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.

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.

SummaryDisarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram‐negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane‐based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway, we used transcriptomics analysis to develop a fluorescence‐based high‐throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen‐43. VUF15259 also impairs β‐barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam‐complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug‐like compounds.

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19The elucidation of the molecular mechanisms of secretion through bacterial protein secretion 20 systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the 21 analysis of the biological role of these secretion systems as well as the identification of 22 inhibitors targeting these systems would greatly benefit from the availability of a simple, quick 23 and quantitative assay to monitor principle secretion and injection into host cells. Here we 24 present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase 25 to assess secretion and injection through the type III secretion system encoded by Salmonella 26 pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise 27 ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion 28 kinetics and is adaptable to a high throughput screening format in 384-well microplates. We 29 further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type 30 III secretion-dependent injection of effector proteins into host cells. 31 32 Importance 33The ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or 34 even into target cells is one of the foundations of interbacterial as well as pathogen-host 35 interaction. While great progress has been made in elucidating assembly and structure of 36 secretion systems, our understanding of their secretion mechanism often lags behind, not last 37 because of the challenge to quantitatively assess secretion function. Here, we developed a 38 luciferase-based assay to enable the simple, quick, quantitative, and high throughput-39 compatible assessment of secretion and injection through virulence-associated type III secretion 40 systems. The assay allows detection of minute amounts of secreted substrate proteins either in 41 the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an 42 enabling technology to elucidate the mechanisms of secretion and injection of type III secretion 43 systems and is likely adaptable to assay secretion through other bacterial secretion systems. 44 PhoA-fusions, instead (18). While these assays proved very valuable to address some specific 78 questions, monitoring of secretion into the periplasm is only sensible for early substrates as 79 switching to the secretion of later substrates does not occur without an assembled needle. High 80 throughput (HTP) assays for screening of T3SS inhibitors exploited the turn-over of the 81 fluorogenic substrate PED6 by a secreted phospholipase fusion (19), the turn-over of the 82 chromogenic cephalosporine nitrocefin by a secreted b-lactamase fusion (20), and the 83 enzymatic uncaging of the fluorogenic substrate Glu-CyFur by a secreted carboxypeptidase 84 fusion (21, 22). 85 Likewise, several reporter assays have been developed to assess the injection of T3SS effectors 86 into eukaryotic host cells. Pioneering work by the Cornelis lab ex...