A high throughput assay to measure Type VII secretion inStaphylococcus aureus

Abstract

AbstractSuccessful colonisation by the opportunistic pathogenStaphylococcus aureusdepends on its ability to interact with other microorganisms.S. aureusstrains 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 acrossS. aureusisolates.