Dataset Information

SufA - a bacterial enzyme that cleaves fibrinogen and blocks fibrin network formation.

ABSTRACT: Finegoldia magna is a member of the normal human bacterial flora on the skin and other non-sterile body surfaces, but this anaerobic coccus is also an important opportunistic pathogen. SufA was the first F. magna proteinase to be isolated and characterized. Many bacterial pathogens interfere with different steps of blood coagulation, and here we describe how purified SufA efficiently and specifically cleaves fibrinogen in human plasma. SufA is both secreted by F. magna and associated with the bacterial surface. Successful gene targeting has previously not been performed in anaerobic cocci, but in order to study the role of the SufA that is present at the bacterial surface, we constructed an F. magna mutant that expresses a truncated SufA lacking proteolytic activity. In contrast to wild-type bacteria that delayed the coagulation of human plasma, mutant bacteria had no such effect. Wild-type and mutant bacteria adhered to keratinocytes equally well, but in a plasma environment only wild-type bacteria blocked the formation of fibrin networks surrounding adherent bacteria. The effective cleavage of fibrinogen by SufA suggests that the interference with fibrin network formation represents an adaptive mechanism of F. magna with potential implications also for pathogenicity.

SUBMITTER: Karlsson C

PROVIDER: S-EPMC2885652 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

SufA - a bacterial enzyme that cleaves fibrinogen and blocks fibrin network formation.

Karlsson Christofer C Mörgelin Matthias M Collin Mattias M Lood Rolf R Andersson Marie-Louise ML Schmidtchen Artur A Björck Lars L Frick Inga-Maria IM

Microbiology (Reading, England) 20090101 Pt 1

Finegoldia magna is a member of the normal human bacterial flora on the skin and other non-sterile body surfaces, but this anaerobic coccus is also an important opportunistic pathogen. SufA was the first F. magna proteinase to be isolated and characterized. Many bacterial pathogens interfere with different steps of blood coagulation, and here we describe how purified SufA efficiently and specifically cleaves fibrinogen in human plasma. SufA is both secreted by F. magna and associated with the ba ...[more]

PMID: 19118364

Similar Datasets

Project description:The fibrin polymers formed in solution during the earliest phase of the fibrinogen-fibrin conversion are shown to be stable soluble molecules at pH7.4 and 0.15m- or 0.3m-NaCl. The various sequential soluble fibrin polymers produced from the fibrinogen-thrombin reaction can be observed by gel chromatography and can be isolated for characterization. The mechanism of fibrin polymerization proposed from the present studies suggests that the initial event is the thrombin activation at only one of the Aalpha-chains in fibrinogen. The resulting highly reactive intermediate is the true fibrin monomer and it rapidly, and irreversibly, self-associates to form the stable fibrin dimer (s(20.w)=12S). Fibrin dimer possesses the N-terminal pattern alanine/glycine/tyrosine (1:1:2) per 340000 molecular weight, and possesses the chain structure [(alpha)Aalpha)(Bbeta)(2)(gamma)(2)](2). The fibrin dimer is a soluble inert molecule, but additional thrombin activation of its remaining intact Aalpha-chains leads to new associations into larger inert soluble fibrin polymers. In this manner progressively larger fibrin oligomers are constructed with thrombin continually in control of the process because of the necessity to repeatedly re-activate the various fibrin polymers in solution. The inert character of the soluble fibrin polymers can be explained by the reciprocal alignment of the associating molecules, which mutually consumes their active surfaces and leaves an intact Aalpha-chain at either end of each fibrin oligomer. The soluble fibrin polymers will proceed to further association only if thrombin activates these remaining Aalpha-chains, otherwise the fibrin molecules are stable indefinitely. The intermolecular associations within the soluble fibrin polymers are essentially irreversible under these nearly physiological conditions. However, the bonding is not covalent. This mechanism accounts for the clinical observations of stable fibrinogen-derived polymers in the plasma from patients undergoing thrombotic processes. Since it is shown that the intermediate fibrin polymers, themselves, are stable soluble molecules, it is no longer necessary, nor warranted, to invoke hypothetical ;fibrinogen-fibrin complexes' to explain observations of fibrin solubility.

Project description:Hydrogels are effective platforms for use as artificial extracellular matrices, cell carriers, and to present bioactive cues. Two common natural polymers, fibrin and alginate, are broadly used to form hydrogels and have numerous advantages over synthetic materials. Fibrin is a provisional matrix containing native adhesion motifs for cell engagement, yet the interplay between mechanical properties, degradation, and gelation rate is difficult to decouple. Conversely, alginate is highly tunable yet bioinert and requires modification to present necessary adhesion ligands. To address these challenges, we developed a fibrin-alginate interpenetrating network (IPN) hydrogel to combine the desirable adhesion and stimulatory characteristics of fibrin with the tunable mechanical properties of alginate. We tested its efficacy by examining capillary network formation with entrapped co-cultures of mesenchymal stromal cells (MSCs) and endothelial cells (ECs). We manipulated thrombin concentration and alginate crosslinking density independently to modulate the fibrin structure, mesh size, degradation, and biomechanical properties of these constructs. In IPNs of lower stiffness, we observed a significant increase in total cell area (1.7 × 105 ± 7.9 × 104 µm2) and decrease in circularity (0.56 ± 0.03) compared to cells encapsulated in stiffer IPNs (4.0 × 104 ± 1.5 × 104 µm2 and 0.77 ± 0.09, respectively). Fibrinogen content did not influence capillary network formation. However, higher fibrinogen content led to greater retention of these networks confirmed via increased spreading and presence of F-actin at 7 days. This is an elegant platform to decouple cell adhesion and hydrogel bulk stiffness that will be broadly useful for cell instruction and delivery. STATEMENT OF SIGNIFICANCE: Hydrogels are widely used as drug and cell delivery vehicles and as artificial extracellular matrices to study cellular responses. However, there are limited opportunities to simultaneously control mechanical properties and degradation while mimicking the complex native adhesion motifs and ligands known to encourage cell engagement with the hydrogel. In this study, we describe a fibrin-alginate interpenetrating network (IPN) hydrogel designed to balance the compliance and provisional qualities of fibrin with the mechanical stability and tunability of alginate to interrogate these contributions on cell response. We used clinically relevant cell sources, a co-culture of endothelial cells and mesenchymal stromal cells, to test its efficacy in supporting capillary formation in vitro. These data demonstrate the promise of this IPN for use in tissue engineering.

Project description:A modular tissue engineering approach may have advantages over current therapies in providing rapid and sustained revascularization of ischemic tissue. In this study, modular protein microbeads were prepared from pure fibrin (FIB) and collagen-fibrin composites (COL-FIB) using a simple water-in-oil emulsification technique. Human endothelial cells and fibroblasts were embedded directly in the microbead matrix. The resulting microbeads were generally spheroidal with a diameter of 100-200?m. Cell viability was high (75-80% viable) in microbeads, but was marginally lower than in bulk hydrogels of corresponding composition (85-90% viable). Cell proliferation was significantly greater in COL-FIB microbeads after two weeks in culture, compared to pure FIB microbeads. Upon embedding of microbeads in a surrounding fibrin hydrogel, endothelial cell networks formed inside the microbead matrix and extended into the surrounding matrix. The number of vessel segments, average segment length, and number of branch points was higher in FIB samples, compared to COL-FIB samples, resulting in significantly longer total vessel networks. Anastomosis of vessel networks from adjacent microbeads was also observed. These studies demonstrate that primitive vessel networks can be formed by modular protein microbeads containing embedded endothelial cells and fibroblasts. Such microbeads may find utility as prevascularized tissue modules that can be delivered minimally invasively as a therapy to restore blood flow to ischemic tissues.Vascularization is critically important for tissue engineering and regenerative medicine, and materials that support and/or promote neovascularization are of value both for translational applications and for mechanistic studies and discovery-based research. Therefore, we fabricated small modular microbeads formulated from pure fibrin (FIB) and collagen-fibrin (COL-FIB) containing endothelial cells and supportive fibroblasts. We explored how cells encapsulated within these materials form microvessel-like networks both within and outside of the microbeads when embedded in larger 3D matrices. FIB microbeads were found to initiate more extensive sprouting into the surrounding ECM in vitro. These results represent an important step towards our goal of developing injectable biomaterial modules containing preformed vascular units that can rapidly restore vascularization to an ischemic tissue in vivo.

Project description:Fibrin is a major component of the provisional extracellular matrix formed during tissue repair following injury, and enables cell infiltration and anchoring at the wound site. Macrophages are dynamic regulators of this process, advancing and resolving inflammation in response to cues in their microenvironment. Although much is known about how soluble factors such as cytokines and chemokines regulate macrophage polarization, less is understood about how insoluble and adhesive cues, specifically the blood coagulation matrix fibrin, influence macrophage behavior. In this study, we observed that fibrin and its precursor fibrinogen elicit distinct macrophage functions. Culturing macrophages on fibrin gels fabricated by combining fibrinogen with thrombin stimulated secretion of the anti-inflammatory cytokine, interleukin-10 (IL-10). In contrast, exposure of macrophages to soluble fibrinogen stimulated high levels of inflammatory cytokine tumor necrosis factor alpha (TNF-α). Macrophages maintained their anti-inflammatory behavior when cultured on fibrin gels in the presence of soluble fibrinogen. In addition, adhesion to fibrin matrices inhibited TNF-α production in response to stimulation with LPS and IFN-γ, cytokines known to promote inflammatory macrophage polarization. Our data demonstrate that fibrin exerts a protective effect on macrophages, preventing inflammatory activation by stimuli including fibrinogen, LPS, and IFN-γ. Together, our study suggests that the presentation of fibrin(ogen) may be a key switch in regulating macrophage phenotype behavior, and this feature may provide a valuable immunomodulatory strategy for tissue healing and regeneration.Statement of significanceFibrin is a fibrous protein resulting from blood clotting and provides a provisional matrix into which cells migrate and to which they adhere during wound healing. Macrophages play an important role in this process, and are needed for both advancing and resolving inflammation. We demonstrate that culture of macrophages on fibrin matrices exerts an anti-inflammatory effect, whereas the soluble precursor fibrinogen stimulates inflammatory activation. Moreover, culture on fibrin completely abrogates inflammatory signaling caused by fibrinogen or known inflammatory stimuli including LPS and IFN-γ. Together, these studies show that the presentation of fibrin(ogen) is important for regulating a switch between macrophage pro- and anti-inflammatory behavior.

Dataset Information

SufA - a bacterial enzyme that cleaves fibrinogen and blocks fibrin network formation.

Publications

SufA - a bacterial enzyme that cleaves fibrinogen and blocks fibrin network formation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets