Staphylococcal SplA and SplB serine proteases target ubiquitin(-like) specific proteases

Abstract

Staphylococcus aureus is a Gram-positive opportunistic pathogen that has colonized nearly 30% of the human population and can cause life-threatening infections. S. aureus exports a variety of virulence factors, such as a novel set of extracellular serine protease-like proteins (Spls). Spls are expressed by most clinical isolates of S. aureus, but their pathophysiological substrates and role during the infection are largely unknown. Here we characterized the substrate and cleavage specificity of recombinantly expressed SplA and SplB proteins. We identified a group of ubiquitin or ubiquitin-like modifying enzymes including deubiquitinating enzymes from human as well as from bacterial sources to be so far unknown SplA and SplB substrates. Distinct cleavage sites within these substrates for SplA (YLY^↓T, FMY^↓N) and SplB (VCD^↓S) were identified by mass spectrometry and confirmed by site-directed mutagenesis of the target proteins. Since many cellular immune signaling pathways are tightly regulated by ubiquitination, the specific cleavage of ubiquitin modifying enzymes strongly suggests a specific role of Spls in manipulating immune signaling and in competing with other bacteria.

Keywords: Staphylococcus aureus; Deubiquitination; Protein degradation; Serine protease-like; SplA; SplB.

Fig. 1

SDS-PAGE analysis of SENP1 cleavage by SplA or SplB. A SENP1 was incubated with the active SplB WT or the inactive SplB mutant S157A (with the catalytic serine exchanged to alanine) in a ~ 1:1 ratio for 3 h at 30 °C in PBS buffer. Control: incubation of Spl WT/S157A or SENP1 in PBS under same conditions. When incubated with SplB WT, a depletion of the SENP1 protein band (~ 35 kDa) was observed with a new band appearing at ~ 28 kDa. This was not the case for the inactive SplB mutant, indicating SENP1 cleavage catalyzed by SplB WT. B Cleavage site identification in SENP1. SplA and SplB predominantly cleaved at the TEV protease-cleavage motif of SENP1 at positions Y21 and Q23, respectively. SENP1 variants were incubated with Spls (2:1 ratio of SENP1 to Spls) for 24 h at 37 °C in PBS buffer. Control: incubation of SENP1 WT/Y21A or Q23A in PBS under same conditions. SplB* = SplB without C-terminal Twin-Strep-tag due to autohydrolysis (see Supplementary Information and Fig. S1 for further information)

Fig. 2

DUBs and ULPs targeted by SplA and SplB. Silver-stained SDS-PAGE of recombinantly expressed and purified Spl targets after incubation in PBS (ctrl), with inactive Spl mutants (SplA_S154A, SplB_S157A) and native Spls (WT) for 24 h at 37 °C. A Cleavage patterns of bacterial DUBs and ULPs catalyzed by Spls. B Cleavage patterns of human/viral DUBs and ULPs catalyzed by Spls

Fig. 3

SplA cleavage sites in RickULP. A SDS-PAGE analysis of RickULP WT cleavage catalyzed by SplA over time in PBS at 37 °C (5:1 ratio of RickULP to SplA). B SDS-PAGE of RickULP WT, Y84A mutant and Y84A_Y162A double mutant cleavage catalyzed by SplA in PBS at 37 °C (2:1 ratio of RickULP to SplA). C Scheme of RickULP WT and Y84A mutant cleavage by SplA at the two determined cleavage sites Y84 and Y162 including the calculated mass of the cleavage products. X = residue Y84 exchanged to alanine.