IdeS, a secreted proteinase of Streptococcus pyogenes, is bound to a nuclease at the bacterial surface where it inactivates opsonizing IgG antibodies.

The important bacterial pathogen Streptococcus pyogenes secretes IdeS (immunoglobulin G-degrading enzyme of S. pyogenes), a proteinase that cleaves human immunoglobulin G (IgG) antibodies in the hinge region resulting in Fc (fragment crystallizable) and F(ab')2 (fragment antigen-binding) fragments and protects the bacteria against phagocytic killing. Experiments with radiolabeled IdeS and flow cytometry demonstrated that IdeS binds to the surface of S. pyogenes, and the interaction was most prominent in conditions resembling those in the pharynx (acidic pH and low salt), the habitat for S. pyogenes. SpnA (S. pyogenes nuclease A) is a cell wall-anchored DNase. A dose-dependent interaction between purified SpnA and IdeS was demonstrated in slot binding and surface plasmon resonance spectroscopy experiments. Gel filtration showed that IdeS forms proteolytically active complexes with SpnA in solution, and super-resolution fluorescence microscopy revealed the presence of SpnA-IdeS complexes at the surface of S. pyogenes. Finally, specific IgG antibodies binding to S. pyogenes surface antigens were efficiently cleaved by surface-associated IdeS. IdeS is secreted by all S. pyogenes isolates and cleaves IgG antibodies with a unique degree of specificity and efficiency. These properties and the finding here that the proteinase is present and fully active at the bacterial surface in complex with SpnA implicate an important role for IdeS in S. pyogenes biology and pathogenesis.

Human IgG Increases Virulence of Streptococcus pyogenes through Complement Evasion.

Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization.

Streptococcus pyogenes Infection and the Human Proteome with a Special Focus on the Immunoglobulin G-cleaving Enzyme IdeS.

Infectious diseases are characterized by a complex interplay between host and pathogen, but how these interactions impact the host proteome is unclear. Here we applied a combined mass spectrometry-based proteomics strategy to investigate how the human proteome is transiently modified by the pathogen Streptococcus pyogenes, with a particular focus on bacterial cleavage of IgG in vivo In invasive diseases, S. pyogenes evokes a massive host response in blood, whereas superficial diseases are characterized by a local leakage of several blood plasma proteins at the site of infection including IgG. S. pyogenes produces IdeS, a protease cleaving IgG in the lower hinge region and we find highly effective IdeS-cleavage of IgG in samples from local IgG poor microenvironments. The results show that IdeS contributes to the adaptation of S. pyogenes to its normal ecological niches. Additionally, the work identifies novel clinical opportunities for in vivo pathogen detection.

Streptococcal inhibitor of complement (SIC) modulates fibrinolysis and enhances bacterial survival within fibrin clots.

Some strains of the bacterial pathogen Streptococcus pyogenes secrete protein SIC (streptococcal inhibitor of complement), including strains of the clinically relevant M1 serotype. SIC neutralizes the effect of a number of antimicrobial proteins/peptides and interferes with the function of the host complement system. Previous studies have shown that some S. pyogenes proteins bind and modulate coagulation and fibrinolysis factors, raising the possibility that SIC also may interfere with the activity of these factors. Here we show that SIC interacts with both human thrombin and plasminogen, key components of coagulation and fibrinolysis. We found that during clot formation, SIC binds fibrin through its central region and that SIC inhibits fibrinolysis by interacting with plasminogen. Flow cytometry results indicated that SIC and plasminogen bind simultaneously to S. pyogenes bacteria, and fluorescence microscopy revealed co-localization of the two proteins at the bacterial surface. As a consequence, SIC-expressing bacteria entrapped in clots inhibit fibrinolysis, leading to delayed bacterial escape from the clots as compared with mutant bacteria lacking SIC. Moreover, within the clots SIC-expressing bacteria were protected against killing. In an animal model of subcutaneous infection, SIC-expressing bacteria exhibited a delayed systemic spread. These results demonstrate that the bacterial protein SIC interferes with coagulation and fibrinolysis and thereby enhances bacterial survival, a finding that has significant implications for S. pyogenes virulence.

Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS (imlifidase) in chronic kidney disease patients.

Safety, immunogenicity, pharmacokinetics, and efficacy of the IgG-degrading enzyme of Streptococcus pyogenes (IdeS [imlifidase]) were assessed in a single-center, open-label ascending-dose study in highly sensitized patients with chronic kidney disease. Eight patients with cytotoxic PRAs (median cytotoxic PRAs of 64%) at enrollment received 1 or 2 intravenous infusions of IdeS on consecutive days (0.12 mg/kg body weight ×2 [n = 3]; 0.25 mg/kg ×1 [n = 3], or 0.25 mg/kg ×2 [n = 2]). IgG degradation was observed in all subjects after IdeS treatment, with <1% plasma IgG remaining within 48 hours and remaining low up to 7 days. Mean fluorescence intensity values of HLA class I and II reactivity were substantially reduced in all patients, and C1q binding to anti-HLA was abolished. IdeS also cleaved the IgG-type B cell receptor on CD19+ memory B cells. Anti-IdeS antibodies developed 1 week after treatment, peaking at 2 weeks. A few hours after the second IdeS infusion, 1 patient received a deceased donor kidney offer. At enrollment, the patient had a positive serum crossmatch (HLA-B7), detected by complement-dependent cytotoxicity, flow cytometry, and multiplex bead assays. After IdeS infusion (0.12 mg/kg ×2) and when the HLA-incompatible donor (HLA-B7+ ) kidney was offered, the HLA antibody profile was negative. The kidney was transplanted successfully.

In vivo enzymatic modulation of IgG antibodies prevents immune complex-dependent skin injury.

IgG antibodies are potent inducers of proinflammatory responses by cross-linking Fc receptors on innate immune effector cells resulting in tissue injury. The recently discovered enzymes endoglycosidase S (EndoS) and IgG-degrading enzyme (IdeS) of Streptococcus pyogenes are able to modulate the interaction between IgG antibodies and the Fc receptors, by hydrolysis of the glycan associated with the heavy chain of the IgG molecule (EndoS), or cleavage in the hinge region of the heavy IgG chain (IdeS). In this work, we investigated their ability to inhibit damage mediated by skin-bound antibodies in vivo in two different experimental models, the Arthus reaction, and epidermolysis bullosa acquisita, an autoimmune blistering skin disease associated with autoantibodies against type VII collagen. We demonstrate that both enzymes efficiently interfere with IgG-mediated proinflammatory processes, offering a great asset to specifically target pathological IgG antibodies in the skin and holding great promise for future applications in human therapy.

Saliva-Induced Clotting Captures Streptococci: Novel Roles for Coagulation and Fibrinolysis in Host Defense and Immune Evasion.

Streptococcal pharyngitis is among the most common bacterial infections, but the molecular mechanisms involved remain poorly understood. Here we investigate the interactions among three major players in streptococcal pharyngitis: streptococci, plasma, and saliva. We find that saliva activates the plasma coagulation system through both the extrinsic and the intrinsic pathways, entrapping the bacteria in fibrin clots. The bacteria escape the clots by activating host plasminogen. Our results identify a potential function for the intrinsic pathway of coagulation in host defense and a corresponding role for fibrinolysis in streptococcal immune evasion.

Functional and structural properties of a novel protein and virulence factor (Protein sHIP) in Streptococcus pyogenes.

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.

Identification of molecular mechanisms used by Finegoldia magna to penetrate and colonize human skin.

Finegoldia magna is a Gram-positive anaerobic commensal of the human skin microbiota, but also known to act as an opportunistic pathogen. Two primary virulence factors of F. magna are the subtilisin-like extracellular serine protease SufA and the adhesive protein FAF. This study examines the molecular mechanisms F. magna uses when colonizing or establishing an infection in the skin. FAF was found to be essential in the initial adherence of F. magna to human skin biopsies. In the upper layers of the epidermis FAF mediates adhesion through binding to galectin-7 - a keratinocyte cell marker. Once the bacteria moved deeper into the skin to the basement membrane layer, SufA was found to degrade collagen IV which forms the backbone structure of the basement membrane. It also degraded collagen V, whereby F. magna could reach deeper dermal tissue sites. In the dermis, FAF interacts with collagen V and fibrillin, which presumably helps the bacteria to establish infection in this area. The findings of this study paint a clear picture of how F. magna interacts with human skin and explain how it is such a successful opportunistic pathogen in chronic wounds and ulcers.

Binding of complement inhibitor C4b-binding protein to a highly virulent Streptococcus pyogenes M1 strain is mediated by protein H and enhances adhesion to and invasion of endothelial cells.

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.

Surface proteins of group G Streptococcus in different phases of growth: patterns of production and implications for the host-bacteria relationship.

Group G Streptococcus (GGS) is a human bacterial pathogen expressing surface proteins FOG and protein G (PG) which interact with several host defence systems, including the complement and contact systems. Selected reaction monitoring mass spectrometry, electron microscopy and protein binding assays were used to track the amounts of FOG and PG intracellularly and on the bacterial surface during different phases of growth. Large and increasing amounts of PG were present on the surface in the stationary growth phase, and this was due to de novo production. In contrast, the amount of FOG did not change substantially during this phase. Apart from PG, a number of housekeeping proteins also increased in abundance in the stationary phase. These results show that GGS protein production is active during the stationary phase and that the bacteria actively remodel their surface and enter a less pro-inflammatory state in this phase.

Streptococcal surface proteins activate the contact system and control its antibacterial activity.

Group G streptococci (GGS) are important bacterial pathogens in humans. Here, we investigated the interactions between GGS and the contact system, a procoagulant and proinflammatory proteolytic cascade that, upon activation, also generates antibacterial peptides. Two surface proteins of GGS, protein FOG and protein G (PG), were found to bind contact system proteins. Experiments utilizing contact protein-deficient human plasma and isogenic GGS mutant strains lacking FOG or PG showed that FOG and PG both activate the procoagulant branch of the contact system. In contrast, only FOG induced cleavage of high molecular weight kininogen, generating the proinflammatory bradykinin peptide and additional high molecular weight kininogen fragments containing the antimicrobial peptide NAT-26. On the other hand, PG protected the bacteria against the antibacterial effect of NAT-26. These findings underline the significance of the contact system in innate immunity and demonstrate that GGS have evolved surface proteins to exploit and modulate its effects.

Streptococcus pyogenes in human plasma: adaptive mechanisms analyzed by mass spectrometry-based proteomics.

Streptococcus pyogenes is a major bacterial pathogen and a potent inducer of inflammation causing plasma leakage at the site of infection. A combination of label-free quantitative mass spectrometry-based proteomics strategies were used to measure how the intracellular proteome homeostasis of S. pyogenes is influenced by the presence of human plasma, identifying and quantifying 842 proteins. In plasma the bacterium modifies its production of 213 proteins, and the most pronounced change was the complete down-regulation of proteins required for fatty acid biosynthesis. Fatty acids are transported by albumin (HSA) in plasma. S. pyogenes expresses HSA-binding surface proteins, and HSA carrying fatty acids reduced the amount of fatty acid biosynthesis proteins to the same extent as plasma. The results clarify the function of HSA-binding proteins in S. pyogenes and underline the power of the quantitative mass spectrometry strategy used here to investigate bacterial adaptation to a given environment.

A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein mediates immune function.

Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis-binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region cannot bypass the M protein's virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, regions for protective antibody targeting.

Binding of albumin promotes bacterial survival at the epithelial surface.

Human serum albumin (HSA) is the dominating protein in human plasma. Many bacterial species, especially streptococci, express surface proteins that bind HSA with high specificity and affinity, but the biological consequences of these protein-protein interactions are poorly understood. Group G streptococci (GGS), carrying the HSA-binding protein G, colonize the skin and the mucosa of the upper respiratory tract, mostly without causing disease. In the case of bacterial invasion, pro-inflammatory cytokines are released that activate the epithelium to produce antibacterial peptides, in particular the chemokine MIG/CXCL9. In addition, the inflammation causes capillary leakage and extravasation of HSA and other plasma proteins, environmental changes at the epithelial surface to which the bacteria need to respond. In this study, we found that GGS adsorbed HSA from both saliva and plasma via binding to protein G and that HSA bound to protein G bound and inactivated the antibacterial MIG/CXCL9 peptide. Another surface protein of GGS, FOG, was found to mediate adherence of the bacteria to pharyngeal epithelial cells through interaction with glycosaminoglycans. This adherence was not affected by activation of the epithelium with a combination of IFN-γ and TNF-α, leading to the production of MIG/CXCL9. However, at the activated epithelial surface, adherent GGS were protected against killing by MIG/CXCL9 through protein G-dependent HSA coating. The findings identify a previously unknown bacterial survival strategy that helps to explain the evolution of HSA-binding proteins among bacterial species of the normal human microbiota.

Antibacterial activity of the contact and complement systems is blocked by SIC, a protein secreted by Streptococcus pyogenes.

Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype.

Histidine-rich glycoprotein promotes bacterial entrapment in clots and decreases mortality in a mouse model of sepsis.

Streptococcus pyogenes is a significant bacterial pathogen in humans. In this study, histidine-rich glycoprotein (HRG), an abundant plasma protein, was found to kill S pyogenes. Furthermore, S pyogenes grew more efficiently in HRG-deficient plasma, and clots formed in this plasma were significantly less effective at bacterial entrapment and killing. HRG-deficient mice were strikingly more susceptible to S pyogenes infection. These animals failed to control the infection at the local subcutaneous site, and abscess formation and inflammation were diminished compared with control animals. As a result, bacterial dissemination occurred more rapidly in HRG-deficient mice, and they died earlier and with a significantly higher mortality rate than control animals. HRG-deficient mice supplemented with purified HRG gave the same phenotype as control animals, demonstrating that the lack of HRG was responsible for the increased susceptibility. The results demonstrate a previously unappreciated role for HRG as a regulator of inflammation and in the defense at the local site of bacterial infection.

Activation of the contact system at the surface of Fusobacterium necrophorum represents a possible virulence mechanism in Lemièrre's syndrome.

Fusobacterium necrophorum causes Lemièrre's syndrome, a serious disease with septic thrombophlebitis of the internal jugular vein, pulmonary involvement, and systemic inflammation. The contact system is a link between inflammation and coagulation, and contact activation by the bacteria could therefore contribute to the abnormal coagulation and inflammation seen in patients with Lemièrre's syndrome. In this study, F. necrophorum was found to bind radiolabeled high-molecular-weight kininogen (HK), a central component of the contact system. Binding was inhibited by the addition of unlabeled HK and domain D5 of HK but not other components of the contact system, indicating a specific interaction mediated through the D5 region. Binding of HK was significantly reduced after pretreatment of the bacteria with trypsin, suggesting that surface proteins are involved in HK binding. Incubation of the bacteria with human plasma resulted in an HK breakdown pattern suggestive of bradykinin release, and bradykinin was also detected in the supernatant. In addition, we show that factor XI (FXI), another component of the contact system, binds to F. necrophorum and that the bound FXI reconstitutes the activated partial thromboplastin time of FXI-deficient plasma. Thrombin activity was detected at the surface of the bacteria following incubation with plasma, indicating that the intrinsic pathway of coagulation is activated at the surface. This activity was completely blocked by inhibitors of the contact system. The combined results show that the contact system is activated at the surface of F. necrophorum, suggesting a pathogenic role for this system in Lemièrre's syndrome.

Heparin-binding protein: a diagnostic marker of acute bacterial meningitis.

BACKGROUND: The early detection of bacterial meningitis is crucial for successful outcome. Heparin-binding protein, a potent inducer of increased vascular permeability, is released from activated neutrophils in severe sepsis. OBJECTIVE: In this study we investigated whether heparin-binding protein levels in cerebrospinal fluid could be used as a diagnostic marker for acute bacterial meningitis. DESIGN: One prospective and one retrospective patient cohort from two university hospitals in Sweden were analyzed. SETTING AND PATIENTS: Cerebrospinal fluid samples were collected from 174 patients with suspected central nervous system infection. Thirty-seven patients with acute community-acquired bacterial meningitis, four patients with neurosurgical bacterial meningitis, 29 patients with viral meningitis or encephalitis, seven patients with neuroborreliosis, and 97 control patients were included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Cerebrospinal fluid samples were analyzed for the concentrations of heparin-binding protein, lactate, protein, glucose, neutrophils, and mononuclear cells. Heparin-binding protein levels were significantly higher (p < .01) in patients with acute bacterial meningitis (median 376 ng/mL, range 12-858 ng/mL) than in patients with viral central nervous system infection (median 4.7 ng/mL, range 3.0-41 ng/mL) or neuroborreliosis (median 3.6 ng/mL, range 3.2-10 ng/mL) or in control patients with a normal cerebrospinal fluid cell count (median 3.5 ng/mL, range 2.4-8.7 ng/mL). In the prospectively studied group, a heparin-binding protein concentration exceeding 20 ng/mL gave a sensitivity of 100%, a specificity of 99.2%, and positive and negative predictive values of 96.2% and 100%, respectively, in diagnosing acute bacterial meningitis. The area under the receiver-operating characteristic curve for heparin-binding protein was 0.994, which was higher than for the other investigated parameters. CONCLUSION: Elevated cerebrospinal fluid levels of heparin-binding protein distinguish between patients with acute bacterial meningitis and patients with other central nervous system infections.

Treatment of invasive streptococcal infection with a peptide derived from human high-molecular weight kininogen.

Sepsis and septic shock remain an important medical problem, emphasizing the need to identify novel therapeutic opportunities. Hypovolemic hypotension, coagulation dysfunction, disturbed microcirculation, and multiorgan failure resulting from vascular leakage are often observed in these severe conditions. In the present study, we find that HKH20, a peptide derived from human high molecular weight kininogen (HK), down-regulates inflammatory reactions caused by Streptococcus pyogenes in a mouse model of sepsis. HK is a component of the pro-inflammatory and pro-coagulant contact system. Activation of the contact system in the bloodstream by S pyogenes leads to massive tissue damage in the lungs of the infected mice, which eventually results in the death of the animals. HKH20 inhibits activation of the contact system and protects mice with invasive S pyogenes infection from lung damage. In combination with clindamycin treatment, the peptide also significantly prolongs the survival of infected mice.