EndoS from

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.

SummaryDisturbed 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 procoagulant and pro-inflammatory state typical of severe S. pyogenes infection.

BackgroundIdeS, 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 FindingsIdeS 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/SignificanceNovel 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.

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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. (Blood. 2010;116(13):2365-2372) IntroductionHistidine-rich glycoprotein (HRG) is an abundant plasma protein synthesized in the liver and also stored in the ␣-granules of platelets, from which it is released on activation. 1 The protein has a diverse array of ligands, including heparin, plasminogen, immunoglobulin, thrombospondin, and fibrinogen. 2 The diversity of ligands probably contributes to the fact that new activities are regularly attributed to HRG in vitro, whereas a definitive biologic function in vivo has been difficult to identify. Based on in vitro studies, HRG has been reported to modulate angiogenesis, 3 phagocytosis, 4 and complement function 5 and to be antibacterial. 6 HRG is negatively charged at physiologic pH but becomes positively charged at low pH or in the presence of zinc. Several the reported biologic effects have also been shown to be dependent on pH or zinc, suggesting that HRG may be a sensitive adaptor molecule to changing environments. 7 The generation of HRG-knockout mice has facilitated the search for the biologic function of HRG. These animals were viable with no apparent abnormalities, but ex vivo studies demonstrated a mild dysregulation of hemostasis with effects on plasma-clotting factors and platelets, and in vivo HRG seemed to have both anticoagulant and antifibrinolytic properties. 8 Recently, it has been shown that HRG-deficient animals have diminished antifungal activity in vivo. 9 In the present study, we have assessed the role of HRG in response to a pathologic situation (ie, bacterial infection). Streptococcus pyogenes is a significant human bacterial pathogen responsible for both superficial and invasive infection. The pathogenesis of S pyogenes infection is a complex multifactorial process, and diverse host systems are involved. [10][11][12] For instance, S pyogenes can counteract phagocytosis, complement activity, antibody recognition, and antimicrobial peptides. S pyogenes infection al...

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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.