Detection of proper Type 3 Secretion System Translocon Assembly using a split Luciferase Assay

Abstract

Type 3 secretion system (T3SS) is used by many Gram‐negative bacteria to colonize the host and inject effector proteins into target cells. Inhibitors targeting T3SS are important to combat the appearance of multidrug‐resistant pathogens. In Pseudomonas aeruginosa, the T3SS secreted proteins PopB (SctE) and PopD (SctB) are required to inject effector proteins into the host cells. PopB and PopD associate with the target membrane and they are proposed to form a translocon pore by which effectors are translocated. This translocon is a potential target for inhibitors because these molecules do not need to overcome the intrinsic problems associated with cell membrane permeability and the presence of efflux pumps.In previous studies, we have reported that the PopD N‐terminus is exposed to host cell cytosol (J. Biol. Chem. 2018 293 8982–8993). We reasoned that the PopD N‐terminal exposure could be used as a reporter for properly assembled translocons. Using a split form of the small NanoLuc (NLuc) luciferase, we have developed an assay to detect the exposure of this N‐terminus to the host cytosol. HeLa cells producing the large truncated version of NLuc are used as targets. These cells are incubated with a P. aeruginosa ΔpopD PAK strain that produces a PopD variant fused with the missing portion of NLuc at the N‐terminus (Nluc10‐PopD). Assembly of a functional translocon is detected by the maturation of NLuc enzyme in the HeLa cell cytosol and the production of luminescence. Failure to assemble a functional translocon did not produce luminescence. The assay has a typical signal‐to‐background ratio (S/B) of 6 ‐ 10 and a Z factor above 0.5, constituting an excellent tool for the screening and detection of small molecule inhibitors of T3SS translocon assembly.