# Staphylococcal SplA and SplB Serine Protease Allelic Variants Exhibit Different Substrate Specificities

## TL;DR

This study shows that different versions of Staphylococcus aureus proteases SplA and SplB cut proteins in distinct ways, affecting how the bacteria interact with human cells.

## Contribution

The study reveals how allelic variants of SplA and SplB differ in substrate specificity and enzymatic activity, adding new insights into bacterial virulence mechanisms.

## Key findings

- Allelic variants of SplA and SplB differ in stability, enzymatic activity, and substrate specificity.
- One SplB variant shows substrate recognition similar to SplE rather than SplB wildtype.
- Variants cleave target proteins RickULP and SseL with distinct patterns.

## Abstract

Staphylococcus aureus is an opportunistic pathogen that is persistently colonizing nearly 30% of the human population and can cause life‐threatening infections. S. aureus secretes a variety of virulence factors, such as a 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 infection are largely unknown. Pathogens use allelic variation of virulence factors to allow an adaption to different host cells and their defense mechanisms. The differences of these variants are marginally characterized so far. Here, we performed a biochemical characterization of selected allelic variants of the S. aureus SplA and SplB. Our data suggests different variants show differences in their stability, enzymatic activity, and substrate specificity. For the recently identified Spl target proteins RickULP and SseL, different cleavage patterns were observed, upon treatment with different Spl allelic variants. One SplB variant strongly differed in its substrate recognition at the P3/P4 position, more closely resembling SplE than SplB wildtype in its substrate selectivity. Our data provide valuable insights into the evolution of bacterial virulence factors and highlight the importance of including allelic variation of virulence factors to fully understand their role in host–pathogen interaction.

A biochemical characterization of Staphylococcus aureus serine proteases SplA and SplB allelic variants (recombinantly produced in E. coli and purified) reveals that allelic variation adds an extra layer of complexity to the functioning of virulence factors in S. aureus colonization and infection. This highlights the importance of considering allelic variation in the study of host‐pathogen interactions.© 2026 WILEY‐VCH GmbH