Streptococcus pyogenes is an important human pathogen that selectively interacts with proteins involved in the humoral defense system, such as immunoglobulins and complement factors. In this report we show that S.pyogenes has the ability to hydrolyze the chitobiose core of the asparagine-linked glycan on immunoglobulin G (IgG) when bacteria are grown in the presence of human plasma. This activity is associated with the secretion of a novel 108 kDa protein denoted EndoS. EndoS has endoglycosidase activity on puri®ed soluble IgG as well as IgG bound to the bacterial surface. EndoS is required for the activity on IgG, as an isogenic EndoS mutant could not hydrolyze the glycan on IgG. In addition, we show that the secreted streptococcal cysteine proteinase SpeB cleaves IgG in the hinge region in a papain-like manner. This is the ®rst example of an endoglycosidase produced by a bacterial pathogen that selectively hydrolyzes human IgG, and reveals a novel mechanism which may contribute to S.pyogenes pathogenesis. Keywords: cysteine proteinase/endo-b-N-acetylglucosaminidase/endoglycosidase/IgG/Streptococcus pyogenes IntroductionAn enzyme expressed by Streptococcus pyogenes capable of releasing the terminal sialic acid residues from glycoproteins such as immunoglobulins was ®rst described by Hayano and Tanaka (1967). Other studies have shown that this release of sialic acid from immunoglobulin G (IgG) is due to a neuraminidase activity produced by S.pyogenes, resulting in autoantigenic and nephritogenic properties in patients with glomerulonephritis (Mosquera et al., 1985). Related endoglycosidases found in Staphylococcus aureus have been shown to be cluster-dispersing enzymes involved in cell separation (Sugai et al., 1995), and glycosidases in Streptococcus oralis that sequentially deglycosylate N-linked glycoproteins promote growth of the bacterium (Byers et al., 1999). It has also been suggested that a b-N-acetylglucosaminidase from Streptococcus pneumoniae contributes to pathogenicity, since N-linked sugar residues are common features of several cell-surface proteins in host tissue (Clarke et al., 1995).Oligosaccharide-cleaving enzymes from other species have been studied in detail, and some are standard tools in glycobiology research. For example, endoglycosidase H from Streptomyces plicatus (Trumbly et al., 1985), and endoglycosidases F 1 , F 2 and F 3 from Flavobacterium meningosepticum (Plummer and Tarentino, 1991;Trimble and Tarentino, 1991) are commonly used. These enzymes all belong to family 18 of chitinases (Henrissat, 1991) and catalyze the hydrolysis of the b-1,4-N-acetyl-D-glucosamine linkages in the chitin core of N-linked oligosaccharides in glycoproteins. These enzymes are referred to as true endoglycosidases (Alexander and Elder, 1989;Tarentino and Plummer, 1994). However, a hydrolase isolated from F.meningosepticum, peptide-N-(N-acetylb-glucosaminyl)asparagine amidase (PNGase F), has activity on the pentasaccharide core region of asparagine-linked glycans, and is therefore referred to as a deglycos...

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IgG antibodies are potent inducers of proinflammatory responses.During autoimmune diseases such as arthritis and systemic lupus erythematosus, IgG autoantibodies are responsible for the chronic inflammation and destruction of healthy tissues by cross-linking Fc receptors on innate immune effector cells. The sugar moiety attached to the asparagine-297 residue in the constant domain of the antibody is critical for the overall structure and function of the molecule. Removal of this sugar domain leads to the loss of the proinflammatory activity, suggesting that in vivo modulation of antibody glycosylation might be a strategy to interfere with autoimmune processes. In this work, we investigated whether removal of the majority of the IgG-associated sugar domain by endoglycosidase S (EndoS) from Streptococcus pyogenes is able to interfere with autoimmune inflammation. We demonstrate that EndoS injection efficiently removes the IgG-associated sugar domain in vivo and interferes with autoantibody-mediated proinflammatory processes in a variety of autoimmune models. Importantly, however, we observed a differential impact of EndoSmediated sugar side chain hydrolysis on IgG activity depending on the individual IgG subclass.autoantibody ͉ endoglycosidase ͉ Fc-receptor ͉ immunotherapy

Streptococcus pyogenes secretes a specific immunoglobulin G (IgG)-protease, SpeB, as well as the IgG glycan-hydrolyzing enzyme EndoS. Here we show that SpeB also degrades IgA, IgM, IgD, and IgE. We also show that EndoS only hydrolyzes the glycan moiety on native but not denatured IgG. Thus, SpeB has a broad immunoglobulin-degrading activity, while EndoS is highly specific for IgG.

Propionibacterium acnes is a common and probably underestimated cause of delayed joint prosthesis infection. Bacterial biofilm formation is central in the pathogenesis of infections related to foreign material, and P. acnes has been shown to form biofilm both in vitro and in vivo. Here, biofilm formation by 93 P. acnes isolates, either from invasive infections (n = 45) or from the skin of healthy people (n = 48), was analysed. The majority of isolates from deep infections produced biofilm in a microtitre model of biofilm formation, whereas the skin isolates were poor biofilm producers (p <0.001 for a difference). This indicates a role for biofilm formation in P. acnes virulence. The type distribution, as determined by sequencing of recA, was similar among isolates isolated from skin and from deep infections, demonstrating that P. acnes isolates with different genetic backgrounds have pathogenic potential. The biofilm formed on plastic and on bone cement was analysed by scanning electron microscopy (EM) and by transmission EM. The biofilm was seen as a 10-mum-thick layer covering the bacteria and was composed of filamentous as well as more amorphous structures. Interestingly, the presence of human plasma in solution or at the plastic surface inhibits biofilm formation, which could explain why P. acnes primarily infect plasma-poor environments of, for example, joint prostheses and cerebrospinal shunts. This work underlines the importance of biofilm formation in P. acnes pathogenesis, and shows that biofilm formation should be considered in the diagnosis and treatment of invasive P. acnes infections.