A high throughput assay to measure Type VII secretion inStaphylococcus aureus

Abstract 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 Firmicutes which functions in bacterial antagonism and virulence. Traditional assays for monitoring protein secretion by the T7SSb rely on concentrating cell culture supernatants for immunoblot analysis, a procedure which is time consuming, low throughput, non-quantitative and has low sensitivity. To overcome some of these disadvantages we have utilised NanoLuc Binary Technology to develop a simple assay to monitor protein secretion via detection of bioluminescence in 384-well plates. High-throughput analysis of secretion in replicate cultures reveals overlapping distributions for T7 + and T7 - populations highlighting a significant limitation of lower-throughput methods. We also demonstrate the utility of the NanoLuc assay in comparing T7-dependent protein secretion across different strains and find differences in the temperature dependence of T7 secretion across S. aureus isolates.