Introduction: Extracellular proteases are important virulence factors in Staphylococcus aureus. This also comprises a set of serin protease-like proteins (Spls) whose role in infection is still poorly understood. In the mouse-adapted S. aureus strain JSNZ, we recently identified a novel secreted serine protease, JSNZ extracellular protease (Jep). Jep shares significant sequence homology and a conserved catalytic triad with the Spls, making it an interesting candidate for investigating the role of serine proteases in murine S. aureus infection models.

Goals: To conduct in-depth analyses of Jep's function, we aim to characterise its expression and secretion in in vitro settings, comparing key features with those of Spls.

Materials and Methods: Using the mouse-adapted S. aureus strain JSNZ, we analysed the expression of jep during growth in TSB and the plasma-mimicking minimal medium RPMI via northern and western blotting. Regulatory motifs were predicted in silico. Additionally, the effect of the 3'-UTR was studied using the conditional mutants JSNZ Δjep pTripleTREP_jep and RN4220 pTripleTREP_jep, allowing the insertion of different jep sequences. Signal peptide cleavage after secretion was investigated using N-terminomics.

Results: Jep shares similarities with SplB in terms of expression and secretion. Expression starts in the transient phase and the protease accumulates in the culture supernatant during the stationary phase. We were also able to prove the same specific cleavage of the signal peptide after secretion. However, Jep reaches a much higher relative proportion in the secretome of murine S. aureus isolates than Spls in known clinical isolates. This is partially achieved by a positive influence of a terminator structure in the 3'-UTR on the stability of jep mRNA.

Summary: Our study provides a baseline characterisation of the newly identified extracellular S. aureus protease Jep, revealing both similarities and differences compared to known Spls. The remarkably high abundance of Jep in the secretome is likely linked to an observed transcript stabilisation by a terminator structure in the 3'-UTR of jep.