Extracellular Fibrinogen-binding Protein Research Articles

Evolution of Invasive and Colonizing Pediatric Staphylococcus aureus Isolates

Abstract Background Staphylococcus aureus is the most common bacterial pathogen isolated in children with invasive bloodstream and musculoskeletal infections. A major barrier to vaccine and novel therapeutic development against S. aureus is its continuous evolution. Many recent and current vaccine candidates target antigens that may have appeared crucial to its pathogenesis, only to see these factors recede from circulating invasive strains. Characterization of clinically relevant S. aureus isolates over time is necessary to find targets for novel therapeutics. Efforts toward new interventions should target factors that remain prevalent in invasive isolates over time. Methods We obtained 131 invasive S. aureus isolates from children admitted to the Monroe Carell Jr. Children's Hospital at Vanderbilt from 2010 to 2022, and 246 colonizing isolates. Whole genome sequences were obtained using the Illumina sequencing platform. Virulence factors and multi-locus sequence type (MLST) were determined using Geneious software and through PubMLST, an open-access curated database. Results There were 10 unique clonal complexes (CC) identified among 320 isolates. CC8 remains the most common invasive clonal complex, though CC8 is significantly less frequent now than in 2010-2012. Diversity of invasive strains has increased substantially, with the emergence of CC5 and CC121 lineages causing invasive disease in children. CC1 was found exclusively in colonization isolates. Virulence factor prevalence has changed significantly over time. Panton-Valentine leucocidin (PVL), enterotoxins K and Q (SEK/SEQ), and the pathogenic arginine catabolic mobile element (ACME), have decreased significantly in invasive S. aureus isolates since 2010, from 53-70% to 21-35% of current isolates. Leukocidin ED (LukED) and surface protein staphylokinase (Sak) are strongly associated with invasive isolates compared to colonization isolates. Conversely, toxic shock toxin (TST) and Staphylococcal enterotoxin B (SEB) were seen more commonly in colonization isolates (12-15%) compared to invasive isolates (5-7%). The genes encoding for gamma hemolysins (HlgA-C), leukocidin AB (LukAB), iron-regulated surface proteins (IsdA, IsdB), staphylococcal binding immunoglobulin protein (sbi), extracellular fibrinogen-binding protein (Efb), and clumping factors A and B (ClfA, ClfB) were identified in all isolates tested. Figure 1 Figure 2 Conclusions Significant shifts have occurred over the past decade in the predominant circulating S. aureus strains and their virulence factor repertoires. The once-dominant CC8 (USA300 clone) has receded, and with that has come a significant reduction in some factors once thought crucial for pathogenesis. These findings also have implications for infection prevention and control practices, as eradication of colonizing strains with low potential for pathogenicity may inadvertently allow for replacement by more virulent strains.

Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats.

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.

Extracellular fibrinogen-binding protein released by intracellular Staphylococcus aureus suppresses host immunity by targeting TRAF3

Many pathogens secrete effectors to hijack intracellular signaling regulators in host immune cells to promote pathogenesis. However, the pathogenesis of Staphylococcus aureus secretory effectors within host cells is unclear. Here, we report that Staphylococcus aureus secretes extracellular fibrinogen-binding protein (Efb) into the cytoplasm of macrophages to suppress host immunity. Mechanistically, RING finger protein 114, a host E3 ligase, mediates K27-linked ubiquitination of Efb at lysine 71, which facilitates the recruitment of tumor necrosis factor receptor associated factor (TRAF) 3. The binding of Efb to TRAF3 disrupts the formation of the TRAF3/TRAF2/cIAP1 (cellular-inhibitor-of-apoptosis-1) complex, which mediates K48-ubiquitination of TRAF3 to promote degradation, resulting in suppression of the inflammatory signaling cascade. Additionally, the Efb K71R mutant loses the ability to inhibit inflammation and exhibits decreased pathogenicity. Therefore, our findings identify an unrecognized mechanism of Staphylococcus aureus to suppress host defense, which may be a promising target for developing effective anti-Staphylococcus aureus immunomodulators.

Extracellular fibrinogen-binding protein (Efb), a key immune evasion protein of Staphylococcus aureus and a potential therapeutic target

Due to the continued growth of antibiotic-resistant Staphylococcus aureus strains, it is necessary to explore alternative targets for future therapeutic applications. For this reason, is important to understand the staphylococcal immune evasion mechanisms with a special focus on extracellular fibrinogen-binding protein (Efb) and Efb related proteins. Therefore, it was conducted a literature review to compile relevant information on this protein. It was found that Efb has three binding sites with biological relevance that could be used as therapeutic targets with specificity for fibrinogen, platelets, and complements. First, the fibrinogen-binding motifs also found in coagulase block neutrophil αMβ2 adherence to fibrinogen and attract fibrinogen to the bacterial surface, forming capsule-like structures that block phagocytosis. Second, Efb is a potent anti-thrombotic agent, probably related to its P-selectin binding capacity. Efb P-selectin binding blocks the interaction of P-selectin with the PSGL-1 receptor, thereby impairs the mechanisms of platelet-mediated leukocyte recruitment to the site of vascular injury. Third, the Efb complement binding domain, also found in other staphylococcal complement inhibitory proteins like Ecb, Sbi, and SCIN, is responsible for the evasion of the complement-mediated immune response. Efb reduces the formation of C3 convertase and the interaction with neutrophils, affect B-cells activation, and maturation. Efb binding sites have a clear implication on the virulence of Staphylococcus aureus in mastitis, wound infection, staphylococcal pneumonia, and infections related to implanted devices, and contributes to staphylococcal persistence in host tissues and abscess formation in the kidneys. Given the biological relevance of Efb binding sites in staphylococcal infections, they are promising vaccine targets. Additionally, due to the inhibitory effect of Efb on platelets and complements, Efb can be a potential therapeutic agent to treat diseases associated with thrombosis and abnormal complement activity.

Inhibition of Complement Retards Ankylosing Spondylitis Progression.

Ankylosing spondylitis (AS) is a chronic axial spondyloarthritis (SpA) resulting in back pain and progressive spinal ankyloses. Currently, there are no effective therapeutics targeting AS largely due to elusive pathogenesis mechanisms, even as potential candidates such as HLA-B27 autoantigen have been identified. Herein, we employed a proteoglycan (PG)-induced AS mouse model together with clinical specimens, and found that the complement system was substantially activated in the spinal bone marrow, accompanied by a remarkable proportion alteration of neutrophils and macrophage in bone marrow and spleen, and by the significant increase of TGF-β1 in serum. The combined treatment with a bacteria-derived complement inhibitor Efb-C (C-terminal of extracellular fibrinogen-binding protein of Staphylococcus aureus) remarkably retarded the progression of mouse AS by reducing osteoblast differentiation. Furthermore, we demonstrated that two important modulators involved in AS disease, TGF-β1 and RANKL, were elevated upon in vitro complement attack in osteoblast and/or osteoclast cells. These findings further unravel that complement activation is closely related with the pathogenesis of AS, and suggest that complement inhibition may hold great potential for AS therapy.

The Staphylococcus aureus polysaccharide capsule and Efb-dependent fibrinogen shield act in concert to protect against phagocytosis.

Staphylococcus aureus has developed many mechanisms to escape from human immune responses. To resist phagocytic clearance, S. aureus expresses a polysaccharide capsule, which effectively masks the bacterial surface and surface-associated proteins, such as opsonins, from recognition by phagocytic cells. Additionally, secretion of the extracellular fibrinogen binding protein (Efb) potently blocks phagocytic uptake of the pathogen. Efb creates a fibrinogen shield surrounding the bacteria by simultaneously binding complement C3b and fibrinogen at the bacterial surface. By means of neutrophil phagocytosis assays with fluorescently labelled encapsulated serotype 5 (CP5) and serotype 8 (CP8) strains we compare the immune-modulating function of these shielding mechanisms. The data indicate that, in highly encapsulated S. aureus strains, the polysaccharide capsule is able to prevent phagocytic uptake at plasma concentrations <10 %, but loses its protective ability at higher concentrations of plasma. Interestingly, Efb shows a strong inhibitory effect on both capsule-negative and encapsulated strains at all tested plasma concentrations. Furthermore, the results suggest that both shielding mechanisms can exist simultaneously and collaborate to provide optimal protection against phagocytosis at a broad range of plasma concentrations. As opsonizing antibodies will be shielded from recognition by either mechanism, incorporating both capsular polysaccharides and Efb in future vaccines could be of great importance.

Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.

Attenuation of Staphylococcus aureus-Induced Bacteremia by Human Mini-Antibodies Targeting the Complement Inhibitory Protein Efb.

Staphylococcus aureus can cause a broad range of potentially fatal inflammatory complications (e.g., sepsis and endocarditis). Its emerging antibiotic resistance and formidable immune evasion arsenal have emphasized the need for more effective antimicrobial approaches. Complement is an innate immune sensor that rapidly responds to bacterial infection eliciting C3-mediated opsonophagocytic and immunomodulatory responses. Extracellular fibrinogen-binding protein (Efb) is a key immune evasion protein of S. aureus that intercepts complement at the level of C3. To date, Efb has not been explored as a target for mAb-based antimicrobial therapeutics. In this study, we have isolated donor-derived anti-Efb IgGs that attenuate S. aureus survival through enhanced neutrophil killing. A phage library screen yielded mini-Abs that selectively inhibit the interaction of Efb with C3 partly by disrupting contacts essential for complex formation. Surface plasmon resonance-based kinetic analysis enabled the selection of mini-Abs with favorable Efb-binding profiles as therapeutic leads. Mini-Ab-mediated blockade of Efb attenuated S. aureus survival in a whole blood model of bacteremia. This neutralizing effect was associated with enhanced neutrophil-mediated killing of S. aureus, increased C5a release, and modulation of IL-6 secretion. Finally, these mini-Abs afforded protection from S. aureus-induced bacteremia in a murine renal abscess model, attenuating bacterial inflammation in kidneys. Overall, these findings are anticipated to pave the way toward novel Ab-based therapeutics for S. aureus-related diseases.

Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins.

Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF’ on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF’, shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0243-7) contains supplementary material, which is available to authorized users.

Method for generation of peptide‐specific igy antibodies directed to Staphylococcus aureus extracellular fibrinogen binding protein epitope

The IgY antibodies offer an attractive alternative to mammalian IgGs in research, diagnosis and medicine. The isolation of immunoglobulin Y from the egg yolks is efficient and economical, causing minimal suffering to animals. Here we present the methodology for the production of IgY antibodies specific to Staphylococcus aureus fibrinogen binding protein (Efb) and its peptidyl epitope (spanning residues 127-140). The Efb is an extracellular, adhesion protein which binds both human fibrinogen and complement C3 protein thus contributing to the high infectious potential of this pathogen. The selected epitope of Efb protein is responsible for the interaction with C3. The immunochemical characterization of both anti-Efb and epitope-specific IgY antibodies revealed their similar avidity, titer, and reactivity profile, although some differences in the hen's immune response to administered antigens is discussed.

Identification of a novel T. cruzi B cell superantigen (INC4P.340)

Abstract Catalytic antibodies represent an innate defense mechanism that allows a subset of antibodies to bind and then hydrolyze their targets, making these antibodies up 1000-fold more efficient than conventional immunoglobulins. Pathogens have evolved B-cell superantigens (BC-SAgs) as an immunevasion strategy designed to neutralize catalytic antibody activity. Exposure to BC-SAgs such as the Staphylococcus aureus extracellular fibrinogen binding protein (Efb) and the HIV gp120 glycoprotein decreased catalytic activity to these antigens, respectively. We describe for the first time the identification and characterization of a Trypanosoma cruzi BC-SAg (Tc24). Immunization with Tc24 in the presence of different adjuvants significantly decreased the ability of purified IgM to hydrolyze Tc24 compared hydrolysis rates observed for IgM purified from unimmunized controls. Infection with T. cruzi resulted in a similar decrease in catalytic activity. In both immunization and infection models, the dose (of either Tc24 or the number of T. cruzi used to infect mice) affected Tc24 hydrolysis rates. Analysis of IgMs purified from infected humans also demonstrated reduced hydrolysis of Tc24 compared to hydrolysis rates observed for IgM purified from uninfected individuals. These data suggest that Tc24 is a BC-SAg that may impact the host's defenses early in the infection process.

Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.

A Novel Peptide Can Mimic Extracellular Fibrinogen-Binding Protein to Block the Activation of Complement System

Extracellular fibrinogen-binding protein (Efb) of Staphylococcus aureus (S. aureus) is a bi-functional protein, which can specifically bind fibrinogen with its N terminus and inhibit deposition of C3b on the surface of S. aureus with its C terminus. Here, we screened the epitopes of Efb using phage display. Four peptides with consensus motif were screened. This consensus motif was identical to C terminus (161-164) of Efb. In the further investigation, it was found the synthesized peptide EC1 (154-165aa of Efb) could specifically bind C3/C3b and subsequently to block the activation of complement. Meanwhile, EC1 could inhibit the interaction between Efb and C3/C3b. Moreover, the interaction between the mutant protein of EmC1 (Efb without EC1) and C3 was decreased. And, the effect on the complement system of the mutant protein was dramatically declined compared with Efb. Our finding suggested that the peptide EC1 could mimic Efb to block complement system activation via binding C3.

Staphylococcus aureus Efb protein expression in Nicotiana tabacum and immune response to oral administration

Staphylococcus aureus (S. aureus) is one of the most widespread agent of diseases in humans and animals. In dairy cows, S. aureus is the most frequently isolated contagious pathogens in mastitis cases and vaccines are one of the potential tools to control the infections, thus decreasing the use of antibiotics. Among all the virulence factors produced by S. aureus, extra cellular fibrinogen binding protein (Efb) is an important one in the pathogenesis of mastitis. Plants are useful bioreactors to produce antigens and the aim of the study was the production of Efb in two cultivars of Nicotiana tabacum as a mean to produce vaccine against S. aureus in plants. A matrix attachment region (MAR) sequence was inserted near the two borders of transfer-DNA in the transformation vector in the two possible orientations. The presence of MAR elements in the transformation system significantly improved transformation efficiency and Efb protein yield up to a 2% level on total soluble protein (TSP). Mice orally immunized with transgenic lyophilized leaves produced an antigen-specific immune response.

Staphylococcus aureus Proteins Sbi and Efb Recruit Human Plasmin to Degrade Complement C3 and C3b

Upon host infection, the human pathogenic microbe Staphylococcus aureus (S. aureus) immediately faces innate immune reactions such as the activated complement system. Here, a novel innate immune evasion strategy of S. aureus is described. The staphylococcal proteins surface immunoglobulin-binding protein (Sbi) and extracellular fibrinogen-binding protein (Efb) bind C3/C3b simultaneously with plasminogen. Bound plasminogen is converted by bacterial activator staphylokinase or by host-specific urokinase-type plasminogen activator to plasmin, which in turn leads to degradation of complement C3 and C3b. Efb and to a lesser extend Sbi enhance plasmin cleavage of C3/C3b, an effect which is explained by a conformational change in C3/C3b induced by Sbi and Efb. Furthermore, bound plasmin also degrades C3a, which exerts anaphylatoxic and antimicrobial activities. Thus, S. aureus Sbi and Efb comprise platforms to recruit plasmin(ogen) together with C3 and its activation product C3b for efficient degradation of these complement components in the local microbial environment and to protect S. aureus from host innate immune reactions.

Protective immunization against Staphylococcus aureus infection in a novel experimental wound model in mice

A novel murine experimental wound infection model was used to assess the efficacy of multi-component immunization against Staphylococcus aureus infection. Necrotic lesions were induced in mice with venom from Bothrops asper and infected with a low inoculum, 1 × 10(2) CFU. The wound infection model therefore more resembles a clinical case of S. aureus infection compared with conventional infection models where far more bacteria are required. Before infection, mice were immunized with four recombinant S.aureus proteins expressed from Escherichia coli: (i) domains 1-3 of Extracellular adherence protein (Eap), (ii) Efb - D (fusion protein combining Extracellular fibrinogen binding protein (Efb) and a fibronectin binding domain (D) of the fibronectin binding protein (FnBP) and (iii) clumping factor A (ClfA). In the immunized group, lower bacterial colonization, undisturbed crust formation and significantly faster wound healing were found compared with the unimmunized control group. Efb and Eap have previously been found to impair wound healing and neutralization of these proteins by antibodies restores a more natural wound healing process. This effect is further also enhanced by the proposed opsonic activity of antibodies against ClfA and FnBP.

Constitutive Production of Catalytic Antibodies to a Staphylococcus aureus Virulence Factor and Effect of Infection

Antibodies that recognize microbial B lymphocyte superantigenic epitopes are produced constitutively with no requirement for adaptive immune maturation. We report cleavage of the Staphylococcus aureus virulence factor extracellular fibrinogen-binding protein (Efb) by catalytic antibodies produced with no exposure to the bacterium and reduction of the catalytic antibody activity following infection. IgG catalytic antibodies that specifically hydrolyzed Efb via a nucleophilic catalytic mechanism were found in the blood of healthy humans and aseptic mice free of S. aureus infection. IgG hydrolyzed peptide bonds on the C-terminal side of basic amino acids, including a bond located within the C3b-binding domain of Efb. Efb digested with the IgG lost its ability to bind C3b and inhibit complement-dependent antibody-mediated red blood cell lysis. In addition to catalysis, the IgG expressed saturable Efb binding activity. IgG from S. aureus-infected mice displayed reduced Efb cleaving activity and increased Efb binding activity compared with uninfected controls, suggesting differing effects of the infection on the antibody subsets responsible for the two activities. IgG from children hospitalized for S. aureus infection also displayed reduced Efb cleavage compared with healthy children. These data suggest a potential defense function for constitutively produced catalytic antibodies to a putative superantigenic site of Efb, but an adaptive catalytic response appears to be proscribed.

Binding of Efb from Staphylococcus aureus to Fibrinogen Blocks Neutrophil Adherence

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, α(M)β(2). A motif in the Fg γ chain previously shown to be central to the α(M)β(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 α(M)β(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.

Inhibiting platelets aggregation could aggravate the acute infection caused by Staphylococcus aureus

Several fibrinogen binding proteins (Fibs) play important roles in the pathogenesis of Staphylococcus aureus (S. aureus). Most Fibs can promote the aggregation of platelets during infection, but the extracellular fibrinogen-binding protein (Efb) is an exception. It is reported that Efb can specifically bind fibrinogen and inhibit the aggregation of platelet with its N terminal. However, the biological significance of platelet aggregation inhibition in the infection caused by S. aureus is unclear until now. Here, we demonstrated that the persistence and aggregation of platelets were important for killing S. aureus in whole blood. It was found that the N terminal of Efb (EfbN) and platelets inhibitors could increase the survival of S. aureus in whole blood. The study in vivo also showed that EfbN and platelets inhibitors could reduce the killing of S. aureus and increase the lethality rate of S. aureus in the acute infection mouse model.

Allosteric inhibition of complement function by a staphylococcal immune evasion protein

The complement system is a major target of immune evasion by Staphylococcus aureus. Although many evasion proteins have been described, little is known about their molecular mechanisms of action. Here we demonstrate that the extracellular fibrinogen-binding protein (Efb) from S. aureus acts as an allosteric inhibitor by inducing conformational changes in complement fragment C3b that propagate across several domains and influence functional regions far distant from the Efb binding site. Most notably, the inhibitor impaired the interaction of C3b with complement factor B and, consequently, formation of the active C3 convertase. As this enzyme complex is critical for both activation and amplification of the complement response, its allosteric inhibition likely represents a fundamental contribution to the overall immune evasion strategy of S. aureus.