Role of group A Streptococcus HtrA in the maturation of SpeB protease

Abstract

The serine protease high-temperature requirement A (HtrA) (DegP) of the human pathogen Streptococcus pyogenes (group A Streptococcus; GAS) is localized to the ExPortal secretory microdomain and is reportedly essential for the maturation of cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Here, we utilize HSC5 (M5 serotype) and the in-frame isogenic mutant HSC5DeltahtrA to determine whether HtrA contributes to the maturation of other GAS virulence determinants. Mutanolysin cell wall extracts and secreted proteins were arrayed by 2-DE and identified by MALDI-TOF PMF analysis. HSC5DeltahtrA had elevated levels of cell wall-associated M protein, whilst the supernatant had higher concentrations of M protein fragments and a reduced amount of mature SpeB protease, compared to wild-type (WT). Western blot analysis and protease assays revealed a delay in the maturation of SpeB in the HSC5DeltahtrA supernatant. HtrA was unable to directly process SpeB zymogen (proSpeB) to the active form in vitro. We therefore conclude that HtrA plays an indirect role in the maturation of cysteine protease SpeB.

The Majority of 9,729 Group A Streptococcus Strains Causing Disease Secrete SpeB Cysteine Protease: Pathogenesis Implications

“…Due to its human health importance, the emm1 epidemic clone has been extensively studied by many investigators (25)(26)(27)(28)(29)(30). The identification of polymorphisms in several genes that confer a SpeB-deficient phenotype has led, in part, to the hypothesis that epidemic emm1 GAS strains are under intense selective pressure to decrease secreted SpeB protease activity during infection (19)(20)(21)(22)(23)(24). Reinforcing this idea, Kansal et al, using a small sample of strains, reported an inverse relationship between SpeB proteolytic activity and disease severity (23).…”

“…Each stage is regulated by multiple GAS accessory factors (Table 1). Disruption of any of these components may render a GAS strain deficient in secreted SpeB protease activity (19)(20)(21)(22)(23)(24)). …”

Type I Signal Peptidase and Protein Secretion in Staphylococcus aureus

“…HtrA is a bifunctional enzyme with both chaperone and protease activities and it is thought to be involved in the folding and maturation of secreted proteins as well as in the degradation of proteins that misfold during secretion (22,30,64,81,94,102), and at least in Bacillus subtilis, htrA is induced by secretion stress (2,44,63). Deletion of htrA conferred only a marginal sensitization to the arylomycin and did not significantly affect stationary-phase protein secretion in the presence or absence of the inhibitor.…”

Analytical strategies for phosphoproteomics

“…Highly complex protein samples can be separated according to their pI and their molecular weight using 2-DE [11][12][13][14][15]. The separated proteins can subsequently be visualized using standard protein stains (e.g., Coomasssie blue and silver staining).…”

“…Since ETD and ECD are optimal methods for fragmentation of multiply charged peptide ions (e.g., charges of 13,14), an alternative protease might be a better choice as trypsin mainly produces doubly charged ions, which are suitable for CID. Lys-C is a logical choice as it generates trypsin-like peptide ions, but only cleaves C-terminal to lysine, thus creating longer peptides.…”