| 1. |
- Lindh, Tova, et al.
(författare)
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Expression of the Bacterial Enzyme IdeS Using a GFP Fusion in the Yeast Saccharomyces cerevisiae
- 2023. - 2nd
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Ingår i: Bacterial pathogenesis : Methods and protocols - Methods and protocols. - 1940-6029. - 9781071632437 ; , s. 131-146
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Bokkapitel (refereegranskat)abstract
- Bacterial proteases are important enzymes used in several technical applications where controlled cleavage of proteins is needed. They are challenging enzymes to express recombinantly as parts of the proteome can be hydrolyzed by their activity. The eukaryotic model organism Saccharomyces cerevisiae is potentially a good expression host as it tolerates several stress conditions and is known to better express insoluble proteins compared to bacterial systems. In this chapter we describe how the protease IdeS from Streptococcus pyogenes can be expressed in S. cerevisiae. The expression of IdeS was followed by constructing a fused protein with GFP and measuring the fluorescence with flow cytometry. The protease presence was confirmed with a Western blot assay and activity was measured with an in vitro assay. To reduce potentially toxic effect on the host cell, the growth and production phases were separated by using the inducible promoter GAL1p to control recombinant gene expression. The protocol provided may be adopted for other bacterial proteases through minor modifications of the fused protein.
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| 2. |
- Nordenfelt, Pontus, et al.
(författare)
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Preface
- 2023. - 2nd
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Ingår i: Bacterial Pathogenesis - Methods and Protocols. - 1064-3745. - 9781493966738 ; 2674
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 3. |
- Albert, Heike, et al.
(författare)
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In vivo enzymatic modulation of IgG glycosylation inhibits autoimmune disease in an IgG subclass-dependent manner
- 2008
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 105:39, s. 15005-15009
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Tidskriftsartikel (refereegranskat)abstract
- 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 EndoS-mediated sugar side chain hydrolysis on IgG activity depending on the individual IgG subclass.
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| 4. |
- Allhorn, Maria, et al.
(författare)
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EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity
- 2008
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: BACKGROUND: The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. RESULTS: We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. CONCLUSIONS: We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.
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| 5. |
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| 6. |
- Allhorn, Maria, et al.
(författare)
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Human IgG/FcgammaR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis.
- 2008
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcgammaR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcgammaR and the Fc domain of IgG depend on the IgG glycosylation state. METHODOLOGY/PRINCIPAL FINDINGS: Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcgammaR and to an erythroleukemic cell line, K562, expressing FcgammaRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcgammaRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot. CONCLUSIONS/SIGNIFICANCE: We provide novel information about bacterial enzymatic modulation of the IgG/FcgammaR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes.
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| 7. |
- Allhorn, Maria, et al.
(författare)
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Sugar-free antibodies--the bacterial solution to autoimmunity?
- 2009
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Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923. ; 1173, s. 664-669
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme EndoS from Streptococcus pyogenes is an immunomodulatory molecule hydrolyzing the conserved glycans in the effector part of immunoglobulin G (IgG). EndoS is remarkably specific for IgG, and hydrolysis has profound effects on IgG effector functions. EndoS pretreatment of IgG, or direct administration to animals with experimental antibody-mediated autoimmune diseases, inhibits development of disease or cures animals from established disease. The properties of EndoS make it a unique experimental tool and an attractive alternative to current therapies of conditions involving pathogenic antibodies. This review describes the discovery of EndoS, the effects of EndoS on IgG effector functions in vitro and in vivo, the biotechnological potential of EndoS, and the outcomes of EndoS treatment in animal models of autoimmunity.
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| 8. |
- Allhorn, Maria, et al.
(författare)
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The IgG specific endoglycosidase EndoS inhibits both cellular and complement mediated autoimmune hemolysis.
- 2010
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Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 115:24, s. 5080-5088
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Tidskriftsartikel (refereegranskat)abstract
- EndoS from Streptococcus pyogenes is an immunomodulating enzyme that specifically hydrolyzes glycans from human IgG and thereby affects antibody effector functions. Autoimmune hemolytic anemia is caused by antibody mediated red blood cell (RBC) destruction and often resists treatment with corticosteroids that also cause frequent adverse effects. We show here that anti-RhD (anti-D) and rabbit anti-human-RBC antibodies (anti-RBC) mediated destruction of RBC, i.e. phagocytosis, complement activation and hemolysis in vitro and in vivo was inhibited by EndoS. Phagocytosis by monocytes in vitro was inhibited by pre-treatment of anti-D with EndoS before sensitization of RBC, and abrogated by direct addition of EndoS to blood containing sensitized RBC. The toxic effects of monocytes stimulated with anti-D-sensitized RBC, as measured by interleukin-8 secretion and oxygen metabolite production, was restrained by EndoS. Agglutination of RBC and complement mediated hemolysis in vitro in whole human blood caused by rabbit anti-RBC was inhibited by EndoS. Development of anemia in mice caused by a murine anti-RBC IgG2a monoclonal autoantibody, and complement activation and erythrophagocytosis by Kupffer cells in the liver, were reduced by EndoS. Our data indicate that EndoS is a potential therapeutic agent that might be evaluated as an alternative to current treatment regimens against antibody mediated destruction of RBC.
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| 9. |
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Bacterial Pathogenesis : Methods and Protocols
- 2017. - 1
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Samlingsverk (redaktörskap) (övrigt vetenskapligt/konstnärligt)abstract
- This volume discusses various methods and protocols used for the experimentation of a wide range of bacterial species, such as Streptococcus pyogenes, Staphylococcus aureus, Streptococcus pneumonia, Listeria monocytogenes, and Mycobacterium marinum. Bacterial Pathogens: Methods and Protocols is divided into 6 parts: Part 1 describes different approaches to identifying and characterizing bacterial effector molecules; Part 2 deals with structural biology of bacterial pathogenesis and how to overcome folding and stability problems with recombinantly expressed proteins; Part 3 details methodology that identifies bacteria in complex communities and how genomes of bacterial pathogens have evolved; Part 4 reflects on the rapid development of advanced imaging techniques that address questions about molecular properties of individual live bacteria, ultrastructure of surfaces, and subcellular localization of bacterial proteins; Part 5 describes methods from in vitro and in vivo modeling of bacterial infections; and Part 6 explores how bacterial pathogens are the true experts of the immune system. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.Cutting-edge and comprehensive, Bacterial Pathogens: Methods and Protocols is a valuable resource for anyone who is interested in this fascinating and evolving field.
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| 10. |
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Bacterial Pathogenesis : Methods and Protocols
- 2023. - 2
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Samlingsverk (redaktörskap) (refereegranskat)abstract
- This detailed volume presents a diverse set of methodological approaches designed to improve our understanding of bacterial infections from a wide range of bacterial species. Beginning with biofilms and subcellular compartments, the book explores transcriptional analysis, methods for studying plasmid dynamics, and tools for phylogenetic analysis of bacterial genomes, as well as bacterial effector proteins interfering with host systems, host response analysis, and in vivo and in vitro infection models. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Bacterial Pathogenesis: Methods and Protocols, Second Edition is a vital resource for researchers in the area of infection biology, as well as but not limited to, those working in the fields of microbiology, immunology, structural biology, molecular biology, genetics, imaging, and computational study.
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