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.

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.

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.

Group G streptococci mediate fibrinogen-dependent platelet aggregation leading to transient entrapment in platelet aggregates.

Platelets have been reported to become activated in response to bacteria and this is proposed to contribute to the acute response to bacterial infection. In the present study, we investigated platelet aggregation in response to group G streptococci (GGS) in vitro in healthy human donors and in vivo in a mouse model of streptococcal sepsis. Platelet aggregation by GGS was dependent on the bacterial surface protein FOG and engagement of the platelet fibrinogen receptor; however, it was independent of IgG and the platelet Fc receptor. Platelets exerted no antibacterial effects on the bacteria, and aggregates formed were markedly unstable, allowing bacteria to rapidly return to the plasma and grow post-aggregation. Thrombocytopenia and platelet activation occurred during invasive infection with GGS, and platelets were demonstrated to contribute to bacterial dissemination during infection. These findings reveal an important role for bacteria-platelet interactions during the pathogenesis of streptococcal infection.

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.

Identification of pili on the surface of Finegoldia magna--a gram-positive anaerobic cocci.

Pili have only been discovered in the major Gram-positive pathogens in the past decade and they have been found to play an important role in colonisation and virulence. Pili have been shown to have many important functions including attachment to host tissues, mediating bacterial aggregation, biofilm formation and binding to proteins in the extracellular matrix. In this study, sortase-dependent pili have been found to be expressed on the surface of Finegoldia magna ALB8. F. magna is a Gram-positive anaerobic coccus that, primarily, is a commensal of the skin and mucous membranes, but has also been isolated from various clinical infection sites and is associated with soft-tissue abscesses, wound infections and bone and prosthetic joint infections. In this study, F. magna ALB8 was found to harbour three sortases at the pilus locus, two of which bear high similarity to class C sortases in Streptococcus pneumoniae. Two putative sortase-dependent pili proteins were found in the locus, with one being identified as the major pilus subunit, Fmp1 (F. magna pilus subunit 1), due to its high similarity to other major pilus proteins in prominent Gram-positive pathogens. The presence of sortase-dependent pili was confirmed experimentally through recombinant production of Fmp1 and production of antiserum. The Fmp1 antiserum was used in Western blot to show the presence of a high molecular weight protein ladder, characteristic of the presence of pili, in trypsin released cell wall surface proteins from F. magna. The presence of sortase-dependent pili was visually confirmed by transmission electron microscopy, which showed the binding of gold labelled anti-Fmp1 to individual pilus proteins along the pilus. Furthermore, pili could also be found to bind and interact with keratinocytes in the epidermal layer of human skin, suggesting an adhesive role for pili on F. magna. Our work represents the first description of pilus structures in F. magna. This discovery further elucidates F. magna physiology and allows for additional analysis of host-bacterial interactions in future studies.

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.

Constitutive and inflammation-dependent antimicrobial peptides produced by epithelium are differentially processed and inactivated by the commensal Finegoldia magna and the pathogen Streptococcus pyogenes.

Epithelial linings serve as physical barriers and produce antimicrobial peptides (AMPs) to maintain host integrity. Examples are the bactericidal proteins midkine (MK) and BRAK/CXCL14 that are constitutively produced in the skin epidermal layer, where the anaerobic Gram-positive coccoid commensal Finegoldia magna resides. Consequently, this bacterium is likely to encounter both MK and BRAK/CXCL14, making these molecules possible threats to its habitat. In this study, we show that MK expression is upregulated during inflammation, concomitant with a strong downregulation of BRAK/CXCL14, resulting in changed antibacterial conditions. MK, BRAK/CXCL14, and the inflammation-dependent antimicrobial ß-defensins human ß-defensin (hBD)-2 and hBD-3 all showed bactericidal activity against both F. magna and the virulent pathogen Streptococcus pyogenes at similar concentrations. SufA, a released protease of F. magna, degraded MK and BRAK/CXCL14 but not hBD-2 nor hBD-3. Cleavage was seen at lysine and arginine residues, amino acids characteristic of AMPs. Intermediate SufA-degraded fragments of MK and BRAK/CXCL14 showed stronger bactericidal activity against S. pyogenes than F. magna, thus promoting survival of the latter. In contrast, the cysteine-protease SpeB of S. pyogenes rapidly degraded all AMPs investigated. The proteins FAF and SIC, released by F. magna and S. pyogenes, respectively, neutralized the antibacterial activity of MK and BRAK/CXCL14, protein FAF being the most efficient. Quantitation and colocalization by immunoelectron microscopy demonstrated significant levels and interactions of the molecules in in vivo and ex vivo samples. The findings reflect strategies used by a permanently residing commensal and a virulent pathogen, the latter operating during the limited time course of invasive disease.

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.

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.

Interaction of Bacteroides fragilis and Bacteroides thetaiotaomicron with the kallikrein-kinin system.

Many bacterial pathogens interfere with the contact system (kallikrein-kinin system) in human plasma. Activation of this system has two consequences: cleavage of high-molecular-mass kininogen (HK) resulting in release of the potent proinflammatory peptide bradykinin, and initiation of the intrinsic pathway of coagulation. In this study, two species of the Gram-negative anaerobic commensal organism Bacteroides, namely Bacteroides fragilis and Bacteroides thetaiotaomicron, were found to bind HK and fibrinogen, the major clotting protein, from human plasma as shown by immunoelectron microscopy and Western blot analysis. In addition, these Bacteroides species were capable of activating the contact system at its surface leading to a significant prolongation of the intrinsic coagulation time and also to the release of bradykinin. Members of the genus Bacteroides have been known to act as opportunistic pathogens outside the gut, with B. fragilis being the most common isolate from clinical infections, such as intra-abdominal abscesses and bacteraemia. The present results thus provide more insight into how Bacteroides species cause infection.

Streptococcal protein SIC activates monocytes and induces inflammation.

Streptococcus pyogenes is a major bacterial pathogen in the human population and isolates of the clinically important M1 serotype secrete protein Streptococcal inhibitor of complement (SIC) known to interfere with human innate immunity. Here we find that SIC from M1 bacteria interacts with TLR2 and CD14 on monocytes leading to the activation of the NF-κB and p38 MAPK pathways and the release of several pro-inflammatory cytokines (e.g. TNFα and INFγ). In human plasma, SIC binds clusterin and histidine-rich glycoprotein, and whole plasma, and these two purified plasma proteins enhanced the activation of monocytes by SIC. Isolates of the M55 serotype secrete an SIC homolog, but this protein did not activate monocytes. M1 isolates are common in cases of invasive S. pyogenes infections characterized by massive inflammation, and the results of this study indicate that the pro-inflammatory property of SIC contributes to the pathology of these severe clinical conditions.

Lack of Opsonic Antibody Responses to Invasive Infections With Streptococcus dysgalactiae.

INTRODUCTION: Streptococcus dysgalactiae can cause severe recurrent infections. This study aimed to investigate antibody responses following S. dysgalactiae bacteraemia and possible development of protective immunity. MATERIALS AND METHODS: Patients with S. dysgalactiae bacteraemia in the county of Skåne between 2017 and 2018 were prospectively included. Acute and convalescent sera were obtained. All isolates were emm typed and enzyme-linked immunosorbent assay (ELISA) was utilised to analyse specific antibody responses to bacteria and antigens. Bactericidal- and phagocytosis assays were applied to further establish antibody function. RESULTS: Sixteen patients with S. dysgalactiae bacteraemia were included of whom one had recurrent episodes of bacteraemia. Using ELISA with S. dysgalactiae isolates and mutants, development of IgG antibodies was demonstrated in few patients. Type-specific antibodies were demonstrated in one patient when recombinant M proteins as antigens, were applied. The type-specific serum mediated a small increase in phagocytosis but did not facilitate increased killing of the S. dysgalactiae isolate, carrying that M protein, in blood or by phagocytic cells. CONCLUSION: S. dysgalactiae bacteraemia sometimes results in increased levels of antibodies to the infecting pathogen. We did not find evidence that these antibodies are effectively opsonising. Apparent failure to produce opsonising antibodies might partially explain why S. dysgalactiae can cause recurrent invasive infections in the same host.

Vaccine-induced, but not natural immunity, against the Streptococcal inhibitor of complement protects against invasive disease.

Highly pathogenic emm1 Streptococcus pyogenes strains secrete the multidomain Streptococcal inhibitor of complement (SIC) that binds and inactivates components of the innate immune response. We aimed to determine if naturally occurring or vaccine-induced antibodies to SIC are protective against invasive S. pyogenes infection. Immunisation with full-length SIC protected mice against systemic bacterial dissemination following intranasal or intramuscular infection with emm1 S. pyogenes. Vaccine-induced rabbit anti-SIC antibodies, but not naturally occurring human anti-SIC antibodies, enhanced bacterial clearance in an ex vivo whole-blood assay. SIC vaccination of both mice and rabbits resulted in antibody recognition of all domains of SIC, whereas naturally occurring human anti-SIC antibodies recognised the proline-rich region of SIC only. We, therefore, propose a model whereby natural infection with S. pyogenes generates non-protective antibodies against the proline-rich region of SIC, while vaccination with full-length SIC permits the development of protective antibodies against all SIC domains.

SufA of the opportunistic pathogen finegoldia magna modulates actions of the antibacterial chemokine MIG/CXCL9, promoting bacterial survival during epithelial inflammation.

The anaerobic bacterium Finegoldia magna is part of the human commensal microbiota, but is also an important opportunistic pathogen. This bacterium expresses a subtilisin-like serine proteinase, SufA, which partially degrade the antibacterial chemokine MIG/CXCL9. Here, we show that MIG/CXCL9 is produced by human keratinocytes in response to inflammatory stimuli. In contrast to the virulent human pathogen Streptococcus pyogenes, the presence of F. magna had no enhancing effect on the MIG/CXCL9 expression by keratinocytes, suggesting poor detection of the latter by pathogen-recognition receptors. When MIG/CXCL9 was exposed to SufA-expressing F. magna, the molecule was processed into several smaller fragments. Analysis by mass spectrometry showed that SufA cleaves MIG/CXCL9 at several sites in the COOH-terminal region of the molecule. At equimolar concentrations, SufA-generated MIG/CXCL9 fragments were not bactericidal against F. magna, but retained their ability to kill S. pyogenes. Moreover, the SufA-generated MIG/CXCL9 fragments were capable of activating the angiostasis-mediating CXCR3 receptor, which is expressed on endothelial cells, in an order of magnitude similar to that of intact MIG/CXCL9. F. magna expresses a surface protein called FAF that is released from the bacterial surface by SufA. Soluble FAF was found to bind and inactivate the antibacterial activity of MIG/CXCL9, thereby further potentially promoting the survival of F. magna. The findings suggest that SufA modulation of the inflammatory response could be a mechanism playing an important role in creating an ecologic niche for F. magna, decreasing antibacterial activity and suppressing angiogenesis, thus providing advantage in survival for this anaerobic opportunist compared with competing pathogens during inflammation.