DOI: 10.5061/dryad.fj6q57443
title: TLR priming licenses NAIP inflammasome activation by immunoevasive ligands

description: NLR family, apoptosis inhibitory proteins (NAIPs) detect bacterial
 flagellin and structurally related components of bacterial type III
 secretion systems (T3SS), and recruit NLR family, CARD domain containing
 protein 4 (NLRC4) and caspase-1 into an inflammasome complex that induces
 pyroptosis. NAIP/NLRC4 inflammasome assembly is initiated by the binding
 of a single NAIP to its cognate ligand, but a subset of bacterial
 flagellins or T3SS structural proteins are thought to evade NAIP/NLRC4
 inflammasome sensing by not binding to their cognate NAIPs. Unlike other
 inflammasome components such as NLRP3, AIM2, or some NAIPs, NLRC4 is
 constitutively present in resting macrophages and not known to be induced
 by inflammatory signals. Here, we demonstrate that Toll-like receptor
 (TLR)-dependent p38 mitogen-activated protein kinase signaling
 up-regulates NLRC4 transcription and protein expression in murine
 macrophages, which licenses NAIP detection of evasive ligands. In
 contrast, TLR priming in human macrophages did not up-regulate NLRC4
 expression, and human macrophages remained unable to detect NAIP-evasive
 ligands even following priming. Critically, ectopic expression of either
 murine or human NLRC4 was sufficient to induce pyroptosis in response to
 immunoevasive NAIP ligands, indicating that increased levels of NLRC4
 enable the NAIP/NLRC4 inflammasome to detect these normally evasive
 ligands. Altogether, our data reveal that TLR priming tunes the threshold
 for the murine NAIP/NLRC4 inflammasome to enable inflammasome responses
 against immunoevasive or suboptimal NAIP ligands. These findings provide
 insights into species-specific TLR regulation of NAIP/NLRC4 inflammasome
 activation.
description: <strong>LDH cytotoxicity assays</strong>
 After infection, cells were spun at 250<em>g </em>for
 10 minutes to pellet cellular debris. Supernatants were removed and used
 to assess cytotoxicity via lactate dehydrogenase (LDH) activity. LDH
 release was quantified using an LDH Cytotoxicity Detection Kit (Roche).
 Samples were incubated for 25 minutes at room temperature and absorbance
 at 490 nm was assessed using a spectrophotometer. Percent cytotoxicity was
 calculated after normalizing to uninfected controls and 100% cell death,
 which is based on 1% triton X-100-treated cells. 
 <strong>ELISA</strong>
 Supernatants from in vitro infections and murine serum from in
 vivo infected mice were used to assess IL-1β and IL-18 levels,
 respectively. For murine IL-1β and IL-18, ELISAs were performed as
 described in Supplemental Materials and Methods. For measuring human
 IL-1β, an ELISA kit from BD Biosciences was used.
 <strong>Quantitative RT-PCR</strong>
 RNA was isolated using TRIzol reagent (ThermoFisher) from either
 5 x 10<sup>6</sup> BMDMs or THP-1s following the
 manufacturer’s protocol. cDNA was prepared from the RNA samples using the
 high-capacity cDNA reverse transcription kit (Applied Biosystems) per
 manufacturer’s protocol. Quantitative PCR was conducted with the QuantBio
 Studio 6 Flex Real-Time PCR system using the PerfeCTa SYBR Green SuperMix
 (QuantaBio). For analysis, mRNA levels of siRNA-treated cells were
 normalized to housekeeping gene <em>GAPDH</em> (murine) or
 <em>HPRT </em>(human) and fold induction was determined using
 the 2<sup>−ΔΔCT</sup> (cycle threshold) method
 Graphing and statistical analyses of data were performed using
 Prism 9 software (GraphPad). Statistical significance was determined using
 the statistical tests indicated in each figure legend. Differences were
 considered statistically significant if the <em>P </em>value
 was less than or equal to 0.05
description: **TLR priming licenses NAIP inflammasome activation by immunoevasive
 ligands**
 [https://doi.org/10.5061/dryad.fj6q57443](https://doi.org/10.5061/dryad.fj6q57443) ## Description of the data and file structure **Figure legends** **Figure 1. C-terminal glutamine residues in flagellin disable recognition by the NAIP/NLRC4 inflammasome.** (A-B) WT, *Nlrc4-/-,* and *Casp1/11-/-* murine BMDMs were infected with ΔyopJ *Yp* expressing either YopE1-100 or YopE1-100 fused to the C-terminal D0 portion of FliC from *S.* Tm or EPEC for four hours at an MOI of 20. (A) % cytotoxicity was measured via lactate dehydrogenase (LDH) release. (B) IL-1β release (ng/mL) was measured by ELISA. (C) BMDMs were infected with the indicated strains for two hours at an MOI of 20. Combined supernatants and cellular lysates were analyzed by immunoblotting for Casp1, GSDMD, and β−actin (loading control). Representative of three independent experiments. (D, E) WT murine BMDMs were infected with the indicated strains for four hours at an MOI of 20. (D) % cytotoxicity was measured via LDH release. (E) IL-1β release (ng/mL) was measured by ELISA. (F) BMDMs were infected with the indicated strains for one hour at an MOI of 20. Combined supernatants and whole cell lysates were analyzed by immunoblotting for Casp1, GSDMD, and β−actin (loading control). Representative of three independent experiments. (A, D) Data shown are the pooled means ± SEM from three independent experiments. Paired *t* test was performed to assess statistical significance. (B, E) Data shown are the pooled means ± SEM from three independent experiments. Statistical significance was measured by performing an unpaired *t* test. ND=not detected; ns=not significant; *=*P*&lt;0.05; **=*P*&lt;0.01; ***=*P*&lt;0.001. **Figure 2. TLR priming licenses NAIP/NLRC4 inflammasome activation by immunoevasive NAIP ligands.** (A and B) WT, *Nlrc4-/-*, *Casp1/11-/-*, and *Naip5-/-* murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4 (Pam3) for 16 hours were infected with the indicated strains for four hours at an MOI of 20. (A) Cell death (% cytotoxicity) was measured via lactate dehydrogenase (LDH) release. (B) IL-1β release (ng/mL) was measured by ELISA. (C) BMDMs were infected with the indicated strains for four hours at an MOI of 20, TCA-precipitated supernatants and whole cell lysates were collected and combined. Samples were analyzed by immunoblot for Casp1, GSDMD, and β−actin (loading control). Image representative of three independent experiments. (D and E) WT murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4 for 16 hours were infected with the indicated strains for two hours at an MOI of 20. (D) % cytotoxicity was measured via LDH release. (E) IL-1β release (ng/mL) was measured by ELISA. (F) BMDMs were infected with the indicated strains for four hours at an MOI of 20. Combined supernatants and whole cell lysates were analyzed by immunoblotting for Casp1, GSDMD, and β−actin (loading control). Image representative of three independent experiments. (A and D) Data shown are pooled means ± SEM from three independent experiments. A paired *t* test was performed to assess statistical significance. (B, G) Data shown are representative of three independent experiments and are the combined means ± SEM. Statistical significance was measured by performing an unpaired t test. ns=not significant; *=*P*&lt;0.05; **=*P*&lt;0.01; ***= *P*&lt;0.001; ****=*P*&lt;0.001. **Figure 3. TLR priming upregulates NLRC4 expression via p38 MAPK signaling.** (A, B) WT murine BMDMs were unprimed or primed for 16hrs with (A) 0.5 μg/mL Pam3CSK4 or (B) 0.25 μg/mL, 0.5 μg/mL, or 1 μg/mL Pam3CSK4. *Nlrc4*,* Naip1*,* Naip2*, *Naip5*, and *Naip6* transcripts were assessed via qRT-PCR and fold change was assessed relative to unstimulated BMDMs (A) NLRC4 protein levels were assessed via immunoblot (B) Data representative of three independent experiments. (C-E) WT murine BMDMs were unprimed or primed with 0.5 μg/mL Pam3CSK4. One hour prior to priming, the BMDMs were treated with 2.5 μM, 5 μM, or 10 μM p38 MAPK inhibitor SB202190 or DMSO vehicle control. (C) *Nlrc4* transcript levels relative to *Gapdh* were assessed via qRT-PCR. Fold change was determined relative to unstimulated control. (D) NLRC4 protein levels were analyzed via immunoblot. NLRC4 protein intensity was quantified relative to loading controls, and average fold change ± SEM in NLRC4 protein levels was assessed relative to unstimulated DMSO-treated cells. Data representative of three independent experiments. (E) % cytotoxicity was measured via LDH release two hours post-infection with the indicated strains at an MOI of 20. (F) Immortalized ER-Hoxb8 *Nlrc4-/-* murine macrophages were transduced with the retroviral mscv2.2 vector alone (mscv2.2) or mscv2.2 overexpressing *Nlrc4* (mscv2.2 *Nlrc4*). WT macrophages, *Nlrc4-/-* macrophages transduced with mscv2.2, or *Nlrc4-/-* macrophages transduced with mscv2.2 *Nlrc4* *were* infected with indicated Yp strains expressing YopE1-100, *S*. Tm FliCD0, and EPEC FliCD0 for two hours at an MOI of 20. % cytotoxicity was measured via LDH release. Only WT ER-Hoxb8 cells were primed for 16 hours with Pam3CSK4. (A, C, E, F) Data shown are representative of three experiments. Means ± SEM are shown (n=3) and statistical significance was measured by performing an unpaired *t* test (A, F) or 1-way ANOVA (C-E). ND=not detected; ns=not significant; **=*P*&lt;0.01; ***=*P*&lt;0.001; ****=*P*&lt;0.0001 **Figure 4. The human NAIP/NLRC4 inflammasome does not respond to TLR priming to detect evasive flagellin.** (A) WT, *Nlrc4-/-*, or *Naip-/-* THP-1 macrophages were primed with 0.1 μg/mL Pam3CSK4 for 16 hours and infected with WT *Yp* expressing either YopE1-100, *S.* Tm FliCD0, or EPEC FliCD0 at an MOI of 60 for six hours. IL-1β release (ng/mL) was measured by ELISA. (B, C) WT THP-1 macrophages were primed with 0.1 μg/mL (B, C) or 0.5 μg/mL (C) Pam3CSK4, 0.05 μg/mL Pam2CSK4, 100 ng/mL LPS, or 100 ng recombinant human IFN-γ for 16 hours. (B) Nlrc4, Naip, and *Il6* transcript levels relative to *Hprt* were assessed via qRT-PCR. Fold change in transcript levels was analyzed relative to unstimulated control. (C) NLRC4 protein levels were analyzed via immunoblot. NLRC4 protein intensity was quantified relative to loading controls, and fold change in NLRC4 protein levels was assessed relative to unstimulated cells. Data representative of three independent experiments. (D) Primary hMDMs from three healthy human donors were primed with 0.1 μg/mL Pam3CSK4 for 16 hours and infected with indicated strains at an MOI of 60 for 6 hours. IL-1β release (pg/mL) was measured by ELISA. Each dot represents the mean of each donor derived from triplicate wells. Bars depict the mean of three donors. (E) Immortalized ER-Hoxb8 *Nlrc4-/-* murine macrophages were transduced with the retroviral mscv2.2 vector alone or mscv2.2 overexpressing human *NLRC4 *(mscv2.2 h*NLRC4*). WT, *Nlrc4-/-* mscv2.2, or *Nlrc4-/-* mscv2.2 *hNLRC4* macrophages were infected with indicated strains for two hours at an MOI of 20. % cytotoxicity was measured via LDH release. Only WT Hoxb8 cells were primed for 16 hours with Pam3CSK4. (F) THP-1 human monocyte-derived macrophages transduced with the MigR1 retroviral vector alone or overexpressing h*NLRC4* were primed with 0.1 μg/mL Pam3CSK4 for 16 hours and infected with WT *Yp* expressing either YopE1-100, *S.* Tm FliCD0, or EPEC FliCD0 at an MOI of 60 for six hours. IL-1β release (ng/mL) was measured by ELISA. (A-F) Data shown are representative of three independent experiments. Means ± SEM are shown (n=3) and statistical significance was measured by performing an unpaired *t* test (A, C, E, F) or paired t test (D) or 1-way ANOVA (B). ND=not detected; ns=not significant; *=*P*&lt;0.05; **=*P*&lt;0.01; ***=*P*&lt;0.001; ****=*P*&lt;0.0001. **Figure 5. TLR priming overcomes evasion of the NAIP/NLRC4 inflammasome during infection.** (A) WT murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4 for 16 hours were infected with WT, *ΔfljB*, or *ΔfljB* *fliCR475QG* *S.* Tm for one hour at an MOI of 20. % cytotoxicity was measured via LDH release. (B) WT or *Naip-/-* THP-1 human macrophages were primed with 0.1 μg/mL Pam3CSK4 for 16 hours and infected with WT, *ΔfljB*, or *ΔfljB* *fliCR475QG* *S.* Tm for six hours at an MOI of 60. IL-1β release (pg/mL) was measured by ELISA. (C, D) Total peritoneal exudate cells were harvested from WT C57Bl/6J mice intraperitoneally injected with heat-killed *S.* Tm (green bars) or PBS (gray bars) and then infected with the indicated *Yp* strains for 2 hours at an MOI of 20. (C) % cytotoxicity was measured via LDH release. (D) IL-1β release (pg/mL) was measured by ELISA. Each dot represents the mean of one mouse derived from triplicate wells. n=7 for PBS-treated mice and n=4 for heat-killed *S.* Tm-treated mice. Bars represent the means of all mice and statistical analyses were performed using a Mann-Whitney test. (E) WT C57BL/6J mice were intraperitoneally infected with 500 CFUs of either *ΔfljB* or *ΔfljB* *fliCR475QG* *S*. Tm for 48 hours. Serum IL-18 levels (pg/mL) were measured by ELISA. Data shown are combined ± SEM from two independent experiments. Unpaired *t* test was performed to assess statistical significance. (A) Data shown are means ± SEM pooled from three independent experiments and a paired *t* test was performed to assess statistical significance. (B) Data shown are representative of one out of at least three independent experiments. Means ± SEM are shown (n=3) and statistical significance was measured by performing an unpaired *t* test. ns=not significant; *=*P*&lt;0.05; **=*P*&lt;0.01. **Figure S1. Design of Yersinia pseudotuberculosis T3SS ligand delivery system.** (A) A heterologous delivery system using the T3SS of the enteric gram-negative pathogen *Yersinia pseudotuberculosis* (*Yp*) to deliver NAIP ligands was made by fusing the C- terminal FliCD0 region to the N-terminal 100 amino acids of the T3SS-secreted *Yersinia* outer protein E (YopE1-100). This fusion protein of YopE1-100 with *S*. Tm or EPEC FliCD0 is under the control of the native yopE promoter. (B) Coomassie stain of exoproteins of Yersinia induced for T3SS and effector expression. Image is representative of three independent experiments. (C) List of mutants generated to assess contribution of C-terminal residues in *S.* Tm or EPEC FliC D0. Red indicates the base pair mutations compared to the WT sequence. **Figure S2. TLR2 and TLR4 priming enhance the inflammasome response to EPEC FliCD0 in mouse macrophages.** (A and B) WT murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4, 0.05 μg/mL Pam2CSK4, 100ng/mL LPS, or 100ng IFN-γ for 16 hours were infected with the indicated strains for four hours at an MOI of 20. (A) % cytotoxicity was measured via lactate dehydrogenase (LDH) release. (B) IL-1β release (ng/mL) was measured by ELISA. (C) Quantification of protein levels of caspase-1 p20and Gasdermin D p30 as shown in Fig. 2C by band intensity, normalized to β−actin expression. (A) Data shown are means ± SEM pooled from three independent experiments for unprimed, Pam3CSK4-primed, Pam2CSK4-primed, or LPS-primed conditions or two independent experiments for IFN-γ-primed condition. A paired *t* test was performed to assess statistical significance. (B) Data shown are representative of three independent experiments. Combined means ± SEM are shown (n=3). Statistical significance was measured by performing an unpaired *t* test. (C) Data shown are pooled from three independent experiments. Statistical significance was measured by a paired *t* test. ND=not detected; ns=not significant; *=*P*&lt;0.05; **=*P*&lt;0.01; ***=*P*&lt;0.001; ****=*P*&lt;0.0001. **Figure S3. TLR-mediated detection of EPEC FliCD0 by the NAIP/NLRC4 inflammasome is independent of NLRP3 and ASC.** (A-F) WT, *Myd88-/-*, *Nlrp3-/-*, or *Asc-/-* murine unprimed BMDMs or primed with 0.5 μg/mL Pam3CSK4 for 16 hours were infected with the indicated strains for four hours at an MOI of 20. (A, C, E) % cytotoxicity was measured via LDH release. (B, D, F) IL-1β release (ng/mL) was measured by ELISA. (A, C, E) Data shown are means ± SEM pooled from three independent experiments. A paired *t* test was performed to assess statistical significance. (B, D, F) Data shown are representative of three independent experiments, means ± SEM are shown (n=3) and statistical significance was measured by performing an unpaired *t* test. ND= not detected; ns=not significant; *=*P*&lt;0.05; **=*P*&lt;0.01; ***=*P*&lt;0.001; ****=*P*&lt;0.0001. **Figure S4. TLR priming increases NLRC4 transcript and protein levels in macrophages.**(A) WT murine BMDMs were unprimed or primed with 0.5 μg/mL Pam3CSK4 for 16 hours. NLRC4 transcript levels relative to *Gapdh*, *Actb*, or *Hprt* were assessed via qRT-PCR. Fold change was quantified relative to unprimed control. (B, C) WT murine BMDMs were unprimed or primed with 0.5 μg/mL Pam3CSK4, 0.05 μg/mL Pam2CSK4, 100ng/mL LPS, or 100ng IFN-γ for 4, 8, or 16 hours. (B) *Nlrc4* transcript levels relative to *Gapdh* were assessed via qRT-PCR. Fold change in *Nlrc4* transcript levels was determined relative to the unstimulated control. (C) NLRC4 protein levels were analyzed via immunoblot. Data representative of three independent experiments. (A, B) Data representative of three independent experiments. Means ± SEM are shown (n=3) and statistical significance was measured by performing a 1-way ANOVA (C-E). ns=not significant; *=P&lt;0.05;**=*P*&lt;0.0001. **Figure S5. p38 activation in response to TLR priming increases NLRC4 expression** (A-C) WT murine BMDMs were unprimed or primed with 0.5 μg/mL Pam3CSK4. One hour prior to priming, BMDMs were treated with 2.5 μM, 5 μM, or 10 μM p38 inhibitor SB202190 or DMSO vehicle control. (A, B) *Il6* or *Nlrc4* transcript levels relative to *Gapdh* were assessed via qRT-PCR. Fold change was analyzed as relative to unstimulated control. (C) NLRC4 protein levels were analyzed via immunoblot. NLRC4 protein intensity was quantified relative to β−actin loading controls, and fold change was assessed relative to unstimulated DMSO treated cells. Data representative of three independent experiments. (D) NLRC4 protein levels in WT or Nlrc4-/- ER-HoxB8 macrophages transduced with mscv2.2 empty vector or mcsv2.2 NLRC4 were assessed via immunoblot. Data representative of two independent experiments. (E) WT or *Nlrc4-/-* ER-HoxB8 macrophages transduced with mcsv2.2 empty vector or mscv2.2 NLRC4 were treated with 25µM p38 inhibitor SB202190 or vehicle control for 1h, then primed with 0.5µg/ml Pam3CSK4 for 16h. Cells were then infected with *Yp* expressing *S*. Tm FliCD0 or EPEC FliCD0. Cytotoxicity was assayed via LDH. (F) Murine BMDMs from either WT or *Nlrc4*S533A mice were unprimed or primed with 0.5µg/mL Pam3CSK4, then infected with *Yp* expressing YopE1-100, *S.* Tm FliCD0, or EPEC FliCD0 for 4 hours. Cytotoxicity was assessed via LDH. (G) THP-1 derived macrophages and WT murine BMDMs were primed with 0.5µg/mL Pam3CSK4 for 0h, 2h, 4h, or 16h. Total and phosphorylated p38 MAPK protein in cell lysates were assessed via immunoblot. Data representative of two independent experiments. (A-C, E-F) Data representative of three independent experiments. Means ± SEM are shown (n=3) and statistical significance was measured by performing a 1-way ANOVA (A-B, D) or an unpaired t test (E-F). ns=not significant; *=*P*&lt;0.05; **=*P*&lt;0.01; ***=*P*&lt;0.001; ****=*P*&lt;0.0001. **Figure S6. S. Tm ΔfljB fliCR475QG mutant does not have impaired flagellar-based motility.** (A) LB plates containing 0.4% agar were stabbed with optical density-normalized overnight cultures of WT, *ΔfljBfliC*, *ΔfljB*, or *ΔfljB fliC*R475QG *S*. Tm. After 12-16 hours incubation at 37°C, the diameter of the colony was measured to quantify motility. Data shown are pooled from 3 independent experiments, mean ± SEM are shown, and statistical significance was measured by performing a paired *t* test. ns=not significant; **=*P*&lt;0.01. (B) Peritoneal macrophages from naïve WT mice were unprimed or primed with 0.5µg/mL Pam3CSK4 for 16h, and then infected with *Yp* expressing YopE1-100, *S*. Tm FliCD0, or EPEC FliCD0 for 4 hours. Supernatant IL-1β levels (ng/mL) were assayed with ELISA. NLRC4 and β-actin (loading control) protein levels were assessed via Western blot. Data shown are representative of two independent experiments. (C) WT C57BL/6J mice were intraperitoneally infected with 500 CFUs of either *ΔfljB* or *ΔfljB fliC*R475QG *S*. Tm for 48 hours, and total spleen CFUs were assessed. Data shown are combined ± SEM from two independent experiments. ### Files and variables #### File: LDH\_raw\_data.xlsx This file contains all raw lactate dehydrogenase (LDH) cell death assay data, including plate reads and calculations. Final calculations are included as percent(%) cytotoxicity. All data is sorted by figure panels into tabs. The plate maps are annotated in the spreadsheet where relevant. #### File: qPCR\_raw\_data.xlsx This file contains mean CT values and calculations from all quantitative polymerase chain reaction (qPCR) experiments. All data is sorted by figure panels into tabs. #### File: WB\_quantification.xlsx This file contains Western blot band intensity quantifications and normalizations. All data is sorted by figure panels into tabs. #### File: IL1b\_raw\_data.xlsx This file contains raw data and calculations from all enzyme-linked immunosorbent assay (ELISA) experiments quantifying the cytokine interleukin 1 beta (IL-1β). All data is sorted by figure panels into tabs. The units of measurements as well as plate maps are annotated in the spreadsheet where relevant. Wells where the reads are below the limit of detection are annotated by "Range?" in the calculations. #### File: In\_Vivo\_Infections.xlsx This file contains raw data relevant to *in vivo* infections (Fig 5E and Fig S6C). The first tab contains raw data and calculations from enzyme-linked immunosorbent assay (ELISA) experiments quantifying the cytokine interleukin 18 (IL-18) as depicted in Fig 5E. The second tab contains CFU data and calculations from Fig S6C. All units of measurements are noted in the spreadsheets. LLR: S. Typhimurium expressing FliC that ends in -LLR; LLQG: S. Typhimurium expressing FliC that ends in -LLQG. #### File: motility\_.pdf This file contains original images taken for the flagellar motility assay in Figure S6A. ## Code/software No Code or Software other than Microsoft Excel is needed to view this data ## Access information All raw data were generated from new experiments described in the manuscript associated with this dataset. **Cell types are as follows**: murine bone marrow derived macrophages (BMDMs, various genotypes used), human monocyte-derived macrophages (hMDMs), and THP-1 cells (various genotypes). Please disregard any other highlighting or colors, as they were used for personal reference. **The various genotypes for BMDMs and THP-1 cells are as follows**: WT (wild-type cells), NAIP-/-, NLRC4-/-, RIPK3-/-, NLRP3-/-, ASC-/-,CASP1/11 -/-, RIPK3C/CASP8 -/-, NAIP5-/-, MyD88 -/-, MigR1 hNLRC4 (THP1 overexpressing human NLRC4), Nlrc4-/- mscv2.2 hNLRC4 (NLRC4 HoxB8 macrophages expressing human NLRC4). **Infection labels:** Plate maps are denoted by a numbering system in which the first number indicates the bacterial strain, followed by a letter to indicate the stimulation conditions, followed by another number to indicate the time point. The corresponding stimulation/infection key for each experiment are available in the data files. For example, in the file **IL1b_raw_data.xlsx** tab **Figure 1E Il1b**, the plate map code “2B4” indicates that well was infected with “yopJ 3277 yopE Stm FlicD0”, primed with Pam3CSK4, and infected for 4 hours. **Abbreviations:** Pam3 = PAM3CSK4, Pam2 = PAM2CSK4, LPS = lipopolysaccharide, IFNg = interferon gamma, WT = wild-type, LDH = lactate dehydrogenase **Strains:** ∆yopJ 3277 pACYC yopE alone = strain 32777 with a mutated yopJ with the YopE promoter sequence with the first 100bp of yopE expressed in pACYC184 ∆yopJ 3277 yopE Stm FlicD0 = strain 32777 with a mutated yopJ with the YopE promoter sequence with the first 100bp of yopE fused to S. Tm FliCD0 expressed in pACYC184 ∆yopJ 32777 yopE EPEC D0 = strain 32777 with a mutated yopJ with the YopE promoter sequence with the first 100bp of yopE fused to EPEC FliCD0 expressed in pACYC184 SL1344= *Salmonella enterica* serovar Typhimurium yopJ 32777 yopE SSAG/SSAI/PRGI/PRGJ = strain 32777 with the YopE promoter sequence with the first 100bp of yopE fused to *S*. Tm ssaG/ssaI/prgI/prgJ expressed in pACYC184 ∆yopJ 32777 yopE ESCI = strain 32777 with the YopE promoter sequence with the first 100bp of yopE fused to EPEC EscI expressed in pACYC184 yopJ 32777 yop Stm LL-/LLQG = strain 32777 with a mutated yopJ with the YopE promoter sequence with the first 100bp of yopE fused to S. Tm FliCD0 R475/R475QG expressed in pACYC184 yopJ 32777 yopE EPEC LLR/LLQR = strain 32777 with a mutated yopJ with the YopE promoter sequence with the first 100bp of yopE fused to EPEC FliCD0 Q547R/G548R expressed in pACYC184