Adherence of Abiotrophia defectiva and Granulicatella species to fibronectin: is there a link with endovascular infections?

During a 6-year period, we isolated three Abiotrophia defectiva, six Granulicatella adiacens and two G. 'para-adiacens' strains from clinical specimens. All A. defectiva strains were isolated from immunocompetent patients with endovascular infections, whereas the Granulicatella spp. strains were isolated from immunosuppressed patients with primary bacteremia. As the capacity of bacteria to adhere to the host extracellular matrix (ECM) has been implicated in the pathogenesis of endovascular infection, we investigated the ability of A. defectiva and Granulicatella spp. isolates to bind different ECM components immobilized in microtiter plates. Adherence tests showed a strong attachment of A. defectiva strains to fibronectin, whereas Granulicatella spp. strains were not adherent. The poor adherence of Granulicatella spp. strains to the ECM could be correlated with a lower propensity to induce endocarditis.     

Adherence of Candida albicans to components of the subendothelial extracellular matrix

Candida albicans yeasts adhered avidly to extracellular matrix (ECM) proteins, type IV collagen, laminin, and fibronectin immobilized on plastic. Type IV collagen showed an increase of adherence of 400% above control values; laminin, 300%; and fibronectin, 150%. In addition, all three (in quantities of 0.02-200 micrograms/well of a culture tray) bound yeasts in a dose-response fashion. Adherence was inhibited when the proteins were preincubated with specific antibody, except with type IV collagen. Soluble laminin or fibronectin inhibited yeast adherence to the same proteins by 36 and 94%, respectively. Soluble fibronectin bound to the yeast surface and in so doing inhibited subsequent yeast adherence to fibronectin by 66%. By comparison, Candida albicans yeasts adhered in smaller numbers to glycosaminoglycans (GAGs). Keratan sulfate, hyaluronic acid, chondroitin sulfate, Type B, and heparin actually decreased yeast adherence compared to control from 10% to 25%.     

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.     

A Collagen-Binding S-Layer Protein in Lactobacillus crispatus

Two S-layer-expressing strains, Lactobacillus crispatus JCM 5810 and Lactobacillus acidophilus JCM 1132, were assessed for adherence to proteins of the mammalian extracellular matrix. L. crispatus JCM 5810 adhered efficiently to immobilized type IV and I collagens, laminin, and, with a lower affinity, to type V collagen and fibronectin. Strain JCM 1132 did not exhibit detectable adhesiveness. Within the fibronectin molecule, JCM 5810 recognized the 120-kDa cell-binding fragment of the protein, while no bacterial adhesion to the amino-terminal 30-kDa or the gelatin-binding 40-kDa fragment was detected. JCM 5810 but not JCM 1132 also bound (sup125)I-labelled soluble type IV collagen, and this binding was efficiently inhibited by unlabelled type IV and I collagens and less efficiently by type V collagen, but not by laminin or fibronectin. L. crispatus JCM 5810 but not L. acidophilus JCM 1132 also adhered to Matrigel, a reconstituted basement membrane preparation from mouse sarcoma cells, as well as to the extracellular matrix prepared from human Intestine 407 cells. S-layers from both strains were extracted with 2 M guanidine hydrochloride, separated by electrophoresis, and transferred to nitrocellulose sheets. The S-layer protein from JCM 5810 bound (sup125)I-labelled type IV collagen, whereas no binding was seen with the S-layer protein from JCM 1132. Binding of (sup125)I-collagen IV to the JCM 5810 S-layer protein was effectively inhibited by unlabelled type I and IV collagens but not by type V collagen, laminin, or fibronectin. It was concluded that L. crispatus JCM 5810 has the capacity to adhere to human subintestinal extracellular matrix via a collagen-binding S-layer.     

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.     

Collagen Binding of Bifidobacterium adolescentis

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity. Received: 15 October 1996 / Accepted: 20 November 1996     

Extracellular matrix proteins modulate endocytosis of the insulin receptor

Internalization of the insulin receptor (IR) is a highly regulated multi-step process whose underlying molecular basis is not fully understood. Here we undertook to study the role of extracellular matrix (ECM) proteins in the modulation of IR internalization. Employing Chinese hamster ovary cells that overexpress IR (CHO-T cells), our results indicate that IR internalization proceeds unaffected even when Tyr phosphorylation of IR substrates, such as IRS-1, is impaired (e.g. in CHO-T cells overexpressing IRS-1 whose pleckstrin-homology domain has been deleted or in CHO-T cells that overexpress the PH/PTB domain of IRS-1). In contrast, IR internalization is affected by the context of the ECM proteins to which the cells adhere. Hence, IR internalization was inhibited 40-60% in CHO-T cells adherent onto galectin-8 (an ECM protein and an integrin ligand of the galectin family) when compared with cells adherent onto fibronectin, collagen, or laminin. Cells adherent to galectin-8 manifested a unique cytoskeletal organization, which involved formation of cortical actin and generation of F-actin microspikes that contrasted with the prominent stress-fibers formed when cells adhered to fibronectin. To better establish a role for actin filament organization in IR endocytosis, this process was assayed in CHO-T cells (adherent onto fibronectin), whose actin filaments were disrupted upon treatment with latrunculin B. Latrunculin B did not affect insulin-induced Tyr phosphorylation of IR or its ability to phosphorylate its substrates; still, a 30-50% reduction in the rate of IR internalization was observed in cells treated with latrunculin B. Treatment of cells with nocodazole, which disrupts formation of microtubules, did not affect IR internalization. These results indicate that proper actin, but not microtubular, organization is a critical requirement for IR internalization and suggest that integrin-mediated signaling pathways emitted upon cell adhesion to different extracellular matrices and the altered cytoskeletal organizations generated thereof affect the itinerary of the insulin receptor.     

Adherence to host extracellular matrix and serum components by Enterococcus faecium isolates of diverse origin

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I (CI) as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm -mediated CI adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (Fn) (20% of the 60 isolates), fibrinogen (17%) and laminin (Ln) (13%), while only one or two of the isolates adhered to collagen type V (CV), transferrin or lactoferrin and none to the other host components tested. Adherence to Fn and Ln was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to Fn and Ln, in addition to CI, may have contributed to the emergence and adaptation of E. faecium , in particular CC17, as a nosocomial pathogen.     

Streptococcus suis serotype 2 binding to extracellular matrix proteins

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.     

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.     

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.     

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.     

Interaction of embryonic corneal epithelium with exogenous collagen,laminin, and fibronectin: Role of endogenous protein synthesis

Previously, we have shown that the embryonic corneal epithelium is capable of interacting with exogenous collagen, laminin, and fibronectin in soluble form, each of which causes isolated epithelium cultured on Millipore filter to stop blebbing, reorganize the basal cytoskeleton, and flatten. Here we examine the involvement of endogenously derived extracellular matrix (ECM) molecules in the interaction of the basal epithelial cell surface with the added ECM molecules. We demonstrate here that the isolated avian corneal epithelium cultured on Millipore filter is capable of synthesizing collagens and laminin, but not fibronectin. To examine whether the epithelium is capable of interacting directly with exogenous ECM components or if there is the necessity for production of a linker molecule, epithelial protein synthesis was inhibited with cycloheximide (CHX). The blebbing epithelium in the presence of CHX was then confronted with soluble ECM molecules added to the medium under the filter; such epithelia are able to interact with, and flatten in response to, both collagen and laminin. However, such inhibited epithelia continue to bled in the presence of fibronectin. We next used l-azetidine-4-carboxylic acid (LACA) to interfere with collagen secretion. Epithelia exposed to LACA are still capable of interacting with collagen and laminin, but not fibronectin, indicating a dependence on collagen secretion. These results suggest that fibronectin requires a linker protein, probably collagen, to interact with the basal epithelial surface, whereas both collagen and laminin may interact directly with the cell surface to transform the basal cytoskeleton into the cortical mat typical of differentiating corneal epithelium in situ.     

Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding.

The localization of the extracellular matrix recognition molecule J1/tenascin was investigated in the crypt-villus unit of the adult mouse ileum by immunoelectron microscopic techniques. In the villus region, J1/tenascin was detected strongly in the extracellular matrix (ECM) between fibroblasts of the lamina propria. It was generally absent in the ECM at the interface between subepithelial fibroblasts and intestinal epithelium, except for some restricted areas along the epithelial basal lamina of villi, but not of crypts. These restricted areas corresponded approximately to the basal part of one epithelial cell. In J1/tenascin-positive areas, epithelial cells contacted the basal lamina with numerous microvillus-like processes, whereas in J1/tenascin-negative areas the basal surface membranes of epithelial cells contacted their basal lamina in a smooth and continuous apposition. In order to characterize the functional role of J1/tenascin in the interaction between epithelial cells and ECM, the intestinal epithelial cell line HT-29 was tested for its ability to adhere to different ECM components. Cells adhered to substratum-immobilized fibronectin, laminin and collagen types I to IV, but not to J1/tenascin. When laminin or collagen types I to IV were mixed with J1/tenascin, cell adhesion was as effective as without J1/tenascin. However, adhesion was completely abolished when cells were offered a mixture of fibronectin and J1/tenascin as substratum. The ability of J1/tenascin to reduce the adhesion of intestinal epithelial cells to their fibronectin-containing basal lamina suggests that J1/tenascin may be involved in the process of physiological cell shedding from the villus.     

Biosynthesized matrix provides a key role for survival signaling in bronchial epithelial cells

The extracellular matrix (ECM) influences a variety of cellular functions, including survival, adhesion molecule expression, differentiation, and migration. The ECM composition of the epithelial basement membrane is altered in asthmatics. In this study, we elucidate the major survival signals received by bronchial epithelial cells in vitro by studying the effects of a variety of ECM factors and soluble growth factors on bronchial epithelial cell survival. Our findings indicate that the insulin family of soluble growth factors provides important survival signals but also that adhesion to ECM is a crucial determinant of bronchial epithelial cell survival. In the BEAS-2B bronchial epithelial cell line, collagens I and IV, laminin, fibronectin, and vitronectin provide significant levels of protection from apoptosis. Tenascin-C has no effect, whereas elastin and collagen V increase apoptosis to above control levels. BEAS-2B cells secrete their own biosynthesized matrix (BSM), which also provides rescue from apoptosis. Protection by collagen I, fibronectin, and vitronectin was found to be via an RGD domain. Laminin-, collagen IV-, and BSM-mediated survival is not RGD dependent. Primary bronchial epithelial cells exhibit a similar pattern of apoptosis rescue to the BEAS-2B cell line, although we did not observe any vitronectin-mediated protection in the primary cells. These data indicate that bronchial epithelial cell survival is dependent both on soluble growth factors and on a variety of ECM-derived signals.     

Surface contact modulation of inflammatory macrophage antibody dependent cytotoxicity and prostanoid release.

Adherence to extracellular matrix proteins modulates the functional and secretory activities of mononuclear phagocytes, although the mechanisms regulating these adherence-dependent changes are poorly understood. In this study, the ability of rat inflammatory peritoneal macrophages (PM) to adhere to an endothelial cell-derived extracellular matrix or a denatured collagen/fibronectin-coated surface and perform antibody dependent cell cytotoxicity (ADCC) and secrete reactive oxygen intermediates was compared with PM adherent to tissue culture plastic. Prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), two major cyclooxygenase products released by inflammatory macrophages, were also measured by PM adherent to the protein coated surfaces. Rat exudate PM were equally adherent to tissue culture plastic or wells coated with either endothelial cell derived matrix or denatured collagen (gelatin)/fibronectin. PM adherent to a denatured collagen/fibronectin-coated wells demonstrated significantly less cytolytic activity (15 +/- 2% lysis) when compared with either tissue culture plastic adherent PM (43 +/- 7% lysis) or PM adherent to extracellular matrix (59 +/- 11% lysis). PM adherent to extracellular matrix released twofold more TxB2 than plastic adherent PM, while PM adherent to denatured collagen/fibronectin released 40% more PGE2 than cells adherent to tissue culture plastic or 80% more PGE2 than PM adherent to the extracellular matrix. PM adherent to denatured collagen/fibronectin release less superoxide anion (27 +/- .9 nmoles/10(6) PM) than PM adherent to either tissue culture plastic (43 +/- 1 nmoles/10(6) PM) or the extracellular matrix (60 +/- 0.5 nmoles/10(6) PM). Furthermore, incubation of plastic adherent PM with exogenous PGE2 reduced superoxide production in a dose-dependent manner. These results demonstrate that the inhibition of ADCC and secretion of reactive oxygen intermediates by PM adherent to a denatured collagen/fibronectin surface correlated with an increased release of the immunosuppressive prostanoid PGE2. Furthermore, the addition of exogenous PGE2 to plastic adherent PM reproduced the depression in ADCC and superoxide anion production observed by PM adherent to a denatured collagen/fibronectin surface. These studies suggest that the increased production and release of PGE2 by inflammatory macrophages adherent to a denatured collagen surface may act to suppress cytotoxic mechanisms and thereby constitutes part of an autocrine feedback mechanism regulating macrophage function during wound injury.     

Embryonal carcinoma cells adhere preferentially to fibronectin and laminin but their endodermal differentiation leads to a reduced adherence to laminin

F9 and PC13 embryonal carcinoma (EC) cells adhered rapidly to growth substrata coated with fibronectin or laminin. When F9 cells were induced to differentiate into visceral or parietal endoderm-like cells, their ability to adhere to laminin diminished, but their adherence to fibronectin remained unchanged. Correspondingly, permanently differentiated teratocarcinoma-derived endoderm cells (PYS-2 and PSA-5e) adhered markedly less efficiently to laminin than to fibronectin. F9 cells adhered to proteolytic fibronectin fragments containing the cell-binding site but not to fragments containing gelatin- or heparin-binding sites. They also adhered slowly to gelatin, but this adhesion was completely blocked by cycloheximide. The results show that the teratocarcinoma stem cells may have specific mechanisms mediating adhesion to fibronectin and laminin and that endodermal differentiation leads to a reduction in their capacity to adhere to laminin but not to fibronectin.     

Adhesion to extracellular materials by neural crest cells at the stage of initial migration

Summary Trunk-level neural anlagen bearing neural crest cells at the stage of initiation of migration were isolated from chick embryos and explanted in serum-free medium onto glass substrates which had previously been treated with extracellular materials. After 0.5–2 h incubation, the expiants were dislodged with a stream of culture medium and the substrate examined for adherent crest cells. Crest cells adhered to collagen gels, and adhered to and spread on adsorbed fibronectin; antiserum to fibronectin prevented adhesion to fibronectin but not to collagen gels. Air-dried collagen gels and collagen solutions were less adhesive, the adhesivity declining with longer drying time and lower collagen concentration. Crest cells adhered poorly to dried gelatin and not at all to adsorbed collagen. Fibronectin increased the adhesion to dried collagen and gelatin. Pretreatment of collagen gels with hyaluronate retarded adhesion. Hyaluronate pretreatment also retarded adhesion to adsorbed fibronectin but only when adsorbed collagen was also present. Pretreatment of collagen gels with the proteoglycan monomer from bovine nasal cartilage had no effect of the adhesion of crest cells, but the proteoglycan almost completely inhibited adhesion to adsorbed fibronectin, but only when absorbed collagen was also present. The results are discussed in terms of the control of migration of neural crest cells by extracellular materials.     

The identification of extracellular matrix (ECM) binding sites on the basal surface of embryonic corneal epithelium and the effect of ECM binding on epithelial collagen production

Previously, we have shown that embryonic corneal epithelia can interact with, and respond to, soluble extracellular matrices (ECM) (laminin, collagen, and fibronectin). The basal surface of epithelia isolated free of the underlying ECM can be seen to be disrupted by numerous blebs that sprout from this formerly smooth surface. Laminin, collagen, or fibronectin added to the culture medium cause the epithelium to reorganize its cytoskeleton and flatten its basal surface. We show here that ECM molecules at concentrations that reorganize epithelial cytoskeletal morphology also increase the amount of collagen produced by the epithelial cells. However, molecules that do not reorganize basal epithelial morphology (concanavalin A, heparin, bovine serum albumin) have no effect on collagen production. We also report that fluorescently labeled laminin, collagen, and fibronectin, when added to the medium surrounding isolated corneal epithelia, bind to and flatten the basal epithelial cell surface. The binding site on the basal surface is protease sensitive and is specific for each ECM molecule. These results are compatible with the idea that the basal epithelial plasmalemma possesses a diverse population of binding sites for ECM that link cell surface matrix to the cytoskeleton, causing a dramatic cytoskeletal reorganization which in turn results in enhanced production of collagen by the cells.     

Role of fibronectin in the adhesion of Acinetobacter baumannii to host cells

Adhesion to host cells is an initial and important step in Acinetobacter baumannii pathogenesis. However, there is relatively little information on the mechanisms by which A. baumannii binds to and interacts with host cells. Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade epithelial cells. Here, we found that A. baumannii adheres more avidly to immobilized fibronectin than to control protein. Free fibronectin used as a competitor resulted in dose-dependent decreased binding of A. baumannii to fibronectin. Three outer membrane preparations (OMPs) were identified as fibronectin binding proteins (FBPs): OMPA, TonB-dependent copper receptor, and 34 kDa OMP. Moreover, we demonstrated that fibronectin inhibition and neutralization by specific antibody prevented significantly the adhesion of A. baumannii to human lung epithelial cells (A549 cells). Similarly, A. baumannii OMPA neutralization by specific antibody decreased significantly the adhesion of A. baumannii to A549 cells. These data indicate that FBPs are key adhesins that mediate binding of A. baumannii to human lung epithelial cells through interaction with fibronectin on the surface of these host cells.