Compound 3 (CAS# 443329-02-0), compound 4 (CAS# 138323-28-1), and INP0007 (CAS# 300668-15-9) were obtained from Chembridge. INP0010 (CAS# 68639-26-9) was obtained from ChemDiv. Compound 20 (CAS# 489402-27-9) was obtained from TimTek. The anti-YscF antibody was raised in rabbits against the Y. pseudotuberculosis YscF peptide KDKPDNPALLADLQH (Morgan et al., 2017). DMSO concentration did not exceed 0.2%, except where indicated.

Bacterial strains used in this paper are listed in Table 1. Y. pseudotuberculosis and Y. enterocolitica were grown, unless otherwise specified, in 2xYT (yeast extract-tryptone) at 26°C shaking overnight. In order to induce the T3SS, overnight cultures were diluted into low calcium medium (2xYT plus 20 mM sodium oxalate and 20 mM MgCl₂) to an optical density (OD₆₀₀) of 0.2 and grown for 1.5 h at 26°C shaking followed by 2–3 h at 37°C to induce Yop synthesis, depending on the assay, as previously described (Auerbuch et al., 2009).

An expression cassette containing the yopH promoter and FLAG (5′ terminus)- and ssrA (3′ terminus)-tagged mCherry was generated using SOEing PCR and cloned into pMMB67EH (Horton et al., 1990; Pettersson et al., 1996; Karzai et al., 2000). The yopH promoter was amplified from pHYopT (gift from J. Bliska) using oligonucleotides SMP425 and SMP426 (Table 2). mCherry (gift from R. Tsien) was amplified using oligonucleotides SMP427 and SMP431. FLAG and ssrA sequences were incorporated into oligonucleotides SMP426/SMP427 and 431, respectively. Oligonucleotides SMP425 and SMP431 were used to generate the pyopH-FLAG-mCherry-ssrA cassette. PCR product was digested with BamHI and EcoRI and cloned into similarly digested pMMB67EH. Site directed mutagenesis using oligonucleotides SMP437 and SMP438 was used to generate the AAV variant of the ssrA tag.

Visualization of Yersinia T3SS cargo secreted in broth culture by Coomassie staining of SDS-PAGE separated proteins was performed as previously described with some modifications (Auerbuch et al., 2009). After low calcium medium cultures were grown for 1.5 h at 26°C, compounds or DMSO were added and the cultures shifted to 37°C for another 2 h. Post-incubation cultures were spun down at 15,000 × g for 10 min at room temperature. Supernatants were transferred to a new eppendorf tube. Ten percent final trichloroacetic acid (TCA) was added and the mixture was vortexed vigorously. Samples were incubated on ice for 20 min and then spun down at 15,000 × g for 15 min at 4°C. The pellet was resuspended in final sample buffer (FSB) with 20% DTT. Samples were boiled for 15 min prior to running on a 12.5% SDS-PAGE gel. Sample loading was normalized for bacterial culture density (OD₆₀₀) measured prior to centrifugation. Densitometric quantification of the bands was done using Image Lab software (Bio-Rad), setting the relevant DMSO-treated YopE band to 1.00.

The miniaturized secretion assay was done by using Y. pseudotuberculosis expressing a YopM-β-lactamase (YopM-Bla) reporter and the chromogenic β-lactamase substrate nitrocefin to detect secreted YopM-Bla (O'Callaghan et al., 1972; Lee et al., 2007; Green et al., 2016). Y. pseudotuberculosis Δyop6 pYopM-Bla and ΔyscNU pYopM-Bla (negative control) were grown overnight at 26°C with 250 rpm shaking in 2xYT supplemented with chloramphenicol (25 μg/ml). The bacterial culture was diluted in low calcium media to OD₆₀₀ 0.2, incubated at 26°C with 250 rpm shaking for 1.5 h, and 25 μl was distributed into a 384-well plate (Nunc^TM, Thermo Fisher). Fifty micromolars of compounds or equivalent volume of DMSO was added to each well and the plate incubated at 37°C with 250 rpm shaking for 2 h. The bacterial plate was spun down for 5 min and 10 μl of supernatant transferred to a new plate containing the same volume of nitrocefin (500 μg/ml). Absorbance at 490 nm was measured 30 min after addition of nitrocefin. Three technical replicates for each sample were carried out for each independent experiment.

Indicated concentrations of compounds, or the equivalent volumes of DMSO, were added to 26°C-grown low calcium medium cultures and treated cultures were shifted to 37°C for 3 h. RNA was isolated using an RNeasy Plus Micro Kit (Qiagen) according to the manufacturer's instructions and 2 μg RNA was used to make cDNA, as previously described (Auerbuch et al., 2009; Miller et al., 2014). SYBR Green PCR master mix (Applied Biosystems) was used for qPCR reactions according to the manufacturer's instructions and a 60°C annealing temperature, using the 16s rRNA gene as a reference for each sample. Three technical replicates were averaged for each sample/primer pair per independent experiment. Primers used are listed in Table 3. Results were analyzed using the Bio-Rad CFX software.

Indicated concentrations of compounds, or the equivalent volumes of DMSO, were added to 26°C-grown low calcium medium cultures and treated cultures were shifted to 37°C for 3 h. Two-hundred microliters of cultures were spun down at 3,000 × g for 5 min, resuspended in 200 μl 1X PBS, and mCherry fluorescence and optical density measured in black, clear bottom 96 well plates (Costar®, Corning Inc.) on a Perkin Elmer Victor X3 plate reader. Two technical replicates were averaged for each sample per independent experiment.

Quantification of YscF staining on the bacterial surface was carried out as described previously (Morgan et al., 2017). Indicated concentrations of compounds, or the equivalent volumes of DMSO, were added to 26°C-grown low calcium medium cultures and treated cultures were shifted to 37°C for 3 h. Bacteria were fixed by the addition of a mix of 800 μl 4% paraformaldehyde, 1 μl 25% glutaraldehyde, and 40 μl 0.5 M NaPO₄ pH 7.4 to 500 μl of bacterial culture for 15 min at room temperature followed by 30 min or longer on ice. Fixed bacteria were gently sedimented (5,000 × g for 5 min), washed four times with PBS and stored in 250 mM glucose, 10 mM Tris-HCl pH 7.5, and 1 mM EDTA. Eight microliters of the fixed cells were added to coverslips and allowed to set until just dried. Coverslips were blocked overnight with PBST with 3% BSA (PBST/BSA) at 4°C. Blocking solution was removed and anti-YscF primary antibody was added at 1:10,000 in PBST/BSA and rocked at 4°C for 4 h. Coverslips were carefully rinsed in ice cold PBST with 0.1% Tween-20 several times and incubated with Alexa fluor 594 or 488 anti-rabbit secondary antibody (Invitrogen) at 1:10,000 in PBST/BSA and rocked at 4°C for 3 h followed by rinsing again in ice cold PBST with 0.1% Tween-20 several times. Coverslips were then stained for nuclear material with Hoechst 33342 (Thermo Scientific) at 1:10,000 in PBST/BSA and left in the dark at room temperature for 30 min. Coverslips were washed in ice cold PBST with 0.1% Tween-20 several times, allowed to dry briefly, mounted onto glass coverslips with Prolong Gold (Thermo Scientific), and sealed with clear nail polish. Images were taken with the Zeiss Axioimager Z2 widefield microscope under 63X/1.4 oil immersion using Zen software, pseudocolored, and merged in FIJI. YscF puncta were counted using IMARIS software.

Y. enterocolitica expressing EGFP-YscD were grown overnight in brain heart infusion (BHI) broth containing nalidixic acid (35 μg/ml) and diaminopimelic acid (80 μg/ml) (Diepold et al., 2010). Overnight cultures were diluted to an OD₆₀₀ of 0.2 in M9 minimal medium supplemented with casamino acids. Cultures were grown for 1.5 h at 26°C. Compounds were added at indicated concentrations or the equivalent volumes of DMSO were added and the cultures switched to 37°C for another 3 h. Two microliters of culture were layered on a patch of 1% agarose in water (Skinner et al., 2013) supplemented with 80 μg/ml diaminopimelic acid, 5 mM EDTA, 10 mM MgCl₂, and either compounds at indicated concentrations or the equivalent volume of DMSO (Diepold et al., 2010). Images were taken with a Zeiss Axioimager Z2 widefield microscope under 63X/1.4 oil immersion using Zen software, pseudocolored, and merged in FIJI. YscD puncta were counted using IMARIS software.

Y. pseudotuberculosis YpIII/pIBX_{N = 1} and YpIII/pIBX were grown overnight in 2xYT media containing kanamycin (30 μg/ml). Overnight cultures were diluted to an OD₆₀₀ of 0.2 in M9 supplemented with casamino acids. Cultures were grown for 1.5 h at 26°C. Compounds were added at indicated concentrations or the equivalent volumes of DMSO were added and the cultures switched to 37°C for another 3 h. Two hundred microliters of each culture was added to clear bottom, white 96 well plates (Costar®, Corning Inc.) and optical density and luminescence were measured using a Perkin Elmer Victor X3 plate reader. Two technical replicates were averaged for each sample per independent experiment.

Measuring translocation of the YopM-β-lactamase (YopM-Bla) reporter effector protein was carried out as previously described (Duncan et al., 2014). A total of 6 × 10³ CHO-K1 cells were plated in each well of a 384-well plate (Corning^TM Falcon^TM) in 50 μl of F-12K medium plus 10% FBS and 1% glutamine, and incubated overnight. The following day, indicated concentrations of compounds, or the equivalent volumes of DMSO, were added to 26°C-grown low calcium medium cultures of Y. pseudotuberculosis expressing YopM-Bla and treated cultures were shifted to 37°C for 3 h. Immediately prior to infection of CHO-K1 cells, the F-12K media was removed and replaced with RPMI plus 10% FBS and compounds or DMSO were added. Y. pseudotuberculosis was added to the CHO-K1 plate at an MOI of 7. Five minutes after this transfer, the plate was centrifuged at 290 × g for 5 min to initiate bacterium-host cell contact and incubated for 1 h at 37°C and 5% CO₂. Thirty minutes prior to the end of the infection, CCF2-AM (Invitrogen) was added to each well, and the plate covered in foil and incubated at room temperature. At the end of the infection, the medium was aspirated and fresh 4% paraformaldehyde added to each well for 20 min to fix the cells. The paraformaldehyde was then aspirated and the DNA dye DRAQ5 (Cell Signaling Technology) in PBS was added to each well. The monolayers were incubated at room temperature for 10 min, washed once with PBS, and visualized using an ImageXpressMICRO automated microscope and MetaXpress analysis software (Molecular Devices). The number of YopM-Bla-positive cells was calculated by dividing the number of blue (CCF2-cleaved) cells by the number of green (total CCF2) cells. Three technical replicates were averaged for each sample per independent experiment.

Overnight cultures of Y. pseudotuberculosis were diluted to an OD₆₀₀ of 0.1 in 2xYT and 200 μl was added to each well of a 96-well plate (Corning^TM Falcon^TM). Compounds were added at 50 μM and kanamycin at 50 μg/ml, plates incubated at 26°C, and the OD₆₀₀ of the cultures measured every hour for 13 h using a VersaMax Tunable Microplate Reader (Molecular Devices). The 96-well plates were intermittently shaken throughout the experiment. One technical replicate was used for each sample per independent experiment.

Following overnight growth, 1 μl of Y. pseudotuberculosis overnight culture was spotted onto motility medium containing either 1% tryptone/0.25% agar or 1% tryptone/0.25% agar supplemented with 5 mM EGTA and 20 mM MgCl₂ in six-well plates. Each well contained either 0.3% DMSO or 50 μM INP0007 or INP0010. The plates were incubated at 26°C for 24 h before the diameter of swimming motility was measured. One technical replicate was used for each sample per independent experiment.

We selected a training set of commercially-available compounds to validate our assay pipeline (Table 4). All compounds selected met two strict requirements: demonstrated inhibitory activity on the Yersinia T3SS and absence of bactericidal activity within 6 h to accommodate the timeframe of the pipeline assays (Figures 1A–C).

Compound 4 (C4), a haloid-containing sulfonamidobenzamide, was identified through a luciferase-based T3SS gene promoter fusion screen as a potent inhibitor of T3SS gene expression (Kauppi et al., 2003). We chose this compound because it was shown, albeit modestly, to inhibit expression of the LcrF master regulator of the T3SS in Yersinia (Kauppi et al., 2003) and we therefore expected that it would have a broad impact on all stages of type III secretion downstream of T3SS expression in our assay pipeline. From the same high throughput screening strategy that identified C4, two members of the salicylidene acylhydrazide class of inhibitors, INP0007 (Kauppi et al., 2003) and INP0010 (Nordfelth et al., 2005), were identified. Despite a large number of studies, the mechanism of action of these compounds remains unclear and there is evidence of off target effects on global virulence gene expression by at least some salicylidene acylhydrazides (Tree et al., 2009). In fact, INP0010 decreased Yersinia growth starting 7 h after initiation of treatment (Figure 1D), consistent with previous reports (Veenendaal et al., 2009). Compound 3 (C3), a malic diamide, was shown to inhibit ExoS secretion in P. aeruginosa and YopE secretion in Yersinia pestis (Aiello et al., 2010). This compound is structurally related to another class of T3SS inhibitors, the phenoxyacetamides, which were proposed to target the PscF needle subunit in Pseudomonas (Bowlin et al., 2014). Based on this structural relatedness, C3 was expected to disrupt T3SS needle assembly without impacting T3SS gene expression. Compound 20 (C20) was identified in a T3SS effector protein-β-lactamase reporter translocation screen, but had no significant effect on the ability of the bacteria to secrete T3SS effectors in the absence of host cells, pointing to the possibility that this compound specifically blocks the bacteria–host cell interaction (Harmon et al., 2010). Therefore, C20 was expected to inhibit the translocation of effector proteins into host cells but not impact T3SS gene expression and assembly.

An advantage of using Ysc-expressing Yersinia for our assay pipeline is that removing calcium from the culture medium enables T3SS effector proteins to be secreted into the supernatant in the absence host cells (Yother and Goguen, 1985; Perry et al., 1986; Straley and Bowmer, 1986; Sample et al., 1987; Forsberg and Wolf-Watz, 1988; Mehigh et al., 1989), providing a useful method of monitoring efficiency of T3SS inhibition in order to validate use of a given inhibitor in our assay pipeline. As expected, all test compounds except C20 exhibited significant inhibition of effector Yop secretion (Figures 1A,B). Importantly, this secretion assay was used routinely to ensure the efficacy of each batch of compound purchased and after 1 month in storage. Therefore, even when a compound had no effect in a particular assay, we could be confident that it was still active as a T3SS inhibitor.

We reasoned that inhibition of the pYV copy number upregulation that occurs during normal induction of type III secretion in Yersinia may impact T3SS gene expression, and therefore affect overall T3SS activity. To test this, we employed a Y. pseudotuberculosis strain with a luciferase reporter gene cluster integrated onto the pYV virulence plasmid (YpIII/pIBX) (Fahlgren et al., 2014). As a negative control, we used a strain in which a replication deficient copy of the virulence plasmid as well as the luciferase gene cluster was integrated into the chromosome (referred to as YpIII/pIBX_{N = 1}) (Wang et al., 2016). In this YpIII/pIBX_{N = 1} strain, the ratio of T3SS and luciferase reporter genes vs. chromosomal genes remains constant even upon induction of type III secretion (Wang et al., 2016). As expected, expression of lcrF, the effector protein yopE, the integral IM ring protein yscD, and the needle subunit yscF were significantly decreased in the YpIII/pIBX_{N = 1} strain compared to the YpIII/pIBX strain (Figure 2A). While these data demonstrate that inhibition of pYV gene copy number could impact pYV gene expression, four of the five compounds in our training set did not significantly affect virulence plasmid copy number as measured by luciferase activity, while INP0007 significantly increased copy number (p = 0.0054; Figure 2B) for reasons that are unclear but that may reflect off target effects. In contrast, luciferase expression could be inhibited by blocking general transcription or translation using rifampicin or chloramphenicol (Figure 2B). Therefore, while inhibiting pYV copy number in Yersinia causes a decrease in T3SS activity, we conclude that none of the compounds in our training set act by inhibiting upregulation of pYV copy number.

In order to determine if the training set compounds negatively impacted T3SS gene expression, we used a Yersinia reporter strain containing an unstable YopH-mCherry-AAV transcriptional reporter (Andersen et al., 1998). C4, INP0007, and INP0010 inhibited YopH-mCherry-AAV expression when T3SS activity was induced by incubation in low calcium medium at 37°C (Figure 3A). In order to validate and extend these results, we measured transcript levels of T3SS and non-T3SS genes by qPCR. Consistent with the YopH-mCherry-AAV data, C3 and C20 had no significant impact on gene expression (Figure 4). However, C4 inhibited expression of all T3SS genes tested, but did not impact expression of two non-T3SS-associated genes: the iron sulfur cluster loading protein erpA and the small ribosomal subunit L9. Interestingly, INP0007 and INP0010 significantly inhibited transcript levels of lcrF, the needle tip subunit lcrV, and effector proteins yopH, yopE, and yopK, but did not inhibit expression of the ATPase yscN, yscF, and yscD under these conditions (Figure 4). To test whether inhibition of secretion via the positive feedback loop indirectly inhibits T3SS gene expression, we measured YopH-mCherry fluorescence and yscD mRNA levels via qPCR during growth at 37°C in high calcium (Figures 3B,C). Under these conditions, Yersinia can assemble the T3SS (Diepold et al., 2010), but no secretion of Yop effectors occurs (Forsberg and Wolf-Watz, 1988); therefore, the positive feedback loop is not active in high calcium. While overall expression levels of YscD and YopH are lower in high calcium compared to low calcium conditions, C4, INP0007, and INP0010 did not significantly decrease YopH-mCherry-AAV fluorescence in high calcium conditions (Figure 3B), suggesting an indirect effect of these compounds on T3SS gene expression through inhibition of secretion in low calcium conditions. Likewise, yscD levels were not decreased by C4 in high calcium (Figure 3C). Importantly, yscD expression was significantly lower in the ΔlcrF mutant compared to WT under high calcium conditions in the absence of compound. These data indicate that C4 is not likely to target LcrF activity, as yscD is predicted to be under LcrF control (Schwiesow et al., 2015).

In order to monitor T3SS assembly, we used fluorescence microscopy to quantify YscD localization in Y. enterocolitica and YscF puncta formation in Y. pseudotuberculosis as a proxy for T3SS basal body and T3SS needle assembly, respectively, as previously described (Davis and Mecsas, 2007; Diepold et al., 2010; Morgan et al., 2017). Only INP0007 significantly impacted YscD-EGFP puncta formation (Figure 5). Importantly, the level of diffuse fluorescence in INP0007-treated Yersinia was greater than that of the non-T3SS-inducing condition (26°C-grown Yersinia; Figure 5A, inset), indicating that while YscD is expressed at 37°C in the presence of INP0007, it is not assembled into basal bodies in the presence of this compound. In contrast, while C3 and C20 had no significant effect on YscF puncta formation as measured with an anti-YscF antibody, C4, INP0007, and INP0010 significantly inhibited needle assembly (Figure 6).

The bacterial flagellar apparatus is composed of a basal body that is structurally related to the injectisome T3SS basal body and mediates secretion of the flagellar hook and filament proteins (Macnab, 2004). Therefore, it is possible that compounds with the ability to inhibit injectisome T3SS basal body assembly may inhibit flagellar assembly and therefore flagellar motility. INP0007 has been shown to inhibit motility in Yersinia (Kauppi et al., 2003), but neither INP0007 nor INP0010 inhibited motility in Salmonella (Negrea et al., 2007). In our standard Yersinia motility agar (Morgan et al., 2017), INP0007 significantly decreased flagellar motility in Y. pseudotuberculosis, while INP0010 decreased motility weakly albeit significantly (Figure 7). Addition of the chelating agent EGTA in the motility agar, as used in the Kauppi et al study (Kauppi et al., 2003), led to an even greater inhibitory effect on motility by INP0010 (Figure 7). The Y. pseudotuberculosis flhDC^Ypestis non-motile strain served as the negative control for the motility assay. Taken together, these data indicate that INP0007 and INP0010 impact both the flagellar and injectisome T3SS.

While the Yop in vitro secretion assay shown in Figure 1 is critical for assessing the impact of a compound on overall Yop secretion, the method used (Coomassie staining of SDS-PAGE separated proteins precipitated from the culture supernatant) is not amenable to a high throughput format. In order to assess Yop secretion in vitro in microtiter plates, Y. pseudotuberculosis expressing a YopM-β-lactamase (YopM-Bla) reporter was used in conjunction with the chromogenic β-lactamase substrate nitrocefin (O'Callaghan et al., 1972; Lee et al., 2007; Green et al., 2016). Consistent with the block in Yop secretion observed in Figure 1, C3, C4, INP0007, and INP0010 significantly blocked YopM-Bla secretion into the culture supernatant while C20 did not (Figure 8). Surprisingly, INP0010 blocked YopM-Bla secretion by only 32% as measured using nitrocefin (Figure 8), yet appeared to have a much more significant effect on native YopM secretion as measured using Coomassie staining (Figure 1). The reason for this discrepancy is unclear; however, we note that INP0010 has cytotoxic effects on bacteria and eukaryotic cells (see below) and is likely to have the greatest off target effects of the compounds in our training set.

One of the most important requirements of a T3SS inhibitor is its ability to prevent translocation of effector proteins into target host cells. We employed a different type of YopM-Bla reporter assay to assess translocation of effector proteins into CHO K1 cells loaded with CCF2, a fluorescent β-lactamase substrate that can enter eukaryotic cells (Dewoody et al., 2011). While C3, C4, C20, INP0007, and INP0010 significantly blocked YopM-Bla translocation at 50 μM, INP0010 appeared to be cytotoxic at this concentration (Figure 9).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.