Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5)

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.     

Identification of Set11 from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Mu50 strain as a ortholog of SSL7 via bioinformatic analysis and determination of its possible targets from human serum using CNBr-pull down assays

Objectives

To identify and characterize staphylococcus exotoxin-like (SET) protein Set11 from Staphylococcus aureus Mu50 strain and its possible targets proteins from human blood/serum.

Results

Set11 is a member of the staphylococcal superantigen-like (SSL) proteins (also called Staphylococcus exotoxin-like (SET) proteins) family that is found in staphylococcal strain Mu50. Its structure and function, however, remain unknown. We performed bioinformatics analysis of Set11: it had 90% sequence identity to SSL7 in NCTC 8325 strain, indicating Set11 is a SSL7 ortholog. SSL7 in ATCC 12598 strain binds complement C5 to inhibit complement system. To investigate if Set11 binds C5, we made the homology model of Set11 and the Set11-C5 complex model based on SSL7 and SSL7-C5 structures, respectively. Structural analysis and sequence alignment reveal that the residues in SSL7 involved in C5 binding are conserved in Set11, indicating C5 as the potential target for Set11. To identify new targets of Set11, we cloned, expressed and purified Set11 and performed CNBr-pull down combined mass spectrum assays using human blood and serum.

Conclusions

We identified Set11 as the ortholog of SSL7 and determined C5, fibronectin 1 isoform 3 preproprotein, albumin, alpha-1-microglobulin precursor and complement C3 processor as the potential target proteins for Set11, indicating new functions of Set11/SSL7.

     

The staphylococcal superantigen-like protein 7 binds IgA and complement C5 and inhibits IgA-Fc alpha RI binding and serum killing of bacteria

The staphylococcal superantigen-like proteins (SSLs) are close relatives of the superantigens but are coded for by a separate gene cluster within a 19-kb region of the pathogenicity island SaPIn2. rSSL7 (formally known as SET1) bound with high affinity (K(D), 1.1 nM) to the monomeric form of human IgA1 and IgA2 plus serum IgA from primate, pig, rat, and horse. SSL7 also bound the secretory form of IgA found in milk from human, cow, and sheep, and inhibited IgA binding to cell surface FcalphaRI (CD89) and to a soluble form of the FcalphaRI protein. In addition to IgA, SSL7 bound complement factor C5 from human (K(D), 18 nM), primate, sheep, pig, and rabbit serum, and inhibited complement-mediated hemolysis and serum killing of a Gram-negative organism Escherichia coli. SSL7 is a superantigen-like protein secreted from Staphylococcus aureus that blocks IgA-FcR interactions and inhibits complement, leading to increased survival of a sensitive bacterium in blood.     

Interaction of human, rat, and mouse immunoglobulin A (IgA) with Staphylococcal superantigen-like 7 (SSL7) decoy protein and leukocyte IgA receptor

Host survival depends on an effective immune system and pathogen survival on the effectiveness of immune evasion mechanisms. Staphylococcus aureus utilizes a number of molecules to modulate host immunity, including the SSL family of which SSL7 binds IgA and inhibits Fcα receptor I (FcαRI)-mediated function. Other Gram-positive bacterial pathogens produce IgA binding proteins, which, similar to SSL7, also bind the Fc at the CH2/CH3 interface (the junction between constant domains 2 and 3 of the heavy chain). The opposing activities of the host FcαRI-IgA receptor ligand pair and the pathogen decoy proteins select for host and pathogen variants, which exert stronger protection or evasion, respectively. Curiously, mouse but not rat IgA contains a putative N-linked glycosylation site in the center of this host receptor and pathogen-binding site. Here, we demonstrate that this site is glycosylated and that the effect of amino acid changes and glycosylation of the CH2/CH3 interface inhibits interaction with the pathogen IgA binding protein SSL7, while maintaining binding of pIgR, essential to the biosynthesis and transport of SIgA.     

Staphylococcal Superantigen-like Protein 10 (SSL10) Inhibits Blood Coagulation by Binding to Prothrombin and Factor Xa via Their γ-Carboxyglutamic Acid (Gla) Domain

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10⁻⁷ m in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca²⁺ and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.     

Staphylococcal phosphatidylinositol‐specific phospholipase C potentiates lung injury via complement sensitisation

Staphylococcus aureus is frequently isolated from patients with community‐acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol‐specific phospholipase C (PI‐PLC); however, the role of PI‐PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI‐PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol‐anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI‐PLC‐positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc‐isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild‐type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc^? mutant. Following treatment with cobra venom factor to deplete complement, the wild‐type strain with PI‐PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI‐PLC‐positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI‐PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.     

Staphylococcus aureus proteins SSL6 and SElX interact with neutrophil receptors as identified using secretome phage display

In order to cause colonization and invasive disease, pathogenic bacteria secrete proteins that modulate host immune defences. Identification and characterization of these proteins leads to a better understanding of the pathological processes underlying infectious and inflammatory diseases and is essential in the development of new strategies for their prevention and treatment. Current techniques to functionally characterize these proteins are laborious and inefficient. Here we describe a high‐throughput functional selection strategy using phage display in order to identify immune evasion proteins. Using this technique we identified two previously uncharacterized proteins secreted by Staphylococcus aureus, SElX and SSL6 that bind to neutrophil surface receptors. SElX binds PSGL‐1 on neutrophils and thereby inhibits the interaction between PSGL‐1 and P‐selectin, a crucial step in the recruitment of neutrophils to the site of infection. SSL6 is the first bacterial protein identified that binds CD47, a widely expressed cell surface protein recently described as an interesting target in anti‐cancer therapy. Our findings provide new insights into the pathogenesis of S. aureus infections and support phage display as an efficient method to identify bacterial secretome proteins interacting with humoral or cellular immune components.     

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.     

Crystal structures of the staphylococcal toxin SSL5 in complex with sialyl Lewis X reveal a conserved binding site that shares common features with viral and bacterial sialic acid binding proteins

Staphylococcus aureus is a significant human pathogen. Among its large repertoire of secreted toxins is a group of staphylococcal superantigen-like proteins (SSLs). These are homologous to superantigens but do not have the same activity. SSL5 is shown here to bind to human granulocytes and to the cell surface receptors for human IgA (FcαRI) and P-selectin [P-selectin glycoprotein ligand-1 (PSGL-1)] in a sialic acid (Sia)-dependent manner. Co-crystallization of SSL5 with the tetrasaccharide sialyl Lewis X (sLe^X), a key determinant of PSGL-1 binding to P-selectin, led to crystal structures of the SSL5–sLe^X complex at resolutions of 1.65 and 2.75 Å for crystals at two pH values. In both structures, sLe^X bound to a specific site on the surface of the C-terminal domain of SSL5 in a conformation identical with that bound by P-selectin. Conservation of the key carbohydrate binding residues indicates that this ability to bind human glycans is shared by a substantial subgroup of the SSLs, including SSL2, SSL3, SSL4, SSL5, SSL6, and SSL11. This indicates that the ability to target human glycans is an important property of this group of toxins. Structural comparisons also showed that the Sia binding site in SSL5 contains a substructure that is shared by other Sia binding proteins from bacteria as well as viruses and represents a common binding motif.     

A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor

Staphylococcus aureus and Staphylococcus epidermidis can cause serious chronic and recurrent infections that are difficult to eradicate. An important pathogenicity factor in these infections caused by S. aureus is its ability to be internalized by non‐professional phagocytes thereby evading the host immune system and antibiotic treatment. Here, we report a novel mechanism involved in staphylococcal internalization by host cells, which is mediated by the major autolysin/adhesins Atl and AtlE from S. aureus and S. epidermidis respectively. In a flow cytometric internalization assay, atl and atlE mutants are significantly reduced in their capacities to be internalized by endothelial cells. Moreover, pre‐incubation of endothelial cells with recombinant Atl dose‐dependently inhibited internalization. As putative Atl‐host cell receptor, the heat shock cognate protein Hsc70 was identified by mass spectrometry. The importance of Hsc70 in internalization was demonstrated by the inhibition of S. aureus internalization with anti‐Hsc70 antibodies. In conclusion, this novel Atl‐ or AtlE‐mediated internalization mechanism may represent a ‘back‐up’ mechanism in S. aureus internalization, while it may represent the major or even sole mechanism involved in the internalization of coagulase‐negative staphylococci and thus may play an important role in the pathogenesis of chronic and relapsing infections with these serious pathogens.     

A new host cell internalisation pathway for SadA‐expressing staphylococci triggered by excreted neurochemicals

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface‐bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal‐pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface‐bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT‐29. We found that TAs and DOP are agonists of the α2‐adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F‐actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F‐actin formation without decreasing the cytoplasmic cAMP level. The neurochemical‐induced internalisation in host cells is independent of the fibronectin‐binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.     

Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

Complement acts as a danger‐sensing system in the innate immune system, and its activation initiates a strong inflammatory response and cleavage of the proteins C3 and C5 by proteolytic enzymes, the convertases. These contain a non‐catalytic substrate contacting subunit (C3b or C4b) in complex with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two‐point attachment between C5 and CVF, where the presence of SSL7 only slightly affects the C5–CVF interface, explaining the IgA dependence for SSL7‐mediated inhibition of C5 cleavage. CVF functions as a relatively rigid binding scaffold inducing a conformational change in C5, which positions its cleavage site in proximity to the serine protease Bb. A general model for substrate recognition by the convertases is presented based on the C5–CVF and C3b–Bb–SCIN structures. Prior knowledge concerning interactions between the endogenous convertases and their substrates is rationalized by this model.     

Staphylococcus aureus protects its immune‐evasion proteins against degradation by neutrophil serine proteases

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti‐staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune‐evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune‐escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.     

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.     

Exploiting dominant‐negative toxins to combat Staphylococcus aureus pathogenesis

Staphylococcus aureus (S. aureus) is a human pathogen that relies on the subversion of host phagocytes to support its pathogenic lifestyle. S. aureus strains can produce up to five beta‐barrel, bi‐component, pore‐forming leukocidins that target and kill host phagocytes. Thus, preventing immune cell killing by these toxins is likely to boost host immunity. Here, we describe the identification of glycine‐rich motifs within the membrane‐penetrating stem domains of the leukocidin subunits that are critical for killing primary human neutrophils. Remarkably, leukocidins lacking these glycine‐rich motifs exhibit dominant‐negative inhibitory effects toward their wild‐type toxin counterparts as well as other leukocidins. Biochemical and cellular assays revealed that these dominant‐negative toxins work by forming mixed complexes that are impaired in pore formation. The dominant‐negative leukocidins inhibited S. aureus cytotoxicity toward primary human neutrophils, protected mice from lethal challenge by wild‐type leukocidin, and reduced bacterial burden in a murine model of bloodstream infection. Thus, we describe the first example of staphylococcal bi‐component dominant‐negative toxins and their potential as novel therapeutics to combat S. aureus infection.     

α‐Hemolysin enhances Staphylococcus aureus internalization and survival within mast cells by modulating the expression of β1 integrin

Mast cells (MCs) are important sentinels of the host defence against invading pathogens. We previously reported that Staphylococcus aureus evaded the extracellular antimicrobial activities of MCs by promoting its internalization within these cells via β1 integrins. Here, we investigated the molecular mechanisms governing this process. We found that S. aureus responded to the antimicrobial mediators released by MCs by up‐regulating the expression of α‐hemolysin (Hla), fibronectin‐binding protein A and several regulatory systems. We also found that S. aureus induced the up‐regulation of β1 integrin expression on MCs and that this effect was mediated by Hla‐ADAM10 (a disintegrin and metalloproteinase 10) interaction. Thus, deletion of Hla or inhibition of Hla‐ADAM10 interaction significantly impaired S. aureus internalization within MCs. Furthermore, purified Hla but not the inactive Hla_H35L induced up‐regulation of β1 integrin expression in MCs in a dose‐dependent manner. Our data support a model in which S. aureus counter‐reacts the extracellular microbicidal mechanisms of MCs by increasing expression of fibronectin‐binding proteins and by inducing Hla‐ADAM10‐mediated up‐regulation of β1 integrin in MCs. The up‐regulation of bacterial fibronectin‐binding proteins, concomitantly with the increased expression of its receptor β1 integrin on the MCs, resulted in enhanced S. aureus internalization through the binding of fibronectin‐binding proteins to integrin β1 via fibronectin.     

Role of sialic acid‐containing glycans of matrix metalloproteinase‐9 (MMP‐9) in the interaction between MMP‐9 and staphylococcal superantigen‐like protein 5

Staphylococcal superantigen‐like proteins (SSL) show no superantigenic activity but have recently been considered to act as immune suppressors. It was previously reported that SSL5 bound to P‐selectin glycoprotein ligand‐1 (PSGL‐1) and matrix metalloproteinase (MMP)‐9, leading to inhibition of leukocyte adhesion and invasion. These interactions were suggested to depend on sialic acid‐containing glycans of MMP‐9, but the roles of sialic acids in the interaction between SSL5 and MMP‐9 are still controversial. In the present study, we prepared recombinant glutathione S‐transferase‐tagged SSL5 (GST‐SSL5) and analyzed its binding capacity to MMP‐9 by pull‐down assay after various modifications of its carbohydrate moieties. We observed that GST‐SSL5 specifically bound to MMP‐9 from a human monocytic leukemia cell line (THP‐1 cells) and inhibited its enzymatic activity in a concentration‐dependent manner. After MMP‐9 was treated with neuraminidase, its binding activity towards GST‐SSL5 was markedly decreased. Furthermore, recombinant MMP‐9 produced by sialic acid‐deficient Lec2 mutant cells showed much lower affinity for SSL5 than that produced by wild‐type CHO‐K1 cells. Treatment of MMP‐9 with PNGase F to remove N‐glycan resulted in no significant change in the GST‐SSL5/MMP‐9 interaction. In contrast, the binding of GST‐SSL5 to MMP‐9 secreted from THP‐1 cells cultured in the presence of an inhibitor for the biosynthesis of O‐glycan (benzyl‐GalNAc) was weaker than the binding of GST‐SSL5 to MMP‐9 secreted from untreated cells. These results strongly suggest the importance of the sialic acid‐containing O‐glycans of MMP‐9 for the interaction of MMP‐9 with GST‐SSL5.

     

Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b

The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 Å limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.