A high throughput assay provides novel insight into type VII secretion in Staphylococcus aureus

Successful colonisation by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. S. aureus strains harbour a T7b-subtype type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions, and the lack of a sensitive assay to measure secretion. Here we have utilised NanoLuc Binary Technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturisation of the assay to 384 well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.