Oonagh Shannon

IgG glycan hydrolysis by a bacterial enzyme as a therapy against autoimmune conditions

EndoS from Streptococcus pyogenes efficiently hydrolyzes the functionally important and conserved N-linked glycan of IgG in human blood. Repeated i.v. administration of EndoS in rabbits completely hydrolyzes the glycans of the whole IgG pool, despite the generation of anti-EndoS antibodies. EndoS administration had no apparent effects on the health of the animals. EndoS hydrolysis of the IgG glycan has profound effects on IgG effector functions, such as complement activation and Fc receptor binding, suggesting that the enzyme could be used as an immunomodulatory therapeutic agent against IgG-mediated diseases. We demonstrate here that EndoS indeed has a protective effect in a mouse model of lethal IgG-driven immune (or idiopathic) thrombocytopenic purpura. EndoS pretreatment of pathogenic antibodies inhibits the development of disease, and the enzyme also rescues mice from already established disease when severe thrombocytopenia and s.c. bleeding have developed. These results identify EndoS as a potential therapeutic agent against diseases where pathogenic IgG antibodies are important and further emphasize antibody glycans as possible targets in future therapies against antibody-mediated autoimmune conditions.

IdeS: A Bacterial Proteolytic Enzyme with Therapeutic Potential

Background IdeS, a proteinase from Streptococcus pyogenes, cleaves immunoglobulin (Ig)G antibodies with a unique degree of specificity. Pathogenic IgG antibodies constitute an important clinical problem contributing to the pathogenesis of a number of autoimmune conditions and acute transplant rejection. To be able to effectively remove such antibodies is therefore an important clinical challenge. Methodology/Principal Findings IdeS was found to specifically and efficiently cleave IgG in human blood in vitro (20 µg of IdeS caused a complete degradation of IgG in one ml of human whole blood in 15 minutes) and to clear IgG from the blood stream of rabbits in vivo (no IgG was detected six hours following an intravenous injection of 5 mg of IdeS) without any side effects. In a mouse model of immune thrombocytopenic purpura (ITP), polyclonal IgG antibodies against platelet surface antigens were used to induce a lethal disease. These profoundly thrombocytopenic animals were treated and cured by a single injection of IdeS. Conclusions/Significance Novel information is provided concerning the IgG-cleaving activity of IdeS in vitro and in vivo. The highly specific and rapid elimination of IgG in vivo, the dramatic effect in a mouse model of ITP, and the lack of side effects in the treated animals, indicate that IdeS could also be used to treat IgG-driven diseases in humans.

Histidine-Rich Glycoprotein Protects from Systemic Candida Infection

Fungi, such as Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG), an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg−/− mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity.

Streptococcal M proteins and their role as virulence determinants.

Group A streptococci (GAS, Streptococcus pyogenes) are exclusive human pathogens that have been extensively studied for many decades. The spectrum of diseases caused by these bacteria ranges from uncomplicated and superficial to severe and invasive infections. In order to give rise to these complications, GAS have evolved a number of surface-bound and secreted virulence factors, of which the M proteins are probably the best characterized. Evidence has emerged that M proteins are multifunctional pathogenic determinants, and over the years many interactions between M proteins and the human host have been reported. The present review article aims to present a state-of-the-art overview of the most important virulence mechanisms employed by M proteins to trigger disease.

Severe streptococcal infection is associated with M protein-induced platelet activation and thrombus formation

Disturbed haemostasis is a central finding in severe Streptococcus pyogenes infection. In particular, microthrombi are found both at the local site of infection and at distant sites. Platelets are responsible for maintaining vascular function and haemostasis. We report here that M1 protein of S. pyogenes triggers immune‐mediated platelet activation and thrombus formation. M1 protein is released from the bacterial surface and forms complexes with plasma fibrinogen. These complexes bind to the fibrinogen receptor on resting platelets. When these complexes also contain immunoglobulin G (IgG) against M1 protein, this will engage the Fc receptor on the platelets and activation will occur. Activation of the platelets leads to platelet aggregation and the generation of platelet‐rich thrombi. Neutrophils and monocytes are in turn activated by the platelets. Platelet thrombi are deposited in the microvasculature, and aggregated platelets, IgG and M1 protein colocalize in biopsies from patients diagnosed with S. pyogenes toxic shock syndrome. This chain of events results in a pro‐coagulant and pro‐inflammatory state typical of severe S. pyogenes infection.

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.

Platelet and Neutrophil Responses to Gram Positive Pathogens in Patients with Bacteremic Infection

Background Many Gram-positive pathogens aggregate and activate platelets in vitro and this has been proposed to contribute to virulence. Platelets can also form complexes with neutrophils but little is however known about platelet and platelet-neutrophil responses in bacterial infection. Methodology/Principal Findings We added isolates of Gram-positive bacteria from 38 patients with a bacteremic infection to blood drawn from the same patient. Aggregometry and flow cytometry were used to assess platelet aggregation and to quantify activation of platelets, neutrophils, and platelet-neutrophils complexes (PNCs) induced by the bacteria. Fifteen healthy persons served as controls. Most isolates of Staphylococcus aureus, beta hemolytic streptococci, and Enterococcus faecalis induced aggregation of platelets from their respective hosts, whereas pneumococci failed to do so. S. aureus isolates induced platelet aggregation more rapidly in patients than in controls, whereas platelet activation by S. aureus was lower in patients than in controls. PNCs were more abundant in baseline samples from patients than in healthy controls and most bacterial isolates induced additional PNC formation and neutrophil activation. Conclusion/Significance We have demonstrated for the first time that bacteria isolated from patients with Gram-positive bacteremia can induce platelet activation and aggregation, PNC formation, and neutrophil activation in the same infected host. This underlines the significance of these interactions during infection, which could be a target for future therapies in sepsis.

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.

Protein C Inhibitor-A Novel Antimicrobial Agent

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor belonging to the family of serpin proteins. Here we describe that PCI exerts broad antimicrobial activity against bacterial pathogens. This ability is mediated by the interaction of PCI with lipid membranes, which subsequently leads to their permeabilization. As shown by negative staining electron microscopy, treatment of Escherichia coli or Streptococcus pyogenes bacteria with PCI triggers membrane disruption followed by the efflux of bacterial cytosolic contents and bacterial killing. The antimicrobial activity of PCI is located to the heparin-binding site of the protein and a peptide spanning this region was found to mimic the antimicrobial activity of PCI, without causing lysis or membrane destruction of eukaryotic cells. Finally, we show that platelets can assemble PCI on their surface upon activation. As platelets are recruited to the site of a bacterial infection, these results may explain our finding that PCI levels are increased in tissue biopsies from patients suffering from necrotizing fasciitis caused by S. pyogenes. Taken together, our data describe a new function for PCI in innate immunity.

Antibodies against a Surface Protein of Streptococcus pyogenes Promote a Pathological Inflammatory Response

Streptococcal toxic shock syndrome (STSS) caused by Streptococcus pyogenes is a clinical condition with a high mortality rate despite modern intensive care. A key feature of STSS is excessive plasma leakage leading to hypovolemic hypotension, disturbed microcirculation and multiorgan failure. Previous work has identified a virulence mechanism in STSS where M1 protein of S. pyogenes forms complexes with fibrinogen that activate neutrophils to release heparin-binding protein (HBP), an inducer of vascular leakage. Here, we report a marked inter-individual difference in the response to M1 protein–induced HBP release, a difference found to be related to IgG antibodies directed against the central region of the M1 protein. To elicit massive HBP release, such antibodies need to be part of the M1 protein–fibrinogen complexes. The data add a novel aspect to bacterial pathogenesis where antibodies contribute to the severity of disease by promoting a pathologic inflammatory response.