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.

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.

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.

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.

Finegoldia magna, an Anaerobic Gram-Positive Bacterium of the Normal Human Microbiota, Induces Inflammation by Activating Neutrophils.

The Gram-positive anaerobic commensal Finegoldia magna colonizes the skin and other non-sterile body surfaces, and is an important opportunistic pathogen. Here we analyzed the effect of F. magna on human primary neutrophils. F. magna strains ALB8 (expressing protein FAF), 312 (expressing protein L) and 505 (naturally lacking both protein FAF and L) as well as their associated proteins activate neutrophils to release reactive oxygen species, an indication for neutrophil oxidative burst. Co-incubation of neutrophils with the bacteria leads to a strong increase of CD66b surface expression, another indicator for neutrophil activation. Furthermore, all tested stimuli triggered the release of NETs from the activated neutrophils, pointing to a host defense mechanism in response to the tested stimuli. This phenotype is dependent on actin rearrangement, NADPH oxidases and the ERK1/2 pathway. Proteins FAF and L also induced the secretion of several pro-inflammatory neutrophil proteins; HBP, IL-8 and INFγ. This study shows for the first time a direct interaction of F. magna with human neutrophils and suggests that the activation of neutrophils plays a role in F. magna pathogenesis.

Streptococcal Enzymes as Precision Tools Against Pathogenic IgG Autoantibodies in Small Vessel Vasculitis.

In primary systemic small vessel vasculitis autoantibodies are common and seem to play an important role in the pathogenesis. Autoantibodies in vasculitis are preferentially directed against components of the immune system or directly against components of the vessel wall. Plasmapheresis is often applied in emergency situationists when the function of vital organs is jeopardized, the level of clinical evidence to apply such therapy, however, varies between low and non-existing. Plasmapheresis is a blunt and unspecific instrument that requires several sessions to achieve a substantial reduction of autoantibody levels. IdeS and EndoS are two relatively recently discovered enzymes produced by S. pyogenes, that have a remarkable capacity to degrade and disarm IgG. They have shown positive results in several in vivo models of autoimmunity, and treatment with IdeS has successfully been used to inactivate HLA alloantibodies in patients undergoing renal transplantation. Both IdeS and EndoS have the potential to become precision tools to replace plasmapheresis in the treatment of vasculitic emergencies and a clinical trial of IdeS in anti-GBM vasculitis is now ongoing.

The Molecular Basis of Human IgG-Mediated Enhancement of C4b-Binding Protein Recruitment to Group A Streptococcus.

Streptococcus pyogenes infects over 700 million people worldwide annually. Immune evasion strategies employed by the bacteria include binding of the complement inhibitors, C4b-binding protein (C4BP) and Factor H in a human-specific manner. We recently showed that human IgG increased C4BP binding to the bacterial surface, which promoted streptococcal immune evasion and increased mortality in mice. We sought to identify how IgG promotes C4BP binding to Protein H, a member of the M protein family. Dimerization of Protein H is pivotal for enhanced binding to human C4BP. First, we illustrated that Protein H, IgG, and C4BP formed a tripartite complex. Second, surface plasmon resonance revealed that Protein H binds IgG solely through Fc, but not Fab domains, and with high affinity (IgG-Protein H: KD = 0.4 nM; IgG-Fc-Protein H: KD ≤ 1.6 nM). Each IgG binds two Protein H molecules, while up to six molecules of Protein H bind one C4BP molecule. Third, interrupting Protein H dimerization either by raising temperature to 41°C or with a synthetic peptide prevented IgG-Protein H interactions. IgG-Fc fragments or monoclonal human IgG permitted maximal C4BP binding when used at concentrations from 0.1 to 10 mg/ml. In contrast, pooled human IgG enhanced C4BP binding at concentrations up to 1 mg/ml; decreased C4BP binding at 10 mg/ml occurred probably because of Fab-streptococcal interactions at these high IgG concentrations. Taken together, our data show how S. pyogenes exploits human IgG to evade complement and enhance its virulence. Elucidation of this mechanism could aid design of new therapeutics against S. pyogenes.

A quantitative Streptococcus pyogenes-human protein-protein interaction map reveals localization of opsonizing antibodies.

A fundamental challenge in medical microbiology is to characterize the dynamic protein-protein interaction networks formed at the host-pathogen interface. Here, we generate a quantitative interaction map between the significant human pathogen, Streptococcus pyogenes, and proteins from human saliva and plasma obtained via complementary affinity-purification and bacterial-surface centered enrichment strategies and quantitative mass spectrometry. Perturbation of the network using immunoglobulin protease cleavage, mixtures of different concentrations of saliva and plasma, and different S. pyogenes serotypes and their isogenic mutants, reveals how changing microenvironments alter the interconnectivity of the interaction map. The importance of host immunoglobulins for the interaction with human complement proteins is demonstrated and potential protective epitopes of importance for phagocytosis of S. pyogenes cells are localized. The interaction map confirms several previously described protein-protein interactions; however, it also reveals a multitude of additional interactions, with possible implications for host-pathogen interactions involving other bacterial species.