Proteolytic processing of the streptococcal IgG endopeptidase IdeS modulates the functional properties of the enzyme and results in reduced immunorecognition.

The important human gram positive bacterial pathogen Streptococcus pyogenes employs various virulence factors to promote inflammation and to facilitate invasive disease progression. In this study we explored the relation of the secreted streptococcal cysteine proteases IdeS and SpeB, and neutrophil (PMN) proteases. We found that SpeB is resistant to proteolytic attack in an inflammatory environment, emphasizing the importance of SpeB for streptococcal pathogenicity, while PMN enzymes and SpeB itself process the IgG degrading endopeptidase IdeS. Processing occurs as NH2-terminal cleavage of IdeS resulting in reduced immunorecognition of the protease by specific antibodies. While the endopeptidase retains IgG cleaving activity, its ability to suppress the generation of reactive oxygen species is abolished. We suggest that the cleavage of NH2-terminal peptides by SpeB and/or neutrophil proteases is a mechanism evolved to prevent early inactivation of this important streptococcal virulence factor, albeit at the cost of impaired functionality.

Rapid IgG heavy chain cleavage by the streptococcal IgG endopeptidase IdeS is mediated by IdeS monomers and is not due to enzyme dimerization.

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.

The streptococcal cysteine protease SpeB is not a natural immunoglobulin-cleaving enzyme.

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.

3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS.

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.

The human protease inhibitor cystatin C is an activating cofactor for the streptococcal cysteine protease IdeS.

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.