Fibronectin-binding Capacity Research Articles

Impact of Vancomycin on sarA-Mediated Biofilm Formation: Role in Persistent Endovascular Infections Due to Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is the most common cause of endovascular infections. The staphylococcal accessory regulator A locus (sarA) is a major virulence determinant that may potentially impact methicillin-resistant S. aureus (MRSA) persistence in such infections via its influence on biofilm formation. Two healthcare-associated MRSA isolates from patients with persistent bacteremia and 2 prototypical community-acquired MRSA strains, as well as their respective isogenic sarA mutants, were studied for in vitro biofilm formation, fibronectin-binding capacity, autolysis, and protease and nuclease activities. These assays were done in the presence or absence of sub-minimum inhibitory concentrations (MICs) of vancomycin. In addition, these strain pairs were compared for intrinsic virulence and responses to vancomycin therapy in experimental infective endocarditis, a prototypical biofilm model. All sarA mutants displayed significantly reduced biofilm formation and binding to fibronectin but increased protease production in vitro, compared with their respective parental strains. Interestingly, exposure to sub-MICs of vancomycin significantly promoted biofilm formation and fibronectin-binding in parental strains but not in sarA mutants. In addition, all sarA mutants became exquisitely susceptible to vancomycin therapy, compared with their respective parental strains, in the infective endocarditis model. These observations suggest that sarA activation is important in persistent MRSA endovascular infection, potentially in the setting of biofilm formation.

The effects of the modulation of the fibronectin-binding capacity of fibrin by thrombin on osteoblast differentiation

Fibrin is a natural provisional matrix involved in wound healing and is widely utilized for tissue regeneration. The biological performance of fibrin is largely dependent on its composition and related structures. In this study, we examined the effect of thrombin, which is engaged with fibrin, on osteoblast differentiation and its mode of action. Fibrin matrices were prepared with different concentrations of thrombin, and MC3T3-E1 pre-osteoblasts were cultured on the fibrin matrices. Thrombin-promoted fibrin-enhanced osteoblast differentiation in a dose-dependent manner, as confirmed by the extent of calcium deposition, alkaline phosphatase activity, and the level of Runx2. The synthetic activating peptide of protease-activated receptor 1 (PAR1), a prototype receptor of thrombin in osteoblast, did not alter the level of Runx2. Instead, the thrombin that was engaged with fibrin in a dose-dependent manner increased the phosphorylation of integrins β1 and β3. The integrin-blocking peptide RGDS reduced the thrombin-enhanced Runx2 in the cells grown on fibrin, whereas the non-functional peptide RGES did not change the level of Runx2. Furthermore, thrombin dose-dependently increased the fibronectin-binding of fibrin. The thrombin-induced integrin phosphorylation and Runx2 expression were also attenuated through the use of a blocking peptide to inhibit the binding of fibronectin to fibrin. The results in this study provide evidence that thrombin engaged with fibrin accelerates osteoblast differentiation via integrins but not PAR1 by modulating the fibronectin-binding capacity of fibrin.

Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia

BackgroundAspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia.ResultsWe used three A. fumigatus isolates which grew as white or brown powdery colonies, to demonstrate the role of melanin. Firstly, sequencing the genes responsible for biosynthesis of melanin (ALB1, AYG1, ARP1, ARP2, ABR1 and ABR2) showed point mutations (missense mutation, deletion or insertion) in the ALB1 gene for pigmentless isolates or in ARP2 for the brownish isolate. The isolates were then shown by scanning electron microscopy to produce numerous, typical conidial heads, except that the conidia were smooth-walled, as previously observed for laboratory mutants with mutations in the PKSP/ALB1 gene. Flow cytometry showed an increase in the fibronectin binding capacity of conidia from mutant isolates, together with a marked decrease in the binding of laminin to the conidial surface. A marked decrease in the electronegative charge of the conidia and cell surface hydrophobicity was also seen by microelectrophoresis and two-phase partitioning, respectively. Ultrastructural studies of mutant isolates detected considerable changes in the organisation of the conidial wall, with the loss of the outermost electron dense layer responsible for the ornamentations seen on the conidial surface in wild-type strains. Finally, analysis of the conidial surface of mutant isolates by atomic force microscopy demonstrated the absence of the outer cell wall rodlet layer which is composed of hydrophobins.ConclusionThese results suggest that, in addition to a protective role against the host's immune defences, melanin is also a structural component of the conidial wall that is required for correct assembly of the cell wall layers and the expression at the conidial surface of adhesins and other virulence factors.

The fibronectin-binding capacity and host cell adherence of Streptococcus pyogenes strains are discordant with each other

Surface exposed fibronectin-binding proteins (FBPs) play an important role in the adherence of S treptococcus pyogenes (group A streptococcus, GAS) to host cells. This pathogen expresses numerous FBPs, of which SfbI, SfbII and PrtF2 are major surface exposed FBPs. However, GAS strains differ in the genetic potential to express these proteins. To test whether this difference reflects in differences in fibronectin (Fn) binding, a set of circulating strains previously examined for adherence to host cells was used. The 68 distinct strains were isolated from throat, skin and blood. They were analyzed for (a) the presence of genes for SfbI, SfbII and PrtF2 and (b) the extent of Fn binding. The results suggest that strains possessing two or more of the genes for these FBPs bound Fn significantly more than strains possessing none or one of the genes. No correlation between the extent of Fn binding and the tissue site of isolation was found. Furthermore, together with our previous studies on adherence capacity of these GAS strains, we found no correlation between Fn binding ability and the avidity of the strains to adhere to epithelial cells. We suggest that while Fn binding is important for adhesion, for many GAS strains the extent of Fn binding is not the critical determinant of adherence.

Impacts of sarA and agr in Staphylococcus aureus strain Newman on fibronectin-binding protein A gene expression and fibronectin adherence capacity in vitro and in experimental infective endocarditis.

We investigated the impacts of sarA and agr on fnbA expression and fibronectin-binding capacity in Staphylococcus aureus in vitro and in experimental endocarditis. Although sarA up-regulated and agr down-regulated both fnbA expression and fibronectin binding in vitro and in vivo, fnbA expression was positively regulated in the absence of both global regulators. Thus, additional regulatory loci contribute to fnbA regulation and fibronectin-binding capacities in S. aureus.

Specific Lectins Map the Distribution of Fibronectin and β1-Integrin on Living MDCK cells

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, β1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated β1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and β1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of β1-integrin. Sialylated β1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated β1-integrin. Furthermore, desialylation of β1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated β1-integrin on MDCK cells.

Monoclonal Antibody Specific to the β1 Integrin-Like Molecule (140 kDa) Immunoprecipitates a Protein Complex of Entamoeba histolytica

One of the most important steps during the invasion of Entamoeba histolytica is its interaction with the extracellular matrix (ECM) and endothelial cells followed by the host tissue destruction (1). In higher eukaryotes, cell attachment, cell spreading, and migration on a variety of extracellular glycoproteins including fibronectin, laminin, vitronectin, and collagen are mediated through cell surface receptors. Many of these cell adhesion receptors belong to a large superfamily called integrins or cytoadhesins (2). A previous report has demonstrated that, like eukaryotic cells, E. histolytica trophozoites also contain a 140-kDa membrane protein that possesses fibronectin binding capacity, and shares some functional and biological properties with the b 1 integrin molecules (3). However, the structural and functional aspects of the amebic receptor molecule are yet to be defined. The development of monoclonal antibodies has become a powerful tool for investigating the cell surface components because of their high specificity. In this preliminary communication, we report on one monoclonal antibody specific to a 140-kDa polypeptide antigen that immunoprecipitates 140-kDa protein complex from detergent-soluble extract of E. histolytica.

Endocardiac infectivity and binding to extracellular matrix proteins of oralAbiotrophiaspecies

Microorganisms of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are members of the oral flora and often isolated from patients with endocarditis, but pathogenicity of oral Abiotrophia species has not been examined yet. In this study, 17 strains isolated from healthy human oral cavities and 7 reference strains (all derived from patients with endocarditis) of Abiotrophia spp. were tested for their abilities to cause infections in damaged heart tissues in catheterized rats and to adhere to extracellular matrix proteins in vitro. The reference strains of A. defectiva and A. adiacens showed high infectivities in the rats. Four oral isolates of these two species showed similarly high infectivities and three had moderate infectivities. Most of 10 oral strains of A. para-adiacens and A. elegans were found to be generally less infective. The highly infective A. adiacens strains showed markedly high fibronectin-binding capacity, suggesting a possible relationship between the fibronectin-binding capacity and damaged heart tissue infectivity of the Abiotrophia species. A. defectiva strains which were also highly infective had moderate levels of binding to fibronectin and other extracellular matrix proteins. Most of A. para-adiacens and A. elegans strains showed low or negligible binding capacities to any extracellular matrix proteins tested.

Endocardiac infectivity and binding to extracellular matrix proteins of oral Abiotrophia species

Microorganisms of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are members of the oral flora and often isolated from patients with endocarditis, but pathogenicity of oral Abiotrophia species has not been examined yet. In this study, 17 strains isolated from healthy human oral cavities and 7 reference strains (all derived from patients with endocarditis) of Abiotrophia spp. were tested for their abilities to cause infections in damaged heart tissues in catheterized rats and to adhere to extracellular matrix proteins in vitro. The reference strains of A. defectiva and A. adiacens showed high infectivities in the rats. Four oral isolates of these two species showed similarly high infectivities and three had moderate infectivities. Most of 10 oral strains of A. para-adiacens and A. elegans were found to be generally less infective. The highly infective A. adiacens strains showed markedly high fibronectin-binding capacity, suggesting a possible relationship between the fibronectin-binding capacity and damaged heart tissue infectivity of the Abiotrophia species. A. defectiva strains which were also highly infective had moderate levels of binding to fibronectin and other extracellular matrix proteins. Most of A. para-adiacens and A. elegans strains showed low or negligible binding capacities to any extracellular matrix proteins tested.

Endocarditis Induction and Fibronectin-Binding Capacity of Abiotrophia Strains

Endocarditis Induction and Fibronectin-Binding Capacity of Abiotrophia Strains

Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis.

Microbial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis.

α4β1 and α5β1 Are Differentially Expressed During Myelopoiesis and Mediate the Adherence of Human CD34+ Cells to Fibronectin in an Activation-Dependent Way

AbstractTo study the receptors involved in the interaction between extracellular matrix proteins and hematopoietic progenitor cells, we analyzed the expression of β1 integrins on CD34+ bone marrow cells by means of immunoflowcytometry. α4β1 and α5β1 were expressed, whereas α1β1, α2β1, α3β1, α6β1, and αvβ1 were virtually absent. Furthermore, we assessed the α4 and α5 expression on committed myeloid progenitor cells. These colony-forming cells were detected in the α4 dull fraction and the α5 dull fraction. During myeloid differentiation, both in vivo and in vitro, a differential expression of α4β1 and α5β1 was observed. α5β1 was found to be lost at the myelocytic-metamyelocytic stage, before the loss of α4β1, at the band stage. Functional studies showed no binding of erythroid progenitor-depleted, CD34+ bone marrow cells to fibronectin. However, protein kinase C activation strongly induced fibronectin binding (68% of the cells). Inhibition experiments with specific antibodies and peptides showed the binding to be mediated by both α4β1 and α5β1. Also, colony-forming cells of granulocytes and macrophages were demonstrated to adhere to fibronectin in an activation-dependent way. During granulocyte colony-stimulating factor-induced in vitro maturation, the activation-dependent fibronectin binding capacity is gradually lost. We conclude that: (1) CD34+ bone marrow cells express α4β1 and α5β1; (2) the expression of α4β1 and α5β1 is differentially expressed during myeloid differentiation; and (3) binding of CD34+ bone marrow cells to fibronectin is activation dependent.

Alpha 4 beta 1 and alpha 5 beta 1 are differentially expressed during myelopoiesis and mediate the adherence of human CD34+ cells to fibronectin in an activation-dependent way

To study the receptors involved in the interaction between extracellular matrix proteins and hematopoietic progenitor cells, we analyzed the expression of beta 1 integrins on CD34+ bone marrow cells by means of immunoflowcytometry. Alpha 4 beta 1 and alpha 5 beta 1 were expressed, whereas alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha v beta 1 were virtually absent. Furthermore, we assessed the alpha 4 and alpha 5 expression on committed myeloid progenitor cells. These colony-forming cells were detected in the alpha 4 dull fraction and the alpha 5 dull fraction. During myeloid differentiation, both in vivo and in vitro, a differential expression of alpha 4 beta 1 and alpha 5 beta 1 was observed. alpha 5 beta 1 was found to be lost at the myelocytic-metamyelocytic stage, before the loss of alpha 4 beta 1, at the band stage. Functional studies showed no binding of erythroid progenitor-depleted, CD34+ bone marrow cells to fibronectin. However, protein kinase C activation strongly induced fibronectin binding (68% of the cells). Inhibition experiments with specific antibodies and peptides showed the binding to be mediated by both alpha 4 beta 1 and alpha 5 beta 1. Also, colony-forming cells of granulocytes and macrophages were demonstrated to adhere to fibronectin in an activation-dependent way. During granulocyte colony-stimulating factor-induced in vitro maturation, the activation-dependent fibronectin binding capacity is gradually lost. We conclude that: (1) CD34+ bone marrow cells express alpha 4 beta 1 and alpha 5 beta 1; (2) the expression of alpha 4 beta 1 and alpha 5 beta 1 is differentially expressed during myeloid differentiation; and (3) binding of CD34+ bone marrow cells to fibronectin is activation dependent.

Mycobacterium leprae produces extracellular homologs of the antigen 85 complex.

The antigen 85 complex is a set of at least three closely related secreted proteins (85A, 85B, and 85C) of 30 to 32 kDa produced by Mycobacterium tuberculosis and other mycobacteria. Their prominence in Mycobacterium leprae, the one obligate intracellular pathogen of the genus, had been assumed on the basis of immunological evidence and proof of the existence of the gene encoding the 85B protein of the complex. We have now observed the production of this family of proteins by M. leprae through analysis of various fractions by Western blotting (immunoblotting) with monospecific rabbit antisera raised against the individual Mycobacterium bovis BCG 85A, 85B, and 85C proteins. A predominant cross-reactive band with an apparent molecular mass of 30 kDa was detected in extracts of nondisrupted whole M. leprae and in soluble fractions prepared from the tissues of M. leprae-infected armadillos. Further studies of the subcellular distribution of this protein within the bacterium confirmed that it is secreted by the organism, an observation that explains past difficulties in detecting the antigen 85 complex in M. leprae. Confirmation that the M. leprae product is a member of the antigen 85 complex was obtained by comparison of peptide fingerprints with those from the BCG product. The pattern of reactivity of the M. leprae antigen 85 complex with anti-M. bovis BCG 85B serum, as well as two-dimensional electrophoresis, established that the 85B component was the predominant member of the complex in M. leprae. The fibronectin-binding capacity of the M. leprae and BCG 85 complexes was reinvestigated by new approaches and is questioned. Nevertheless, the results obtained with the native proteins reinforce previous reports, derived primarily from the use of homologous proteins, that the antigen 85 complex is one of the dominant protein immunogens of the leprosy bacillus.

Use of biospecific interactions of collagen, fibronectin and their fragments in affinity chromatography

Various aspects of the application of fibronectin—collagen biospecific interactions in affinity chromatography are described. A new biospecific method for one-stage isolation of collagen peptides containing fibronectin-binding sites is proposed. The α 1CB 7-peptide of type-I collagen cyanogen bromide cleavage was isolated by means of affinity chromatography on adsorbents containing an immobilized gelatin-binding domain (45 000 relative molecular mass) of fibronectin. The method gives highly purified preparations of α 1CB 7-peptide. This peptide, as well as some other collagen molecular fragments (α-chains, β-components, α 1CB 8-peptide), were immobilized on Sepharose, and the properties of such affinity adsorbents obtained were studied. Adsorbents with immobilized α-chains and α 1CB 7-peptide had a fibronectin-binding capacity 1.5–2.0 times higher than commercial gelatin-Sepharose. Large-scale production of highly purified fibronectin from human plasma, using affinity chromatography on immobilized individual α-chains of collagen, was developed.

Interaction of sub-epithelial connective tissue components with Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis

• Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from bovine mastitis were examined for their ability to interact with 125I-labelled fibronectin, fibrinogen and type II collagen. Their relative surface hydrophobicity and production of extracellular capsule were also investigated. Almost all S. aureus strains bound fibronectin (mean value 23%), fibrinogen (mean value 12%) and type II collagen (mean value 16%). CNS bound fibronectin (mean value 6%) and type II collagen (mean value 7%), but not fibrinogen (mean value 2%). • The specificity of binding of these proteins to S. aureus strain F1440 and to coagulase-negative Staphylococcus chromogenes strain BO52 was studied by adding an excess of unlabelled proteins. Fibronectin and collagen binding were observed to be specific, varying between 50 and 75%, whereas the specificity of fibrinogen binding to S. aureus strain F1440 was lower (26%). Most of the S. aureus strains (63%) showed very high surface hydrophobicity (autoaggregation) or lower hydrophobicity (29% of the strains) and the rest were hydrophilic. None of the CNS strains autoaggregated, 44% were classified as hydrophilic strains. Hydrophilic strains (except the reference strains) did not show extracellular capsule production. However, the encapsulated (reference) strains showed low binding to these proteins as compared to their unencapsulated variants. • Pre-treatment of S. aureus strain F1440 and S. chromogenes strain BO52 with trypsin decreased their fibronectin binding capacity and surface hydrophobicity, whereas pre-treatment with bovine milk (except on collagen binding to strain F1440) did not significantly affect binding to these proteins. • These data indicate that S. aureus and CNS isolated from bovine udder infection have the ability to bind to tissue matrix and plasma proteins which may be exposed in the traumatized or toxin-damaged udder epithelial lesions.

In-vivo studies on Haemaccel-fibronectin interaction in man.

An enzyme-linked immunoassay has been recently set up for direct measurement of the binding capacity of plasma fibronectin to gelatin. This binding capacity could be completely inhibited in vitro by an eight-fold excess of gelatin, of Haemaccel, but not of Geloplasma. On the contrary, the levels of immunoreactive fibronectin measured by laser nephelometry did not change, in presence of 10 to 1000 micrograms ml-1 of gelatin, of Haemaccel or of Geloplasma. When infused into normal volunteers, Haemaccel provoked a strong and immediate inhibition of the plasma fibronectin binding capacity to gelatin. This inhibition was dose-dependent and maximal after infusion of 500 ml of Haemaccel. Twenty-four hours after this infusion, there was a progressive recovery of the gelatin-binding capacity, which was almost completely achieved 96 h later. The formation of complexes between Haemaccel and fibronectin was demonstrated by gel filtration chromatography and by affinity chromatography. Immunoreactive plasma fibronectin levels remained unchanged up to 24 h after infusion of 500 ml of Haemaccel. A transient decline to 50% of its initial value then occurred the second day after the infusion. Therefore, a delay existed between the formation of fibronectin-Haemaccel complexes and their elimination from the bloodstream. This delay decreased when smaller volumes of Haemaccel were infused, which strongly suggests that plasma fibronectin is cleared by means of Haemaccel and does not seem to play a role of opsonin in these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibronectin interacts with Clq, a subcomponent of the first component of complement

Human Clq, a subcomponent of the first component of complement interacts with human fibronectin. Using ELISA methodology fixation of Clq to solid phase fibronectin, as well as fibronectin to solid phase Clq has been demonstrated. Cl in its native macromolecular form displays little reactivity for fibronectin, nor does Cl reconstituted from Clq, Clr and Cls in the presence of Ca 2+ ions. Heating of Clq above its thermal transition temperature (51°C) induces an increased binding capacity for fibronectin. On the other hand, a mixture of the dissociated A, B and C chains of Clq is less active than native Clq. The binding of fibronectin appears to be mediated by the A chain. Studies with Clq deprived of its globular parts by peptic digestion indicate that the collagen-like regions of Clq are involved in fibronectin binding. In contrast, collagenase treatment of Clq abrogates its fibronectin binding capacity.