present in the sera of syphilis patients and experimentally infected rabbits. Recombinant TP0136 (rTP0136) protein was purified and shown to be strongly antigenic during human and experimental rabbit infection. The TP0136 protein was exposed on the surface of the bacterial outer membrane and bound to the host extracellular matrix glycoproteins fibronectin and laminin. In addition, the TP0136 open reading frame was shown to be highly polymorphic among T. pallidum subspecies and strains at the nucleotide and amino acid levels. Finally, the ability of rTP0136 protein to act as a protective antigen to subsequent challenge with infectious T. pallidum in the rabbit model of infection was assessed. Immunization with rTP0136 delayed ulceration but did not prevent infection or the formation of lesions. These results demonstrate that TP0136 is expressed on the outer membrane of the treponeme during infection and may be involved in attachment to host extracellular matrix components.     

Role of glycosaminoglycan and fibronectin in endothelial cell growth

This study was undertaken to clarify the biological significance of fibronectin and GAG synthesized in human endothelium. The synthesis of GAG and fibronectin was proved morphologically and biochemically in endothelial cells. The conditioned media of fibroblasts and smooth muscle cells have promoted endothelial cell proliferation and production of extracellular matrix in the endothelium. These results suggest that the extracellular matrix which is composed of fibronectin and GAG may play an important role in endothelial cell regeneration.     

In vitro model of Treponema pallidum invasiveness.

The purpose of this investigation was to develop an in vitro model with which invasion of tissues by pathogenic Treponema pallidum could be studied. Double-sided culture chambers were created by mounting abdominal walls excised from mice between two halves of small dialysis cells. The integrity of tissue barriers was confirmed by dye exclusion. T. pallidum subsp. pallidum, including intrinsically radiolabeled organisms, was introduced into one side of each chamber, and fractions from the other side were evaluated over time by dark-field microscopy and scintillation counting. Tissues were evaluated by scanning electron microscopy and immunologic staining. Motile T. pallidum, but not nonpathogenic, host-indigenous Treponema phagedenis biotype Reiter, was able to pass from one side of the chamber to the other side within 10 h. Up to 12% of the inoculum crossed the chamber within 24 h. Spirochetes were found within tissue in the greatest numbers between 6 and 8 h postinoculation. The murine abdominal wall has epithelium only on the peritoneum side, and results showed that T. pallidum required an epithelial surface on the entry side of the double-chambered cell in order to traverse the tissue barrier. This new in vitro technique may be of value in studying spirochete virulence and host resistance.     

Inhibition of hydrophobic protein-mediated Candida albicans attachment to endothelial cells during physiologic shear flow

Adhesion interactions during hematogenous dissemination of Candida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1 beta activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.     

Virulent Treponema pallidum promotes adhesion of leukocytes to human vascular endothelial cells.

Perivasculitis and endothelial cell abnormalities are characteristic histopathologic features of syphilis, a sexually transmitted disease caused by Treponema pallidum. To extend earlier studies demonstrating that T. pallidum activates endothelial cells, we now show that virulent T. pallidum, but not heat-killed T. pallidum or nonpathogenic Treponema phagedenis, promotes increased adherence of lymphocytes and monocytes to human umbilical vein endothelial cells. Lymphocytes and monocytes are the two cell types prominent in the histopathology of syphilis. Recognition that T. pallidum can stimulate endothelial cells to bind leukocytes provides important insights into the early mechanisms of syphilis immunopathogenesis.     

Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405

There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type I, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.     

Parasitism by Virulent Treponema pallidum of Host Cell Surfaces

The interaction between virulent Treponema pallidum extracted from infected rabbit testes and animal cells in culture was examined. The extent of treponemal attachment to monolayers of normal rabbit testicular and HEp-2 cells was dependent upon the incubation temperature and retained motility of the spirochetes. The specific orientation of treponemes to host cell surfaces was demonstrated by dark-field microscopic examination of wet-mount preparations and scanning and transmission electron microscopy. Once attached, T. pallidum organisms remained actively motile yet anchored in place by their terminal tapered structures. After several hours of co-incubation, maximal attachment was attained, and the degree of parasitism seemed regulated not only by available surface sites on individual host cells but also by the proposed membrane response of parasitized cells to continued exposure to treponemes. The avirulent strain, Treponema phagedenis biotype Reiter, did not adhere to monolayer cultures. Characterization of host cell determinants that permitted surface colonization by T. pallidum was attempted. Also, properties of virulent treponemes that enabled surface parasitism were monitored by measuring the effects of enzymes, detergents, and metabolic inhibitors on the host-parasite interaction. Results reinforced the specific nature of the treponemal attachment mechanism. Furthermore, the ability of convalescent rabbit sera to reduce attachment of treponemes to host cells suggested that surface structures on T. pallidum could be masked or inactivated by host components, thus providing a potentially effective research approach for investigating the pathogenesis of syphilis and screening appropriate vaccine candidates.     

Herpes simplex virus inhibits endothelial cell attachment and migration to extracellular matrix proteins.

Herpes simplex virus (HSV) infection may be involved in various endothelial-injury syndromes, including vasculitis and atherosclerosis. In a previous study, it was reported that HSV-infected human umbilical endothelial cells are more vulnerable to detachment mediated by granulocyte-secreted proteases. To elucidate the molecular basis of this observation, the authors examined the interaction of infected endothelial cells with the purified basement membrane proteins, fibronectin, laminin, and type IV collagen. HSV-infected endothelial cells exhibited defects in their ability to adhere, spread, and migrate on all three matrix components. This defective adhesion could be partially overcome by increasing concentrations of fibronectin; in contrast, no abrogation of deficient binding occurs with increased levels of laminin or collagen type IV. This suggests that endothelial cells may use different surface constituents for binding to the three proteins and use multiple "receptors" for adhesion to the fibronectin molecule--"receptors" that are variably affected by HSV infection. The authors investigated this supposition by assaying adhesion of normal and infected endothelial cells to two non-overlapping cell-adhesion promoting fragments of fibronectin: 1) a 75 kd motility-promoting fragment which contains the arginyl-glycyl-aspartylserine (RGDS) adhesion sequence, and 2) a 33 kd carboxyl-terminal heparin binding fragment, which promotes cell adhesion by an RGDS-independent mechanism. Normal endothelial cells adhered and spread on both purified fragments. In contrast, while infected endothelial cells could adhere, albeit rather poorly, to high coating concentrations of the 75 kd fragment, these cells did not bind to the 33 kd heparin binding fragment of fibronectin at all. These results support the concept that endothelial cells adhere to multiple domains of fibronectin, and that HSV infection preferentially abrogates binding to the heparin-binding domain, while leaving relatively intact receptors for the RGDS-containing domain. In support, soluble RGDS significantly blocked fibronectin adhesion of infected, but not control, endothelial cells. It is concluded that HSV infection inhibits the interaction of endothelial cells with basement membrane proteins and weakens their tethering to substratum. This tethering is inadequate for proper cell spreading or movement to occur and may result in both excessive endothelial lift-off and impaired vascular repair in HSV infections.     

Effect of sugars and metabolic intermediates on the attachment of Treponema pallidum to rabbit cells

The effect of various energy sources and metabolic intermediates on the attachment of Treponema pallidum to baby rabbit genital organ (BRGO) cells in culture was examined. Pyruvate and glucose enhanced the motility of T. pallidum in vitro. Pyruvate increased significantly the attachment of treponemes to BRGO cells when compared with the other substrates but all substrates tested stimulated DNA synthesis by cultured BRGO cells. Thus, the effect of pyruvate on attachment may be due to an effect on the treponemes. Prior exposure of the BRGO cells to the glucose analogue 2-deoxyglucose greatly inhibited the attachment of T. pallidum whereas three other analogues had no effect. The inhibitory effect of 2-deoxyglucose was partially reversed by the presence of pyruvate in the attachment assay. These results suggest that energy metabolism of both T. pallidum and host cells may be required for the initial interaction of T. pallidum with its host in vivo.     

Enhanced levels of attachment of fibronectin-primed Treponema pallidum to extracellular matrix.

Freshly extracted Treponema pallidum organisms treated with exogenous human fibronectin (Fn) (Fn-primed treponemes) showed a 6- to 15-fold increased level of attachment to Fn-coated cover slips and to extracellular matrix (ECM) when compared with unprimed treponemes. Treponemes primed with collagen or laminin showed no similar enhanced binding to immobilized Fn or ECM. Preexposure of immobilized Fn and ECM to anti-Fn serum but not to anti-collagen or anti-laminin serum prevented treponemal adherence. Also, the presence of proteoglycanlike molecules such as dextran sulfate or heparan sulfate inhibited Fn-primed treponemal attachment to Fn or ECM. In contrast Fn-primed treponemes did not exhibit elevated levels of attachment to eucaryotic cell monolayers. To understand the increased tropism of Fn-primed T. pallidum organisms for Fn and ECM-like surfaces, we radiolabeled freshly extracted treponemes with [35S]methionine and examined them for the presence of surface immunoreactive Fn. Magnetic protAspheres and glass beads coated with monospecific anti-Fn serum bound only 20 to 30% of radiolabeled treponemes. Nonadherent treponemes failed to bind to gelatin-agarose, further confirming the absence of surface Fn or Fn-like material. Fn-free organisms, however, did attach to Fn-coated cover slips and to cell monolayers like treponemes of the original population. Incubation of Fn-free treponemes with human Fn resulted in almost total binding of organisms to anti-Fn antibody on glass beads and also produced increased attachment to Fn-coated cover slips and ECM. These results suggest that enhanced interactions between T. pallidum and the host are dependent on the presence of Fn on syphilis spirochetes and the specific location and orientation of Fn in vivo.     

Release of fibronectin fragments from endothelial cell monolayers exposed to activated leukocytes: relationship to plasma fibronectin levels after particle infusion

Fibronectin is found in a soluble form in plasma as well as in an insoluble form in tissues. It is produced by cultured endothelial cells and can be localized in vitro and in vivo between adjacent endothelial cells as well as underneath endothelial cells in association with their collagenous matrix, where it is believed to influence cell adhesion. Fibronectin is susceptible to proteolytic cleavage by enzymes released from activated leukocytes. In the present study, cultured rat endothelial cells developed a fibrillar fibronectin network in their extracellular matrices in addition to releasing soluble, intact fibronectin (440 kDa) into their culture medium. Exposure of monolayers of cultured endothelial cells to activated polymorphonuclear leukocytes (PMNs) results in disruption of the fibrillar matrix fibronectin, damage to the endothelial cell monolayer, and presence of fibronectin fragments in the culture medium. In addition, acute leukocyte activation and peripheral leukopenia in vivo as induced by the intravenous infusion of foreign test particles also resulted in the appearance of low-molecular-weight fibronectin fragments in plasma. In the in vivo studies, the appearance of fibronectin fragments preceded the release of intact 440-kDa fibronectin in the plasma after its acute depletion by particle injection. Thus, activated leukocytes, adherent to an endothelial surface in vitro and in vivo, may result in degradation of matrix fibronectin and the release of fibronectin fragments into the extracellular environment. In vivo fibronectin fragments in blood may serve as a stimulus for the subsequent synthesis and/or release of intact plasma fibronectin.     

Endothelium as a target for antiphospholipid antibodies

Several, and not necessarily alternative, pathogenic mechanisms have been suggested to sustain the thrombophilic diathesis of the anti-phospholipid syndrome. Among them, interference of anti-phospholipid antibodies with cell acting in the coagulation cascade likely plays a major role. Anti-phospholipid antibodies have been shown to react with endothelial cells mainly by reacting with beta 2 glycoprotein I expressed on the cell membrane surface. Beta 2 glycoprotein I can adhere to endothelial cell surface through the Annexin II receptor and through negatively charged structures (heparin-like molecules) that are bound by the phospholipid-binding site of the molecule. The autoantibody binding involves a yet unknown receptor that activates a signalling pathway able to translocate NFkappaB from the cytoplasm to the nucleus and to activate genes for adhesion molecule, pro-inflammatory cytokine and Tissue Factor up-regulation. The ultimate effect is the induction of a pro-inflammatory and a pro-coagulant endothelial phenotype that has been reproduced both in vitro and in vivo experimental models. Additional effects of anti-phospholipid-mediated endothelial cell activation are the interference with the protein C/S system, with the Annexin V binding, the up-regulation of endothelin I synthesis and the induction of apoptosis. Altogether these effects cooperate in switching endothelium from an anti-coagulant to a pro-coagulant surface.     

Outer membrane protein YadA of enteropathogenic yersiniae mediates specific binding to cellular but not plasma fibronectin.

The binding of bacteria or bacterial products to host proteins of tissue extracellular matrix may be a mechanism of tissue adherence. We investigated interactions of the plasmid-encoded outer membrane protein YadA, which confers pathogenic functions on enteropathogenic yersiniae, with fibronectin. Attachment of YadA-positive and YadA-negative recombinant Yersinia enterocolitica strains to cartilage-derived human cellular fibronectin and human plasma fibronectin in the solid phase revealed that YadA mediates binding of yersiniae to cellular fibronectin in a saturable, concentration-dependent manner. The interaction could be inhibited by an anti-YadA-specific anti-serum. An anti-beta 1-integrin antibody and the synthetic peptide G-R-G-D-S-P, representing the binding site for alpha 5 beta 1-integrin on fibronectin, did not block attachment of YadA-positive yersiniae to cellular fibronectin, indicating a binding site for YadA on cellular fibronectin independent of the R-G-D-S-containing site. By contrast, YadA failed to mediate binding to plasma fibronectin immobilized on nitrocellulose or plastic surfaces. These observations provide evidence for the hypothesis that the binding region for YadA in cellular fibronectin is not present in plasma fibronectin. This study is the first report on differential binding of bacteria to splicing variants of fibronectin. Further experiments might answer the question whether binding of YadA to cellular fibronectin contributes to the pathogenesis of yersiniae, both to the initial adhesion of the bacteria to the matrices of the host and to the arthritogenic potential of enteropathogenic yersiniae.     

Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans.

Mycoplasma penetrans adhered to cultured human cells, forming clusters that localized to specific areas of the host cell surface. Adherence and cluster formation were inhibited by anti-M. penetrans antibodies, suggesting the involvement of specific adhesin-receptor interactions. Ultrastructural studies showed that after 2 h of infection, mycoplasmas attach to and penetrate the host cell surface. M. penetrans bound selectively to immobilized fibronectin, an interaction which was not inhibited by a 70-kDa fragment containing a heparin-gelatin-binding domain of fibronectin, other matrix glycoproteins, or an RGD tripeptide, suggesting the recognition of other specific binding sites on the fibronectin molecule. A ca. 65-kDa fibronectin-binding protein of M. penetrans was eluted following Sepharose-fibronectin affinity chromatography. Confocal, light, and immunofluorescence microscopy demonstrated that the interaction of M. penetrans with target cells triggers a signal that causes recruitment of several cytoskeletal components, including tubulin and alpha-actinin, and aggregation of phosphorylated proteins. Detergent-soluble mycoplasma proteins with apparent molecular masses of 18, 28, 32, 36, 39, and 41 kDa selectively bound to glutaraldehyde-fixed HEp-2 cells. Our findings offer new insights into understanding the interaction of this human mycoplasma with host target cells.