Abstract
Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor. Although subject to several studies, its role during infection is still under debate, and its proteolytic properties remain insufficiently characterized. Here, we revisited this protease through a set of complementary approaches relying on state of-the-art HPLC-MS methods. After conceiving an efficient protocol to recombinantly express SpeB, the zymogen of the protease and its activation were characterized. Employing proteome-derived peptide libraries, a strong preference for hydrophobic and aromatic residues at P2 alongside negatively charged amino acids at P3′ to P6′ was revealed. To identify relevant in vivo substrates, native proteins were obtained from monocytic secretome and plasma to assess their cleavage under physiological conditions. Besides corroborating our findings concerning specificity, more than 200 cleaved proteins were identified, including proteins of the extracellular matrix, proteins of the immune system, and proteins involved in inflammation. Finally, the cleavage of IgG subclasses was studied in detail. This study precisely depicts the proteolytic properties of SpeB and provides a library of potential host substrates, including their exact cleavage positions, as a valuable source for further research to unravel the role of SpeB during streptococcal infection.
Highlights
Streptococcus pyogenes, or group A streptococcus (GAS), is an exclusively human pathogen responsible for a number of mild and severe pathological conditions, including necrotizing fasciitis, toxic shock syndrome, and rheumatic fever [1]
Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor
Substantial efforts have been undertaken over the last several decades to study this key protease, we revisited this topic employing a number of complementary stateof-the-art HPLC-MS workflows to provide a comprehensive and unbiased characterization
Summary
Streptococcus pyogenes, or group A streptococcus (GAS), is an exclusively human pathogen responsible for a number of mild and severe pathological conditions, including necrotizing fasciitis, toxic shock syndrome, and rheumatic fever [1]. The diversity of diseases caused by this pathogen ranges from toxin-mediated pathologies (over immunemediated ones) to those resulting from the direct infection. The protease is conserved in virtually all GAS strains [4], and it was shown that the pathogen is dependent on its expression to cause disease [5]. While the early phase of GAS infection may be characterized by a generally reduced proteolytic activity, SpeB is thought to be expressed at a later stage of infection under nutrient-restricted conditions to facilitate bacterial evasion into the surrounding tissue [9]. The complex and tight regulation of SpeB activity by GAS is thoroughly reviewed by Carroll et al [10]
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