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

Abstract

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.

A sensitive and reproducible cell-based assay via secNanoLuc to detect neutralizing antibody against adeno-associated virus vector capsid

“…20,21 Moreover, NanoLuc conjugated with an interleukin (IL)-6 signal peptide (secNanoLuc) was shown to enable the detection of protein expression in the supernatant. [22][23][24] We generated the AAV5 and AAV8 vectors harboring EGFP, luciferase, or secNanoLuc reporters under the control of the CAG promoter (Figure 1A) and transduced the CHO-K1 or Huh-7 cells at different MOI values (Figures 1B-1D). We detected only 5%-15% of EGFP-positive cells even in the high vector genome (MOI of 10,000) (Figure 1B).…”

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution

“…These results show that the activity of the LITESEC systems can be efficiently toggled. Besides the Western blot, we used a sensitive bioluminescencebased luciferase assay 42 to quantify the export efficiency of the reporter protein YopE 1-53 -NanoLuc-FLAG ( Fig. 7b).…”

“…manufacturer instructions, similar to ref. 42 . Five microliters of supernatant was mixed with 25 µl H 2 O and 30 µl of NanoLuc detection reagent (Nano-Glo Luciferase Assay Substrate in Luciferase Assay Buffer, PROMEGA Corporation, Madison).…”

Keeping in Touch with Type-III Secretion System Effectors: Mass Spectrometry-Based Proteomics to Study Effector–Host Protein–Protein Interactions

“…A potential strategy based on BioID would be to tag effectors with a PBL at their endogenous locus (endogenous BioID, eBioID, Figure 4). Secretion of tagged T3Es and T4Es have been proven successful in case of small tags and certain reporters [109,111,115]. However, viewing the limited unfolding capacity of the T3SS (or T4SS [116]) and the stable β-barrel structure of fluorescent proteins (e.g., GFP), fusion of fluorescent proteins to T3Es was shown to block T3SS-mediated secretion [13], thereby generally excluding the use of tightly folded proteins as T3E fusions.…”