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Binding of Efb from Staphylococcus aureus to fibrinogen blocks neutrophil adherence.

ABSTRACT: In addition to its pivotal role in hemostasis, fibrinogen (Fg) and provisional fibrin matrices play important roles in inflammation and regulate innate immune responses by interacting with leukocytes. Efb (the extracellular fibrinogen-binding protein) is a secreted Staphylococcus aureus protein that engages host Fg and complement C3. However, the molecular details underlying the Efb-Fg interaction and the biological relevance of this interaction have not been determined. In the present study, we characterize the interaction of Efb with Fg. We demonstrate that the Fg binding activity is located within the intrinsically disordered N-terminal half of Efb (Efb-N) and that the D fragment of Fg is the region that mediates Efb-N binding. More detailed studies of the Efb-N-Fg interactions using ELISA and surface plasmon resonance analyses revealed that Efb-N exhibits a much higher affinity for Fg than typically observed with Fg-binding MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), and data obtained from ELISA analyses using truncated Efb-N constructs demonstrate that Efb-N contains two binding sites located within residues 30-67 and 68-98, respectively. Efb-N inhibits neutrophil adhesion to immobilized Fg by binding to Fg and blocking the interaction of the protein with the leukocyte integrin receptor, ?(M)?(2). A motif in the Fg ? chain previously shown to be central to the ?(M)?(2) interaction was shown to be functionally distinguishable from the Efb-N binding site, suggesting that the Fg-Efb interaction indirectly impedes Fg engagement by ?(M)?(2). Taken together, these studies provide insights into how Efb interacts with Fg and suggest that Efb may support bacterial virulence at least in part by impeding Fg-driven leukocyte adhesion events.

SUBMITTER: Ko YP

PROVIDER: S-EPMC3059020 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Binding of Efb from Staphylococcus aureus to fibrinogen blocks neutrophil adherence.

Ko Ya-Ping YP Liang Xiaowen X Smith C Wayne CW Degen Jay L JL Höök Magnus M

The Journal of biological chemistry 20110119 11

In addition to its pivotal role in hemostasis, fibrinogen (Fg) and provisional fibrin matrices play important roles in inflammation and regulate innate immune responses by interacting with leukocytes. Efb (the extracellular fibrinogen-binding protein) is a secreted Staphylococcus aureus protein that engages host Fg and complement C3. However, the molecular details underlying the Efb-Fg interaction and the biological relevance of this interaction have not been determined. In the present study, we ...[more]

PMID: 21247890

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Project description:UnlabelledCoagulase (Coa) and Efb, secreted Staphylococcus aureus proteins, are important virulence factors in staphylococcal infections. Coa interacts with fibrinogen (Fg) and induces the formation of fibrin(ogen) clots through activation of prothrombin. Efb attracts Fg to the bacterial surface and forms a shield to protect the bacteria from phagocytic clearance. This communication describes the use of an array of synthetic peptides to identify variants of a linear Fg binding motif present in Coa and Efb which are responsible for the Fg binding activities of these proteins. This motif represents the first Fg binding motif identified for any microbial protein. We initially located the Fg binding sites to Coa's C-terminal disordered segment containing tandem repeats by using recombinant fragments of Coa in enzyme-linked immunosorbent assay-type binding experiments. Sequence analyses revealed that this Coa region contained shorter segments with sequences similar to the Fg binding segments in Efb. An alanine scanning approach allowed us to identify the residues in Coa and Efb that are critical for Fg binding and to define the Fg binding motifs in the two proteins. In these motifs, the residues required for Fg binding are largely conserved, and they therefore constitute variants of a common Fg binding motif which binds to Fg with high affinity. Defining a specific motif also allowed us to identify a functional Fg binding register for the Coa repeats that is different from the repeat unit previously proposed.ImportanceStaphylococcus aureus infections are a major health problem that affects an estimated 50 million people globally and causes the death of about 20,000 Americans each year. A number of experimental vaccines have been developed during the past years. However, these vaccines have all failed in clinical trials. The ability of S. aureus to form an Fg shield surrounding and protecting bacterial cells from clearance may explain why the vaccines are failing. Furthermore, S. aureus coagulase can induce the formation of a fibrin(ogen) shield in experimental abscess models which surrounds and protects bacteria in the microcolony from clearance. In this study, we identified for the first time a microbial Fg binding motif. Variants of this motif are present in coagulase and Efb. Our results provide a molecular basis for the rational design of inhibitors that could potentially prevent the formation of the obstructing Fg shield.

Project description:Staphylococcus aureus can target a variety of tissues, causing life-threatening infections. The basis for this diversity stems from the microorganism's ability to spread in the vascular system throughout the body. To survive in blood, S. aureus coats itself with a fibrinogen (Fg)/fibrin shield. The protective shield is assembled by the coordinated actions of a number of Fg-binding bacterial proteins that manipulate the host's blood coagulation system. Several of the Fg binders appear redundant, sharing similar functional motifs. This observation led us to screen for the presence of novel proteins with significant amino acid identities to von Willebrand factor-binding protein (vWbp), a key component in the shield assembly machinery. One identified protein showed significant sequence identity with the C-terminal region of vWbp, and we consequently named it vWbp homologous protein (vhp). The vhp gene lies within a cluster of genes that encode other virulence factors in S. aureus. Although each isolate only contains one copy of the vhp gene, S. aureus has at least three distinct alleles, vhpA, B, and C, that are present in the core genome. All three vhp isoforms bind Fg with high affinity, targeting a site located in the D fragment of Fg. We further identified an ∼79 amino acid-long, conserved segment within the C-terminal region of vWbp that shares high sequence identities (54 to 67%) with the vhps and binds soluble Fg with high affinity. Further analysis of this conserved motif and the intact vhps revealed intriguing differences in the Fg binding behavior, perhaps suggesting that these proteins have similar but discrete functions in the shield assembly. IMPORTANCE The life-threatening diseases caused by multidrug-resistant Staphylococcus aureus strains are a worldwide medical problem due to treatment limitations and the lack of an effective vaccine. The ability of S. aureus to coat itself with a protective fibrinogen (Fg)/fibrin shield allows the organism to survive in blood and to disseminate and cause invasive diseases. This process represents a promising target for novel antistaphylococcal treatment strategies but is incompletely understood. S. aureus expresses a number of Fg-binding proteins. Some of these proteins have apparently redundant functions. Proteins with similar functions often share a structural or functional motif with each other. In this study, we identified a protein homologous to the C-terminal of von Willebrand factor-binding protein (vWbp), a key contributor in the Fg shield assembly that also binds Fg. Further analysis allowed us to identify a common Fg-binding motif.

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Binding of Efb from Staphylococcus aureus to fibrinogen blocks neutrophil adherence.

Publications

Binding of Efb from Staphylococcus aureus to fibrinogen blocks neutrophil adherence.

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