| 1. |
- Bratanis, Eleni, et al.
(author)
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A novel broad-spectrum elastase-like serine protease from the predatory bacterium bdellovibrio bacteriovorus facilitates elucidation of site-specific IgA glycosylation pattern
- 2019
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In: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10:MAY
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Journal article (peer-reviewed)abstract
- The increased interest in predatory bacteria due to their ability to kill antibiotic resistant bacteria has also highlighted their inherent plethora of hydrolytic enzymes, and their potential as natural sources of novel therapeutic agents and biotechnological tools. Here, we have identified and characterized a novel protease from the predatory bacterium Bdellovibrio bacteriovorus: BspE (Bdellovibrio elastase-like serine protease). Mapping preferential sites of proteolytic activity showed a single proteolytic cleavage site of native plasma IgA (pIgA) in the Fc-tail; as well as in the secretory component (SC) of secretory IgA (SIgA). Proteolysis of other native immunoglobulins and plasma proteins was either absent (IgG1 and 2, IgM, albumin and orosomucoid) or unspecific with multiple cleavage sites (IgG3 and 4, IgE, IgD). BspE displayed a broad activity against most amino acid bonds in shorter peptides and denatured proteins, with a slight preference for hydrolysis C-terminal of Y, V, F, S, L, R, P, E, and K. BspE autoproteolysis results in numerous cleavage products sustaining activity for more than 6 h. The enzymatic activity remained stable at pH 5.0-9.0 but was drastically reduced in the presence of MnCl2 and completely inhibited by ZnCl2. The hydrolysis of pIgA was subsequently utilized for the specific glycan characterization of the released pIgA Fc-tail (Asn459). Besides contributing to the basic knowledge of Bdellovibrio biology and proteases, we propose that BspE could be used as a potential tool to investigate the importance, and biological function of the pIgA Fc-tail.
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| 2. |
- Bratanis, Eleni
(author)
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Bacterial antibody hydrolyzing enzymes – as bacterial virulence factors and biotechnological tools
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Antibodies are an essential part of the human immune system, and antibody mediated immunity has been an area of interest for many researchers for almost a century. An accumulation of knowledge regarding antibody structure, glycosylation and receptor interactions has contributed to the current understanding of antibody mediated immunity. It has more recently become evident how bacteria and other microorganisms evade host recognition and eradication through specific antibody degradation or modification. The importance of antibody glycosylation and how glycan modification can fine-tune the elicited immune response has also contributed to the development of antibody-based drugs with improved clinical efficacy. In turn these insights have paved the way and created a need for the development of biotechnological methods and tools to specifically engineer antibodies with defined properties, for analysis to ensure quality and safety, and for improved antibody purification.This thesis highlights the importance of glycosylation for antibody function and presents different aspects and applications of antibody modifications by bacteria. We show, for the first time, activity of the IgG-specific Streptococcal endoglycosidase EndoS during Streptococcus pyogenes infection, clearly demonstrating that EndoS contributes to S. pyogenes pathogenesis and bacterial survival in the context of adaptive immunity. Further this thesis presents the use of bacterial enzymes as antibody modifying tools and their potential as binding reagents for selective antibody purification. The identification and characterization of two novel proteases, BspK and BspE exhibiting unique IgG and IgA cleavage profiles respectively, from Bdellovibrio bacteriovorus highlights the potential of using Bdellovibrio as a source for the identification of novel enzymes with biotechnological applications. Finally, I present the development of a novel method for selective antibody purification, using the inactive variants of the bacterial enzymes EndoS and EndoS2, ensuring the purification of native, correctly folded and modified antibodies.
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| 3. |
- Nägeli, Andreas, et al.
(author)
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Streptococcus pyogenes evades adaptive immunity through specific IgG glycan hydrolysis
- 2019
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In: Journal of Experimental Medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 216:7, s. 1615-1629
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Journal article (peer-reviewed)abstract
- Streptococcus pyogenes (Group A streptococcus; GAS) is a human pathogen causing diseases from uncomplicated tonsillitis to life-threatening invasive infections. GAS secretes EndoS, an endoglycosidase that specifically cleaves the conserved N-glycan on IgG antibodies. In vitro, removal of this glycan impairs IgG effector functions, but its relevance to GAS infection in vivo is unclear. Using targeted mass spectrometry, we characterized the effects of EndoS on host IgG glycosylation during the course of infections in humans. Substantial IgG glycan hydrolysis occurred at the site of infection and systemically in the severe cases. We demonstrated decreased resistance to phagocytic killing of GAS lacking EndoS in vitro and decreased virulence in a mouse model of invasive infection. This is the first described example of specific bacterial IgG glycan hydrolysis during infection and thereby verifies the hypothesis that EndoS modifies antibodies in vivo. This mechanisms of immune evasion could have implications for treatment of severe GAS infections and for future efforts at vaccine development.
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