N-terminomics identifies substrates of the secreted Staphylococcus aureus protease Jep missed by classical label-free bottom-up proteomics

The identification of protease substrates in complex samples, such as plasma or cell culture supernatants, can be challenging, particularly when the substrate is not degraded but cleaved at specific sites by proteolytic processing. In such cases, classical label-free bottom-up mass spectrometry reaches its limits in identifying proteins of interest, as the abundance of most peptides remains unchanged. However, N-terminomics can overcome this issue by enriching the N-termini prior to mass spectrometric analysis1. With this approach, the full potential of mass spectrometry can be focused on the peptides of particular interest. Moreover, in addition to identifying protease substrates, this method can provide information on protease-specific cleavage motifs.

In our study, we focused on the novel serine protease Jep, which is found almost exclusively in mouse-adapted Staphylococcus aureus strains. In contrast to clinical S. aureus isolates, mouse-adapted strains were demonstrated to persistently colonise mice under physiological conditions. Consequently, these strains are crucial for improving current mouse models for investigating S. aureus colonization and infection2, which is necessary for the development of new prophylactic and therapeutic strategies against this notorious hospital pathogen. To understand the role of Jep as a highly host-specific factor is a pivotal step in defining the model.

We have shown that the deletion of jep in the mouse-adapted S. aureus strain JSNZ led to hypervirulence in a murine bacteraemia model. However, no substrates were known so far, and a classical label-free bottom-up proteomic approach did not reveal any differences in the proteome pattern comparing the deletion strain with the wild-type strain. Various potential substrate groups were subsequently screened for Jep substrates using N-terminomics. Tested samples include bacterial culture supernatants of JSNZ wild-type and a jep deletion mutant as well as mouse plasma and murine lung epithelial cells incubated with purified Jep protease. Most interestingly, this revealed alterations in the N-termini of a number of secreted bacterial proteins, including known virulence factors such as the subunits of LukAB. Our results suggest that the protease Jep influences virulence rather by targeted proteolytic processing of specific secreted virulence factors than by protein degradation. This example illustrates the power of N-terminomics in the investigation of proteases, to reveal effects that cannot be covered by classical label-free bottom-up proteomic approaches.

(1) Kleifeld O, Doucet A, auf dem Keller U, et al. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat Biotechnol. 2010.

(2) Trübe P, Hertlein T, Mrochen DM, et al. Bringing together what belongs together: Optimizing murine infection models by using mouse-adapted Staphylococcus aureus strains. Int J Med Microbiol. 2019.