How PDGFRB Snuck Up On Us All

aureus survival in human blood by promoting production of anti-opsonophagocytic virulence factors that inhibit serum- and complement-mediated mechanisms. FIGURE 2 AirSR regulated exported proteins inhibit complement and antibody mediated opsonophagocytic killing of S. aureus. Sterile TSB and culture supernatants of ATc (250 ng/ml) induced control (Control Cult. Sup) and AirR overproducing S. aureus (WAirR Cult. ... Identification of Overproduced Exported Proteins Resulting from AirR Overproduction in S. aureus To identify which exported protein(s) are overexpressed resulting from AirR overproduction, we prepared the exported proteins from cell-free culture supernatants of ATc induced pYH4 control and WAirR strains. The exported proteins were visually compared using SDS-PAGE, and protein bands that were obviously over-represented or bands that only appeared in the WAirR lane were processed for peptide identification by mass spectrometry (see Materials and Methods). The subsequently identified proteins were cross-referenced with published studies to identify proteins that are involved in innate immune suppression via inhibition of the humoral and/or innate cellular response. Peptides from the cysteine endopeptidase, staphopain B (SspB) dominated one of the over-represented bands from ATc induced WAirR culture supernatants. More than 80% of the processed active form of SspB was identified by mass spectrometry (Supplementary Figure S2). AirR Overproduction Results in Increased Functional Staphopain B Production To examine if the overproduced SspB is functional, we conducted gelatin zymography assays using the cell-free culture supernatants, as SspB is able to degrade collagen (Ohbayashi et al., 2011). Coomassie Blue staining and gelatin zymography analyzed was used to analyze an equal volume of PDGFRB concentrated culture supernatant from each induced strain. A single band in the induced WAirR lane and the disappearance of other proteins relative to the pYH4 control lane was detected by Coomassie Blue (Figure ?Figure3A3A). Gelatin zymography analysis of the same samples revealed very little gelatin degradation in the control strain, while a large, prominent band of gelatin degradation appeared in the induced WAirR sample (Figure ?Figure3B3B). Importantly, both the Coomassie Blue stained protein band and gelatin degradation in the zymogram resolve at the same molecular weight from the induced WAirR, suggesting the gelatin degradation is the result of this protein. These data highly suggest that the overproduction of AirR results in the overproduction of functional cysteine endopeptidase SspB. FIGURE 3 Zymographic and immunoblot analysis of concentrated control and WAirR exported proteins. Equal volumes of concentrated culture supernatant were resolved by (A) 12% SDS-PAGE and proteins stained with Coomassie Blue or (B) 0.1% gelatin-12% SDS-PAGE and ...