The staphylococcal type VII secretion system delays inflammatory cell death during macrophage infection

The human pathogen Staphylococcus aureus is a facultatively intracellular bacterium that can replicate and survive within 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 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 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 ({Delta}essC), a central T7SS transporter. Interestingly, {Delta}essC-infected macrophages displayed increased bacterial escape compared to wild type (WT)-infected cells, implicating a role for T7SS in delaying macrophage cell death. Changes in cell death were accompanied by a reduction in efferocytosis of {Delta}essC-infected cells in vitro. Investigation of cell death signalling markers in WT and {Delta}essC-infected macrophages demonstrated that the T7SS delayed induction of key cell death pathways, necroptosis and pyroptosis, and a dual RNAseq analysis of WT- and {Delta}essC-infected macrophages indicated a T7SS-dependent modulation of bacterial oxidative stress and selected host chemokines. We also demonstrated that T7SS effectors, EsxA and EsxC individually delayed macrophage cell death. Interestingly, {Delta}esxC mutants demonstrated accelerated calcium fluxes, indicating that the T7SS-mediated suppression of cellular calcium signalling may cause the delay in 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.