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 lack 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 secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells. Importance The ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

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. The 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 Introduction 45 The ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or 46 even into target cells is one of the foundations of interbacterial as well as pathogen-host 47 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 labelling enzyme tags because labelling can only be done with effectors that have already been 103 translated before host cell contact. 104 We aimed to develop a T3SS assay based on effector-luciferase fusions to enable a simple, 105 quantitative, and HTP-compatible assessment of principle secretion and injection. The 106 advantage of luciferase-reporters is a very high S/N and sensitivity. In addition, luciferase-based 107 assays benefit from the lack of product (light) accumulation, simplifying the analysis of 108 secretion and injection rates. We developed a secretion assay utilizing NanoLuc (NLuc) 109 luciferase, an engineered 19 kDa glow-type luciferase from the deep-sea shrimp Oplophorus 110 gracilirostris that converts furimazine, emitting blue light (31). The NLuc-based secretion assay 111 allowed quantification of minute amounts of secreted effectors either in the supernatant of the 112 bacterial culture or within eukaryotic host cells. The assay's ultra-high sensitivity, its wide 113 dynamic range and quick response dynamics qualify it as an enabling technology to elucidate 114 the mechanisms of secretion and injection of T3SS and is likely adaptable to assay secretion 115 through other bacterial secretion systems.

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Assessment of effector-luciferase fusion proteins as reporters for type III secretion 119 In order to identify a luciferase compatible with type III secretion through the T3SS encoded 120 by Salmonella pathogenicity island 1 (SPI-1, T3SS-1), we evaluated six different commercially 121 available luciferases as effector-fused secretion reporters: Cypridinia luciferase (CLuc),

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The activity of the secreted luciferases in filtered culture supernatants of the S. Typhimurium 137 wild type and of the ΔsctV mutant, respectively, was assessed by luminometry using the speci- always higher for SipA-luciferase fusions (Fig. 1C).

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Since the SipA-NLuc fusion showed the best S/N, we introduced SipA-NLuc-myc into the 142 chromosome of a S. Typhimurium wild type strain and of a ∆sctV mutant for further analysis. 143 First, we compared the expression and secretion of plasmid and chromosome-encoded SipA-144 NLuc, respectively, and as a reference also of the secreted translocator SctE, by SDS PAGE, 145 Western blotting and immunodetection. SipA-NLuc was expressed well from the chromosome 146 even though, not unexpectedly, at lower levels compared to its expression from the plasmid 147 (Fig. 1D). The extent of T3SS-dependent secretion of plasmid and chromosome-encoded SipA-148 NLuc was indistinguishable (Fig. 1D). 149 We next evaluated the S/N of the secreted SipA-NLuc fusion when expressed from plasmid or  showing that split-NLuc can serve as a secretion reporter when NLuc fails.

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In summary, we could show that luciferases are versatile reporters for T3SS and that effector-  in very small volumes, in brief intervals, and with very short handling times (10 min after col-219 lection of supernatant). Our results also show that induction and assembly of the megadalton 220 injectisome is a very quick process that gets bacteria rapidly armed for attack.  To assess the robustness of this assay and the variation across the plate, we filled an entire 384-236 well plate with 50 µl of a S. Typhimurium, SipA-NLuc culture and allowed it to grow for 5 h at 237 37°C. Luminescence of secreted, wall-bound SipA-NLuc was assessed after washing out bac-238 teria as described above. The assay proved very robust with a coefficient of variation of 7% 239 over the entire plate and with little edge effects (Fig. 3C, Table S1). We then performed a proof-

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of-concept inhibitor screen by assessing the effect of a range of 37 different bioactive com-241 pounds on the activity of the T3SS in the 384-well format (Table S2, Fig. 3D). Each well of the 242 plate was printed with 0.5 µl of a compound in 100% DMSO, to which 50 µl of a S. 243 Typhimurium, SipA-NLuc culture was added. Again, the culture was allowed to grow for 5 h, 244 after which secretion of SipA-NLuc was assessed by luminometry. The assay showed a highly 245 dynamic response from 10 % to 120 % secretion activity compared to the DMSO-treated wild 246 type control (Fig. 3D). Detection of SipA-NLuc was most strongly reduced by the flavonoids 247 quercetin (30 µg/ml, 90% reduction) and scutellarin (10 µg/ml, 75% reduction), which con-  Over all, the SipA-NLuc assay proved to be highly adaptable to a high throughput screening 253 format in 384-well plates, featuring a high S/N, a low error across the plate, a great reproduci-254 bility and requiring only short hands-on time.   In a first and simple approach, we infected HeLa cells in 96-well plates at an MOI = 50 with 301 SipA-NLuc-expressing S. Typhimurium, using wild type bacteria and secretion-deficient ΔsctV 302 mutants. After infection for 60 min, attached bacteria were gently washed off with PBS using a 303 microplate washer and subsequently, the HeLa cell-associated luminescence was measured us-304 ing live cell buffer (Fig. 5A). The non-secreting ∆sctV mutants (Fig. 5A) showed a HeLa cell-305 associated luminescence of 8% of the wild type, corresponding to a S/N = 12 (Fig. 5C). To 306 determine whether the HeLa cell-associated signal was truly resulting from injected SipA-307 NLuc, we assessed injection in a set of mutants that are capable of secreting SipA but incapable 308 of injecting it into host cells: a needle tip-deficient ΔsctA, a translocon-deficient ΔsctEBA, and 309 a gatekeeper-deficient ΔsctW mutant. While secretion of SipA-NLuc into the culture superna-310 tant was increased between 2 and 5-fold in ∆sctA, ∆sctEBA, and ∆sctW mutants (Fig. 5B), 311 which are reportedly unlocked for secretion of late substrates like SipA (47, 48), the HeLa cell-312 associated luminescence was strongly reduced to 9-24% of the wild type when infecting with 313 these mutants (Fig. 5C). From these results we can conclude that the luminescence signal ob-314 tained from infection with wild type S. Typhimurium resulted to more than 90% from injected NLuc-based injection assay, none of the T3SS mutant strains yielded any detectable lumines-328 cence in the split NLuc assay (Fig. 5F), making this assay highly suitable for monitoring the 329 specific injection of T3SS effectors into host cells. This setup even allowed us to follow the 330 kinetics of SipA-HiBiT injection over time directly in a microplate reader (Fig. 5G).  We demonstrated that the NLuc-secretion assay is highly suited to study the kinetics of secretion 368 due to its superior sensitivity. Our simple assay setup only allowed deduction of secretion 369 kinetics from the accumulation of NLuc in the culture supernatant but culturing bacteria in a HiBiT of the injected effector also localizes to the cytoplasm. However, the split-NLuc injection 406 assay may also be utilized to analyze the localization and topogenesis of effector proteins inside 407 host cells by targeting LgBiT to specific organelles instead (Fig. 6). Furthermore,

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In summary, our data show that NLuc-fusions of secreted substrate proteins can be used as a 412 robust, versatile, cheap, simple and quick reporter for T3SS secretion and injection that will 413 enable future in-depth elucidation of T3SS function (Fig. 6). The NLuc reporter is likely to be 414 adaptable to other bacterial secretion systems as well.     For the inhibitor screen, 0.5 µl of each compound (Table S2) was added to 50 µl bacterial culture 471 prior to incubation at 37°C for 5 h, and the plate was processed as described above.   Buffer was exchanged to PBS by using the Amicon Ultra system (Merck).

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All data represent means (± standard deviation) of three independent measurements.         HiBiT was inserted behind residue 331 of SctP, so that the Shine-Dalgarno sequence of sctQ, which is overlapping with the gene of SctP, was unaffected. Note that SctE is not secreted when expressing SctP-NLuc fusions, i.e. SctP-NLuc cannot complement the needle length regulating function of SctP, thus substrate specificity switching to the secretion of intermediate substrates is not induced. (B) Luminescence of the indicated SctP-NLuc/HiBiT-fusions secreted into the culture supernatant. Data represent the mean (± standard deviation) of three technical replicates. Note that SctP 331 -NLuc can be detected in the culture supernatant by luminometry but not by Western blotting. Also note that split-NLuc generally gives lower luminescence than regular NLuc. (C) Immunodetection of the indicated SctA-NLuc fusions on Western blot of SDS PAGE-separated culture supernatants and whole cell lysates. The numbers (184, 220, 275) mean that NLuc was inserted behind these residues of SctA. The insertion positions where chosen based on the structure of S. Typhimurium SctA-1. Secreted SctA 275 -NLuc reproducibly appeared as a double band for unknown reasons. (D) Luminescence of the indicated SctA-NLuc and SipA-NLuc-fusions secreted into the culture supernatant. Data represent the mean (± standard deviation) of three technical replicates. Note that SctA C -NLuc can be detected in the culture supernatant by luminometry but not by Western blotting. Also note that internal fusions of NLuc are acommodated well, with SctA 275 -NLuc providing even stronger signal than SipA-NLuc. Abbreviations: sup: culture supernatant, wc: whole cell lysates, C: C-terminus, RLU: relative luminescence units, NLuc: NanoLuc luciferase, T3SS: type III secretion system