Researchers have a real-time look at how protein secretion works, using a Type III secretion system

Movement of proteins to the bacterial cell surface, the environment outside the cell, and even into target cells is critical for bacterial communities and pathogen-host interactions. The export of proteins, from Gram-negative bacteria in particular, is challenging, because two membranes (inner and outer) must be passed.

In Gram-negative bacteria, researchers have discovered nine different systems, referred to as type I–IX secretion systems (T1SS–T9SS). Three of the discovered secretion systems can put effector proteins into target cells that are of prokaryotic or eukaryotic origin.

The type III secretion machine. 

Built with two bases, one in the inner membrane and one in the outer membrane. These anchor the machine to the bacterial cell envelope. The machine also has some components in the cytoplasm. These parts of the machine usually receive cargo that is addressed to the extracellular environment. There is also an export apparatus in the inner membrane, which may unfold and repackages exports. Finally, there is a needle filament through which secreted substrates reach the host cell.

The process of secretion happens in a specific order. Early substrates aren’t secreted but build the needle filament. Then intermediate substrates come together to create the tip of the needle and the translocon pore. Finally, the late substrates move through the tip of the needle and are injected inside the target cell.

Researchers struggledd to understand how the secretion mechanism works because technology has not been developed to assess how secretion happens. In this report researchers demonstrate that the NanoLuc luciferase assay can be used to discover how secretion happens in the Type III system in Salmonella. The fusion of NanoLuc and the Type III secretion substrate move into the culture supernatant, which allows researchers to remove the bacteria and quantify the secreted substrates based on luminometry.

“The NanoLuc‐based secretion assay features a very high signal‐to‐noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384‐well microplates. We further developed a split NanoLuc‐based assay that enables the real‐time monitoring of type III secretion‐dependent injection of effector–HiBiT fusions into host cells stably expressing the complementing NanoLuc–LgBiT,” says the abstract of the study, published as part of the Wiley Online Library.