The role of the staphylococcal type vii secretion system in the modulation of host responses

The staphylococcal type VII secretion system (T7SS) is responsible for the export of several virulence factors. Some T7SS effectors are associated with long-term persistence of Staphylococcus aureus in murine models and modulation of epithelial and dendritic cell death, although the molecular mechanisms underlying T7SS-mediated modulation of cellular processes remain unclear. The aim of this study was to investigate the role of staphylococcal T7SS during macrophage infection. Isogenic mutants lacking either single or multiple secretion substrates of the T7SS (EsxC, EsxB, EsaE and EsxD) or EssC, an ATPase driving protein export were studied in comparative in vitro assays using THP-1 macrophages and in vivo mouse models. Time-lapse microscopy over the course of 24 hrs revealed that intracellular S. aureus caused the formation of pore-induced intracellular traps (PIT)-like structures, indicative of lytic cell death, in macrophages. Wild type (WT) S. aureus infections resulted in an early peak in cell death and inflammatory cytokine release which was absent or markedly shifted with T7SS mutant infections. WT induced early cell death was attributed to the T7SS promotion of the necroptosis pathway. Importantly T7SS mutants were not defective at causing necroptosis, but promotion of this pathway appeared to occur at later timepoints, suggesting other S. aureus virulence factors can also cause the onset of necroptosis in macrophages. Interestingly, in low-dose skin infections, T7SS mutants survived better than the WT and demonstrated enhanced dissemination to distal sites. However, WT was more successful in high-dose infections which indicates dose dependent changes in the effect of the T7SS. Finally, using interdisciplinary techniques a microfluidic air-way-blood-vessel-on-a-chip was designed, fabricated and evaluated for the investigation of S. aureus-host interactions under shear stress. Overall, this project provided insight into specific roles of the T7SS in macrophages and questions whether that the T7SS is always beneficial for S. aureus during infection