Defining the human lung pathodegradome of the V8 protease from <i>Staphylococcus aureus</i>

<i>Staphylococcus aureus</i> is a globally relevant human pathogen and key driver of human morbidity and mortality. During infection, it employs a suite of extracellular proteases to subvert host defenses and promote disease progression. Key among these, the V8 serine protease (SspA) stands out as a master manipulator of the immune response; however, host targets for this enzyme remain incompletely defined. Herein, we use a high-efficiency N-Terminomic approach developed by our group (TAGS-CR) to capture ~320 biologically relevant V8 targets in the human lung proteome. These substrates present a role for V8 in modulating the complement system, with specific targets like Complement C3 carrying significant importance in this niche. In addition, neutrophil effector functions such as ROS production, migration, phagocytosis, and degranulation were disrupted by the action of V8. Similarly, epithelial barrier integrity may suffer as a consequence of V8 targeting foundational proteins, such as zona occludens and alpha actinin proteins, coupled with supporting actin molecules. Notably, our data provide new insights into V8 as a key player exploiting nutritional immunity by disrupting iron-complexed proteins such as transferrins, hemopexin, and hemoglobin. In addition to its capacity to intensify inflammation, V8 also undermines the function of key immune regulators, including pro-inflammatory sink molecules like gelsolin. Collectively, this comprehensive proteolytic mapping expands our understanding of how <i>S. aureus</i> can manipulate human biology across infection-relevant niches and offers valuable insight into conserved molecular mechanisms that underlie bacterial virulence.