Analysis of SPI-1 Dependent Type III Secretion and Injection Using a NanoLuc Luciferase-Based Assay.

Studies of bacterial protein secretion have relied on a variety of reporters that allow the tracking of secreted proteins. However, the lack of truly quantitative and highly sensitive reporters has hindered, in particular, the investigation of the kinetics of protein secretion. In this chapter, we describe a luminescence-based assay using NanoLuc luciferase to analyse secretion and injection into host cells of type III secretion (T3S) substrates encoded on Salmonella pathogenicity island-1 (SPI-1). This method has a very high sensitivity and high signal-to-noise ratio. Moreover, the simplicity of the protocol and the rapid determination and quantification of the luminescence makes it ideal for the monitoring of the kinetics of secretion but also convenient for high-throughput screenings. The protocols presented here include (1) Salmonella SPI-1 secretion assay, where the T3S substrates-NanoLuc fusions are detected by luminometry in the bacterial supernatant, and (2) Salmonella injection assays, using the split-Nanoluc (HiBiT/LgBiT) to monitor the injection of T3S substrates-HiBiT fusions into the host cells stably expressing LgBiT.