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 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.     

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.     

Identification and characterization of the C3 binding domain of the Staphylococcus aureus extracellular fibrinogen-binding protein (Efb)

The secreted Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) is a virulence factor that binds to both the complement component C3b and fibrinogen. Our laboratory previously reported that by binding to C3b, Efb inhibited complement activation and blocked opsonophagocytosis. We have now located the Efb binding domain in C3b to the C3d fragment and determined a disassociation constant (Kd) of 0.24 microM for the Efb-C3d binding using intrinsic fluorescence quenching assays. Using truncated, recombinant forms of Efb, we also demonstrate that the C3b binding region of Efb is located within the C terminus, in contrast to the fibrinogen binding domains that are located at the N-terminal end of the protein. Enzyme-linked immunosorbent assay-type binding assays demonstrated that recombinant Efb could bind to both C3b and fibrinogen simultaneously, forming a trimolecular complex and that the C-terminal region of Efb could inhibit complement activity in vitro. In addition, secondary structure analysis using circular dichroism spectroscopy revealed that the C-terminal, C3b binding region of Efb is composed primarily of alpha-helices, suggesting that this domain of Efb represents a novel type of C3b-binding protein.     

A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo

Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well‐characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA‐based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost‐effective method that can be used to prevent unnecessary animal experiments.     

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus,Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.     

A small regulatory RNA alters Staphylococcus aureus virulence by titrating RNAIII activity

Staphylococcus aureus is an opportunistic human and animal pathogen with an arsenal of virulence factors that are tightly regulated during bacterial infection. The latter is achieved through a sophisticated network of regulatory proteins and regulatory RNAs. Here, we describe the involvement of a novel prophage-carried small regulatory S. aureus RNA, SprY, in the control of virulence genes. An MS2-affinity purification assay reveals that SprY forms a complex in vivo with RNAIII, a major regulator of S. aureus virulence genes. SprY binds to the 13th stem-loop of RNAIII, a key functional region involved in the repression of multiple mRNA targets. mRNAs encoding the repressor of toxins Rot and the extracellular complement binding protein Ecb are among the targets whose expression is increased by SprY binding to RNAIII. Moreover, SprY decreases S. aureus hemolytic activity and virulence. Our results indicate that SprY titrates RNAIII activity by targeting a specific stem loop. Thus, we demonstrate that a prophage-encoded sRNA reduces the pathogenicity of S. aureus through RNA sponge activity.     

Human anti-peptidoglycan-IgG-mediated opsonophagocytosis is controlled by calcium mobilization in phorbol myristate acetate-treated U937 cells

Recently, we demonstrated that human serum amyloid P component (SAP) specifically recognizes exposed bacterial peptidoglycan (PGN) of wall teichoic acid (WTA)-deficient Staphylococcus aureus ΔtagO mutant cells and then induces complement-independent phagocytosis. In our preliminary experiments, we found the existence of human serum immunoglobulins that recognize S. aureus PGN (anti-PGNIgGs), which may be involved in complement-dependent opsonophagocytosis against infected S. aureus cells. We assumed that purified serum anti-PGN-IgGs and S. aureus ΔtagO mutant cells are good tools to study the molecular mechanism of anti-PGN-IgG-mediated phagocytosis. Therefore, we tried to identify the intracellular molecule(s) that is involved in the anti-PGN-IgG-mediated phagocytosis using purified human serum anti-PGN-IgGs and different S. aureus mutant cells. Here, we show that anti-PGN-IgG-mediated phagocytosis in phorbol myristate acetate-treated U937 cells is mediated by Ca²⁺ release from intracellular Ca²⁺ stores and anti-PGN-IgGdependent Ca²⁺ mobilization is controlled via a phospholipase Cγ-2-mediated pathway. [BMB Reports 2015; 48(1): 36-41]     

Extracellular fibrinogen-binding protein, Efb, from Staphylococcus aureus blocks platelet aggregation due to its binding to the alpha-chain.

Extracellular fibrinogen-binding protein (Efb) secreted by Staphylococcus aureus has previously been shown to contribute to pathogenesis in a rat wound infection model. Also antibodies against Efb exhibited a protective effect in a mouse mastitis model. The interaction between Efb and fibrinogen is divalent, with one binding site within the N-terminal repeat region in Efb and one at the C terminus. In this study we show that the distal D domain of fibrinogen contains at least one of the binding domains recognized by Efb. Efb stimulates fibrinogen binding to ADP-activated platelets. Furthermore, Efb inhibits ADP-induced, fibrinogen-dependent platelet aggregation in a concentration-dependent manner. This implies that Efb modifies platelet function by amplifying a non-functional interaction between fibrinogen and platelets. Efb recognizes the A alpha-chain of the D fragment of fibrinogen. The RGD sequence on the A alpha-chain is located close to the region recognized by Efb and contains a putative binding site for the platelet integrin GPIIb/IIIa receptor complex involved in platelet aggregation.     

Characteristics of new Staphylococcus aureus-RBC adhesion mechanism independent of fibrinogen and IgG under hydrodynamic shear conditions

Staphylococcus aureus infection begins when bacterial cells circulating in blood adhere to components of the extracellular matrix or endothelial cells of the host and initiate colonization. S. aureus is known to exhibit extensive interactions with platelets. S. aureus is also known to bind to red blood cells (RBCs) in the presence of plasma proteins, such as fibrinogen and IgG. Herein we report a new binding mechanism of S. aureus to RBC independent of those plasma proteins. To characterize the new adhesion mechanism, we experimentally examine the binding kinetics and molecular constituents mediating the new adhesive interactions between S. aureus and RBCs under defined shear conditions. The results demonstrate that the receptors for fibrinogen (clumping factor A) and IgG (protein A) of S. aureus are not involved in the adhesion. S. aureus binds to RBCs with maximal adhesion at the shear rate 100 s^–1 and decreasing adhesion with increasing shear. The heteroaggregates formed after shear are stable when subjected to the shear rate 2,000 s^–1, indicating that intercellular contact time rather than shear forces controls the adhesion at high shear. S. aureus binding to RBC requires plasma, and 10% plasma is sufficient for maximal adhesion. Plasma proteins involved in the cell-cell adhesion, such as fibrinogen, fibronectin, von Willebrand factor, IgG, thrombospondin, laminin, and vitronectin are not involved in the observed adhesion. The extent of heteroaggregation is dramatically reduced on RBC treatment with trypsin, chymotrypsin, or neuraminidase, suggesting that the receptor(s) mediating the heteroaggregation process is a sialylated glycoprotein on RBC surface. Adhesion is divalent cation dependent and also blocked by heparin. This work demonstrates a new mechanism of S. aureus-RBC binding under hydrodynamic shear conditions via unknown RBC sialoglycoprotein(s). The binding requires plasma protein(s) other than fibrinogen or IgG and does not involve the S. aureus adhesins clumping factor A or protein A. adhesion; red blood cell     

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.     

A Scabies Mite Serpin Interferes with Complement-Mediated Neutrophil Functions and Promotes Staphylococcal Growth

Background

Scabies is a contagious skin disease caused by the parasitic mite Sarcoptes scabiei. The disease is highly prevalent worldwide and known to predispose to secondary bacterial infections, in particular by Streptococcus pyogenes and Staphylococcus aureus. Reports of scabies patients co-infected with methicillin resistant S. aureus (MRSA) pose a major concern for serious down-stream complications. We previously reported that a range of complement inhibitors secreted by the mites promoted the growth of S. pyogenes. Here, we show that a recently characterized mite serine protease inhibitor (SMSB4) inhibits the complement-mediated blood killing of S. aureus.

Methodology/Principal Findings

Blood killing of S. aureus was measured in whole blood bactericidal assays, counting viable bacteria recovered after treatment in fresh blood containing active complement and phagocytes, treated with recombinant SMSB4. SMSB4 inhibited the blood killing of various strains of S. aureus including methicillin-resistant and methicillin-sensitive isolates. Staphylococcal growth was promoted in a dose-dependent manner. We investigated the effect of SMSB4 on the complement-mediated neutrophil functions, namely phagocytosis, opsonization and anaphylatoxin release, by flow cytometry and in enzyme linked immuno sorbent assays (ELISA). SMSB4 reduced phagocytosis of S. aureus by neutrophils. It inhibited the deposition of C3b, C4b and properdin on the bacteria surface, but did not affect the depositions of C1q and MBL. SMSB4 also inhibited C5 cleavage as indicated by a reduced C5b-9 deposition.

Conclusions/Significance

We postulate that SMSB4 interferes with the activation of all three complement pathways by reducing the amount of C3 convertase formed. We conclude that SMSB4 interferes with the complement-dependent killing function of neutrophils, thereby reducing opsonization, phagocytosis and further recruitment of neutrophils to the site of infection. As a consequence secreted scabies mites complement inhibitors, such as SMSB4, provide favorable conditions for the onset of S. aureus co-infection in the scabies-infected microenvironment by suppressing the immediate host immune response.     

Interaction between fibronectin and purified staphylococcal clumping factor

Clumping of Staphylococcal aureus was observed in the presence of fibrinogen as well as fibronectin. In order to elucidate the mechanism of this clumping, binding of radiolabelled fibrinogen and fibronectin to S. aureus cultures was studied. Cultures of S. aureus reacted with ¹²⁵I-labelled fibrinogen as well as fibronectin. The binding of labelled fibrinogen to S. aureus could be completely inhibited by unlabelled fibronectin, whereas the binding of labelled fibronectin was only partially inhibited by unlabelled fibrinogen. This suggested an interaction of fibronectin with clumping factor which is the binding protein for fibrinogen in staphylococci. The clumping factor was purified from S. aureus strain K 807 by affinity chromatography on fibrinogen-Sepharose followed by HPLC. The purified clumping factor inhibited the binding of fibrinogen and fibronectin to staphylococci. In western blots the purified clumping factor reacted with fibrinogen as well as fibronectin. Thus, the direct interaction of clumping factor with fibronectin might be responsible for the clumping of staphylococci in fibrinogen depleted plasma or serum.     

Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway

Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.     

The Staphylococcus aureus Protein Sbi Acts as a Complement Inhibitor and Forms a Tripartite Complex with Host Complement Factor H and C3b

The Gram-positive bacterium Staphylococcus aureus, similar to other pathogens, binds human complement regulators Factor H and Factor H related protein 1 (FHR-1) from human serum. Here we identify the secreted protein Sbi (Staphylococcus aureus binder of IgG) as a ligand that interacts with Factor H by a—to our knowledge—new type of interaction. Factor H binds to Sbi in combination with C3b or C3d, and forms tripartite Sbi∶C3∶Factor H complexes. Apparently, the type of C3 influences the stability of the complex; surface plasmon resonance studies revealed a higher stability of C3d complexed to Sbi, as compared to C3b or C3. As part of this tripartite complex, Factor H is functionally active and displays complement regulatory activity. Sbi, by recruiting Factor H and C3b, acts as a potent complement inhibitor, and inhibits alternative pathway-mediated lyses of rabbit erythrocytes by human serum and sera of other species. Thus, Sbi is a multifunctional bacterial protein, which binds host complement components Factor H and C3 as well as IgG and β₂-glycoprotein I and interferes with innate immune recognition.     

TLR2 mediates phagocytosis and autophagy through JNK signaling pathway in Staphylococcus aureus-stimulated RAW264.7 cells

Toll-like receptor 2 (TLR2) is involved in phagocytosis and autophagy to enhance host innate immune response to bacterial infection. TLR2 has been reported to participate in the recognition of Staphylococcus aureus (S. aureus). However, the role of TLR2 in phagocytosis and autophagy in S. aureus-stimulated macrophages and the underlying mechanisms as yet remain unclear. In the present study, stimulation of mouse macrophage cell line RAW264.7 with S. aureus activated multiple signaling pathways including mitogen-activated protein kinases (MAPKs), myeloid differentiation factor 88 (MyD88), phosphatidylinositide 3-kinase (PI3K) and Rac1 and triggered autophagy process. Knockdown of TLR2 by siRNA significantly reduced phagocytosis and autophagy of macrophages upon S. aureus infection. Interestingly, TLR2 siRNA markedly attenuated S. aureus-induced phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 or extracellular regulated protein kinase (ERK) in macrophages. Similarly, SP600125, a JNK inhibitor, also down-regulated phagocytosis and autophagy in S. aureus-stimulated macrophages. Furthermore, TLR2 siRNA and SP600125 simultaneous treatment showed similar phagocytosis and autophagy compared to that in TLR2 siRNA treatment alone. Collectively, our results indicate that TLR2 may be critical for phagocytosis and autophagy through JNK signaling pathway, and provide an underlying mechanistic link between innate immune receptor and induction of phagocytosis and autophagy in S. aureus-stimulated macrophages.     

Evidence for Steric Regulation of Fibrinogen Binding to Staphylococcus aureus Fibronectin-binding Protein A (FnBPA)

The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for “latch” strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.     

N-Acetylglucosaminylation of Serine-Aspartate Repeat Proteins Promotes Staphylococcus aureus Bloodstream Infection

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.     

Two Distinct Coagulase-Dependent Barriers Protect Staphylococcus aureus from Neutrophils in a Three Dimensional in vitro Infection Model

Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options.