A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems.

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate-NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. 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.

The inner rod of virulence-associated type III secretion systems constitutes a needle adapter of one helical turn that is deeply integrated into the system's export apparatus.

Type III secretion injectisomes are essential virulence factors for many pathogenic bacteria by mediating the transport of effector proteins into eukaryotic host cells. The secretion conduit of injectisomes is formed by a helical assembly of three hydrophobic proteins (SctR, SctS and SctT), an inner rod (SctI) and a needle filament (SctF). SctI is thought to play a role in switching between the secretion of different substrate classes and assembly of the inner rod has been implicated in regulating the length of the needle filament. While high-resolution structures of the hydrophobic components and of the needle filament have been solved, little is known about the structure and the assembly of the inner rod, which impedes the deeper assessment of its function. Here we show by exhaustive in vivo photocrosslinking that SctI engages in extensive interactions with SctR and SctT throughout its entire length. Our data imply that the inner rod serves as an adapter between the export apparatus and the needle filament by forming one helical turn. We show that assembly of the inner rod does not play a role in needle length control nor in substrate specificity switching. Instead, our findings imply that inner rod assembly must precede assembly of the needle filament.

Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells.

Virulence-associated type III secretion systems (T3SS) serve the injection of bacterial effector proteins into eukaryotic host cells. They are able to secrete a great diversity of substrate proteins in order to modulate host cell function, and have evolved to sense host cell contact and to inject their substrates through a translocon pore in the host cell membrane. T3SS substrates contain an N-terminal signal sequence and often a chaperone-binding domain for cognate T3SS chaperones. These signals guide the substrates to the machine where substrates are unfolded and handed over to the secretion channel formed by the transmembrane domains of the export apparatus components and by the needle filament. Secretion itself is driven by the proton motive force across the bacterial inner membrane. The needle filament measures 20-150 nm in length and is crowned by a needle tip that mediates host-cell sensing. Secretion through T3SS is a highly regulated process with early, intermediate and late substrates. A strict secretion hierarchy is required to build an injectisome capable of reaching, sensing and penetrating the host cell membrane, before host cell-acting effector proteins are deployed. Here, we review the recent progress on elucidating the assembly, structure and function of T3SS injectisomes.

Blue Native PAGE Analysis of Bacterial Secretion Complexes.

Bacterial protein secretion systems serve to translocate substrate proteins across up to three biological membranes, a task accomplished by hydrophobic, membrane-spanning macromolecular complexes. The overexpression, purification, and biochemical characterization of these complexes is often difficult, impeding progress in understanding the structure and function of these systems. Blue native (BN) polyacrylamide gel electrophoresis (PAGE) allows for the investigation of these transmembrane complexes right from their originating membranes, without the need for long preparative steps, and is amenable to the parallel characterization of a number of samples under near-native conditions. Here we present protocols for sample preparation, one-dimensional BN PAGE and two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE, as well as for downstream analysis by staining, immunoblotting, and mass spectrometry on the example of the type III secretion system encoded on Salmonella pathogenicity island 1.