The staphylococcal type VII secretion system delays macrophage cell death through modulating multiple cell death pathways

The major human pathogen Staphylococcus aureus is a facultatively intracellular bacterium that can replicate and survive within a range of host cells including macrophages. While macrophage-mediated immune modulation is pivotal in S. aureus pathogenesis, bacterial manipulation of macrophage pathways remains poorly understood. The specialised type VII secretion system (T7SSb) is an important virulence-associated factor and immune modulator during staphylococcal infection, although, unlike its mycobacterial counterparts, its role in controlling staphylococcal-macrophage interactions remains unclear. Employing high-resolution time-lapse imaging, we demonstrated an induction of cell death corpses or pore-induced cellular traps (PITs) during S. aureus infection of macrophages in vitro , with a higher number of PITs forming in macrophages infected with S. aureus lacking EssC (Δ essC ), a central T7SS transporter. Interestingly, Δ essC -infected macrophages displayed increased bacterial escape compared to wild type (WT)-infected cells, implicating a role for T7SS in delaying macrophage cell death. Δ essC -infected macrophages were also efferocytosed less compared to WT in vitro . We investigated the induction of cell death signalling markers in WT and Δ essC -infected macrophages and demonstrated that the T7SS delayed the induction of key cell death pathways, necroptosis and pyroptosis. A dual RNAseq analysis of WT- and Δ essC -infected macrophages further indicated a T7SS-dependent increase in ferroptosis later in infection, along with modulation of chemokines. We demonstrated that individual T7SS effectors, EsxA and EsxC, which were induced within macrophages, could individually delay macrophage cell death. Furthermore, in a murine skin infection model, we showed that effector and transporter mutants have distinct S. aureus infection outcomes. Our findings suggest for the first time a key role for T7SSb proteins in controlling macrophage cell death, which impacts staphylococcal survival within the host during infection.