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Studies on secreted cysteine proteases of Streptococcus pyogenes: IdeS and SpeB
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Ulrich von Pawel-Rammingen)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The pathogen Streptococcus pyogenes is a significant cause of human morbidity and mortality. Most of the work in this thesis is focused on streptococcal virulence factor IdeS, but the thesis also features work on SpeB, another streptococcal virulence factor. Both IdeS and SpeB are secreted cysteine proteases and both have previously been shown to degrade human IgG. IgG is the only known substrate for IdeS while SpeB is a more promiscuous protease with a larger number of identified substrates. A significant part of the data presented in this thesis is the result of designing and optimizing methods to detect and accurately measure the proteolytic degradation of IgG. Methods aimed at measuring the binding interactions between enzyme and substrate have also been frequently utilized. I show that IdeS is a monomeric protease, as opposed to previously published data that suggested it to be dimeric. IdeS cleaves the two heavy chains of IgG in a two-step reaction and I demonstrate that the first cleavage is magnitudes faster than the second one. This means that IdeS is a more efficient enzyme than previously thought. The difference in rate cannot completely be explained by a loss of affinity between IdeS and IgG after the cleavage of the first heavy chain. The velocity of IdeS is further increased by the presence of human Cystatin C, via an unknown mechanism. Cystatin C is normally a protease inhibitor and it having an opposite effect is puzzling.The synthesis and evaluation of novel inhibitors are also described. Peptide analogues mimicking the sequence surrounding the scissile bond on IgG - with an amino acid replaced with a more rigid motif - act as specific, but low-affinity, inhibitors of IdeS. The peptide analogues’ inhibitory capacity for SpeB and papain was also assayed.When it comes to SpeB, I show that it does not have IgG as a substrate under physiological conditions, in contrast to what was previously thought. This thesis does not only present findings on the IgG degrading capacity of IdeS and SpeB but also include data on fundamental enzymatic properties for these proteases.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2014. , 49 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1646
Keyword [en]
Streptococcus pyogenes, Group A Streptococci, GAS, virulence factor, IdeS, SpeB, cysteine protease, protease inhibitor, IgG, immune evasion
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
URN: urn:nbn:se:umu:diva-88223ISBN: 978-91-7601-048-8 (print)OAI: oai:DiVA.org:umu-88223DiVA: diva2:714455
Public defence
2014-05-27, Sal E04, by 6E, Norrlands universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2014-05-06 Created: 2014-04-28 Last updated: 2014-05-06Bibliographically approved
List of papers
1. Rapid IgG heavy chain cleavage by the streptococcal IgG endopeptidase IdeS is mediated by IdeS monomers and is not due to enzyme dimerization
Open this publication in new window or tab >>Rapid IgG heavy chain cleavage by the streptococcal IgG endopeptidase IdeS is mediated by IdeS monomers and is not due to enzyme dimerization
2013 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, no 12, 1818-1822 p.Article in journal (Refereed) Published
Abstract [en]

Streptococcus pyogenes employs an IgG specific endopeptidase, IdeS, to counteract the effector functions of specific IgG. The physiological significant step in disarming specific IgG is the cleavage of one IgG heavy chain. So far, characterizations of IdeS enzymatic activity have employed techniques that failed to differentiate between the first and the second cleavage step. The present data demonstrate that IdeS is active as a monomer and that IdeS activity follows classical Michaelis-Menten kinetics arguing against the previously proposed formation of a functional IdeS dimer. Our results show that IdeS inactivates IgG 100-fold faster than previously reported. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Place, publisher, year, edition, pages
Elsevier, 2013
Keyword
IgG, Cysteine protease, Monomer/dimer, GAS, Virulence factor, Streptococcus pyogenes
National Category
Cell and Molecular Biology Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-78961 (URN)10.1016/j.febslet.2013.04.039 (DOI)000320427400020 ()
Available from: 2013-07-29 Created: 2013-07-29 Last updated: 2017-12-06Bibliographically approved
2. The human protease inhibitor cystatin C is an activating cofactor for the streptococcal cysteine protease IdeS
Open this publication in new window or tab >>The human protease inhibitor cystatin C is an activating cofactor for the streptococcal cysteine protease IdeS
2008 (English)In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 15, no 9, 960-968 p.Article in journal (Refereed) Published
Abstract [en]

Human cystatin C is considered the physiologically most important inhibitor of endogenous papain-like cysteine proteases. We present here an unexpected function of cystatin C. Instead of acting as an inhibitor, cystatin C acts as a facultative, endogenous cofactor for the papain-like IgG-cleaving enzyme IdeS of the human pathogen Streptococcus pyogenes. IdeS activity is not dependent on cystatin C, but is significantly enhanced in the presence of cystatin C. We report a protease inhibitor that accelerates the activity of its putative target protease and a unique example of how a host protease inhibitor is "hijacked" by a bacterial protease to increase its activity. This finding has important implications for the view on protease-inhibitor interactions, and is relevant to consider in the therapeutic use of protease inhibitors.

Keyword
Chembio; microbio; proteins
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-20793 (URN)10.1016/j.chembiol.2008.07.021 (DOI)000259918200011 ()18804033 (PubMedID)
Available from: 2009-03-25 Created: 2009-03-25 Last updated: 2017-12-13Bibliographically approved
3. The streptococcal cysteine protease SpeB is not a natural immunoglobulin-cleaving enzyme
Open this publication in new window or tab >>The streptococcal cysteine protease SpeB is not a natural immunoglobulin-cleaving enzyme
2013 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 81, no 6, 2236-2241 p.Article in journal (Refereed) Published
Abstract [en]

The human bacterial pathogen Streptococcus pyogenes has developed a broad variety of virulence mechanisms to evade the actions of the host immune defense. One of the best-characterized factors is the streptococcal cysteine protease SpeB, an important multifunctional protease that contributes to group A streptococcal pathogenesis in vivo. Among many suggested activities, SpeB has been described to degrade various human plasma proteins, including immunoglobulins (Igs). In this study, we show that SpeB has no Ig-cleaving activity under physiological conditions and that only Igs in a reduced state, i.e., semimonomeric molecules, are cleaved and degraded by SpeB. Since reducing conditions outside eukaryotic cells have to be considered nonphysiological and IgG in a reduced state lacks biological effector functions, we conclude that SpeB does not contribute to S. pyogenes virulence through the proteolytic degradation of Igs.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-74504 (URN)10.1128/IAI.00168-13 (DOI)000318855100040 ()
Available from: 2013-07-02 Created: 2013-07-01 Last updated: 2017-12-06Bibliographically approved
4. 3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS
Open this publication in new window or tab >>3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS
Show others...
2012 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 6, 2549-2560 p.Article in journal (Refereed) Published
Abstract [en]

A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-55368 (URN)10.1021/jm201517a (DOI)000301767000004 ()
Available from: 2012-05-31 Created: 2012-05-14 Last updated: 2017-12-07Bibliographically approved
5. Insights into substrate recognition and biochemical properties of the streptococcal IgG endopeptidase IdeS
Open this publication in new window or tab >>Insights into substrate recognition and biochemical properties of the streptococcal IgG endopeptidase IdeS
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-88464 (URN)
Available from: 2014-05-06 Created: 2014-05-06 Last updated: 2014-05-06Bibliographically approved

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