Salmonella Effector SpvB Inhibits NF-κB Activity via KEAP1-Mediated Downregulation of IKKβ

Bacterial pathogens have a broad arsenal of genes that are tightly regulated and coordinated to facilitate adaptation to alter host inflammatory response and prolong intracellular bacterial survival. Salmonella enterica serovar Typhimurium utilizes a type III secretion system (T3SS) to deliver effector molecules into host cells and regulate signal transduction pathways such as NF-κB, thereby resulting in salmonellosis. SpvB, a pSLT-encoded cytotoxic protein secreted by Salmonella pathogenicity island-2 T3SS, is associated with enhanced Salmonella survival and intracellular replication. In this report, we characterized the effects of SpvB on NF-κB signaling pathway. We showed that SpvB has a potent and specific ability to prevent NF-κB activation by targeting IκB kinase β (IKKβ). Previous studies from our laboratory showed that SpvB decreases Nrf2 through its C-terminal domain. Here we further demonstrated that KEAP1, a cytoplasmic protein that interacts with Nrf2 and mediates its proteasomal degradation, is involved in SpvB-induced downregulation of IKKβ expression and phosphorylation. Reduction of KEAP1 by small-interfering RNA prevented the suppression of IKKβ and its phosphorylation mediated by SpvB. These findings revealed a novel mechanism by which Salmonella modulates NF-κB activity to ultimately facilitate intracellular bacterial survival and proliferation and delay host immune response to establish infection.


INTRODUCTION
Early sensing of pathogenic bacteria by the host numerous innate and adaptive immune mechanisms is important to limit microbial intrusion. By eliciting inflammation, pathogenic bacteria use highly evolved mechanisms to counteract host defense mechanisms (Baumler and Sperandio, 2016). During the long-standing associations with the host, pathogens have developed Abbreviations: co-IP , co-immunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; IL-8, interleukin 8; IL-1R, interleukin-1 receptor; IKKb, IkB kinase b; LB, Luria-Bertani; MAPK, mitogen-activated protein kinases; MOI, multiplicity of infection; NC siRNA, negative control siRNA; IkBa, NF-kB inhibitor alpha; NLRs, NOD-like receptors; NF-kB, nuclear factor kB; IKKs, nuclear factor-kB kinases; SPI-1, pathogenicity islands-1; SPI-2, pathogenicity islands-2; RT-qPCR, Quantitative real-time PCR; S. Typhimurium, Salmonella enterica serovar Typhimurium; spv, Salmonella plasmid virulence; STAT3, signal transducer and activator of transcription 3; SEM, standard error of the mean; TLRs, toll-like receptors; TNFR, tumor necrosis factor receptor; T3SS, type III secretion system; WT, wild-type. complex adaptations to modulate host inflammatory response to promote their own growth and virulence. A facultative intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), which is a leading cause of food-and waterborne diseases in mammals, has acquired adaptations that interfere with host defense mechanisms (Besser, 2018). One important adaptation of Salmonella is the type III secretion system (T3SS), which delivers effector molecules into host cells. There are two T3SSs encoded by Salmonella pathogenicity islands-1 and -2 (SPI-1 and SPI-2), which deliver more than 40 effectors into target cells to facilitate bacterial invasion, survival, and proliferation (Agbor and Mccormick, 2011). Several effector proteins have been shown to interfere with the activity of host inflammatory signaling pathways, including nuclear factor kB (NF-kB) and mitogen-activated protein kinases (MAPK), which result in attenuated host inflammatory response and enhanced bacterial replication within host cells (Kurtz et al., 2017).
NF-kB signaling pathway is a major innate immune signaling pathway responsible for early detection of pathogens such as Salmonella (Pinaud et al., 2018). Under naive conditions, NF-kB remains inactive in the cytoplasm bound to the NF-kB inhibitor alpha (IkBa) protein. Following stimulation with multiple exogenous and endogenous substances, which are sensed by tumor necrosis factor receptor (TNFR), interleukin-1 receptor (IL-1R), toll-like receptors (TLRs), and NOD-like receptors (NLRs) (Verstrepen et al., 2008;Caruso et al., 2014;Keestra-Gounder et al., 2015), different pathways are triggered to lead to phosphorylation of the inhibitor of nuclear factor-kB kinases (IKKs). The IKK complex contains three subunits including IKKa, IKKb, and a regulatory protein, NEMO (or IKKg). In particular, IKKb activation is dependent on the phosphorylation of the kinase domain. IKKb activation can further phosphorylate IkBa and lead to its degradation, thereby releasing p50/p65 for import into nucleus to initiate transcription of the target genes (Bhoj and Chen, 2009). Numerous components of the NF-kB signaling pathway are targeted by some of the Salmonella effectors (such as SseK1, SspH1, GtgA, and AvrA) that result in inhibition of the NF-kB pathway (Collier-Hyams et al., 2002;Haraga and Miller, 2003;Sun et al., 2016;Gunster et al., 2017). Although several effectors delivered by S. Typhimurium T3SS can efficiently attenuate NF-kB activation, inhibit the host inflammatory response, and promote bacterial replication in host cells and tissues, the functions and the underlying mechanisms of most effectors remain obscure.
Salmonella plasmid virulence (spv) gene, a highly conserved 8-kb region in the pSLT virulence plasmid from non-typhoid Salmonella strains of clinically significant serovars, has been implicated in intracellular survival and growth (Fabrega and Vila, 2013). The spv region contains a positive transcriptional regulator gene spvR, a negative regulator gene spvA, and three structural genes spvB, spvC, and spvD (Guiney and Fierer, 2011;Passaris et al., 2018). Previous studies have found that all the spv effectors are secreted via the T3SS, and spv-containing Salmonella exhibit enhanced intracellular proliferation in intestinal and extraintestinal tissues of the natural host (Libby et al., 1997), suggesting that they have the potential to target molecules and mechanisms to attenuate host inflammatory response to establish a successful infection. Each of the spv effectors has been shown to target specific signal transduction pathways in a variety of cell types (Passaris et al., 2018).
Here, we found that spv could inhibit NF-kB activation during Salmonella infection, and SpvB, a member of the spv effectors, is critical for spv-mediated NF-kB inhibition and IkBa degradation. The detailed investigation of the NF-kB signaling pathway suggested that SpvB acts at the level of IKKb activation. We showed that SpvB prevents the expression and phosphorylation of IKKb, and this was associated with SpvB-mediated upregulation of an E3 ligase KEAP1. Together, these findings indicated that Salmonella effector protein SpvB inhibits the activation of NF-kB signaling pathway via repressing the activity of IKKb by targeting KEAP1. Our studies revealed a novel inhibitory mechanism used by Salmonella to disrupt host inflammatory response to enhance its proliferation and virulence.

Bacterial Strains and Growth Condition
Wild-type (WT) S. Typhimurium strain SL1344 was kindly provided by Professor Qian Yang (Nanjing Agricultural University, Nanjing, China). The DspvB strain was constructed in our previous work (Yang et al., 2019). Dspv, DspvA, DspvD and DspvBD mutant strains were constructed with the l-Redmediated recombination system as previously described (Datsenko and Wanner, 2000). DspvBD,pspvB and DspvBD, pspvD strains were complemented with DspvBD in plasmid pBAD/gIIIA (Invitrogen, Carlsbad, CA, USA) encoding the full-length of spvB or spvD gene. The primers used in this study are listed in Table 1. Bacteria were grown in Luria-Bertani (LB) medium (Hangwei, Hangzhou, China) at 37°C and supplemented with ampicillin (100 mg/ml) or kanamycin (50 mg/ml) as appropriate.
For siRNA knockdown of KEAP1, cells were transfected with KEAP1 siRNA or negative control siRNA (NC siRNA) using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h according to the manufacturer's instructions. Knockdown efficiency was assessed by Western blot with KEAP1 antibody.

Bacterial Infection of Cells
Overnight S. Typhimurium cultures were diluted 1:100 in fresh LB medium and subcultured for an extra 3 h. The bacteria were then washed three times with PBS and quantified spectrophotometrically by determining the optical density at 600 nm along with viable plate counts. HeLa cells were seeded in complete DMEM without antibiotics 24 h prior to use. Cells were infected with the different S. Typhimurium strains at the multiplicity of infection (MOI) described in the figure legends. One hour later, cells were washed in PBS and treated with DMEM containing 10% heat-inactivated FBS and amikacin (100 mg/ml) (Sigma, Saint Louis, Missouri, USA) to kill extracellular bacteria. 2 h later, cells were washed and maintained in DMEM containing 10% heat-inactivated FBS and low concentration of amikacin (10 mg/ml) (Sigma).

Immunofluorescence Microscopy
Following infection, cells seeded on glass coverslips were washed three times with PBS prior to be fixed in 4% paraformaldehyde for 20 min and permeabilized in 0.3% Triton X-100 for 10 min. After being blocked with 3% BSA, the coverslips were incubated with primary antibodies at 4°C overnight, developed with the appropriate Alexa Fluor ® 555 secondary antibody (Thermo Fisher Scientific) for 1 h and then stained with Hoechst 33258 (Beyotime) for 10 min. All photomicrographs were taken with a Nikon Eclipse Ni-U fluorescence microscope (Nikon Corporation, Tokyo, Japan) with NIS-Elements F (Nikon Corporation).

Luciferase Reporter Assays
HeLa cells were seeded at a density of 5 x 10 4 cells per well in 24well plates for 16 h. Cells were transfected with pNFkB-Luc (Firefly luciferase, experimental reporter) and phRL-TK reporter (Renilla luciferase, internal control) plasmids (Clontech, USA) using Lipofectamine 2000 (Thermo Fisher Scientific). After 24 h, the cells were infected with different Salmonella strains and NF-kB activity was measured using the Dual Luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Immunoprecipitation
293T cells were seeded in 6-well plates for 16 h. Cells were transfected with indicated DNA with Lipofectamine 2000. Three wells were used per condition. Cells were lysed in RIPA Buffer (Beyotime) containing a protease and phosphatase inhibitor mini tablet (Thermo Fisher Scientific). Lysates were centrifuged at 16000 g and the supernatants were harvested and incubated with protein A/G Plus-agarose (Santa Cruz Biotechnology) by rotation for 2 h at 4°C. The supernatants were then incubated with anti-HA or anti-FLAG antibody overnight at 4°C for immunoprecipitation. Immunoprecipitated complexes were then washed three times, probed to anti-FLAG, anti-EGFP or anti-HA and subjected to immunoblotting.

Statistical Analysis
Each experiment was performed at least three times and data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed with IBM SPSS statistics 22. Differences between two groups were evaluated by independent Student's ttest. p values less than 0.05 were considered statistically significant and are presented as *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant.

RESULTS
spv Inhibits NF-kB Activation During Salmonella Infection First, we investigated whether the Salmonella spv locus contributes to the intracellular Salmonella pathogenesis via interfering with the NF-kB pathway. Cells were infected with either the wild-type (WT) S. Typhimurium or the Dspv mutant S. Typhimurium strain to study the kinetics of p65 phosphorylation and IKBa degradation. We showed that cells infected with the Dspv strain displayed a significantly higher level of p65 phosphorylation compared with cells infected with the WT strain since 1 h post-infection ( Figure 1A). Consistently, lower expression of IkBa was found in Dspv-infected cells compared with WT-infected cells at 2 h post-infection ( Figure  1B). To confirm these results, immunofluorescence was used to assess the subcellular distribution of p-P65. The results showed that spv decreases the accumulation of p65 in the nucleus ( Figure  1C). These findings indicated that spv inhibited the activation of NF-kB during Salmonella infection.

SpvB Is Critical for spv-Mediated NF-kB Inhibition and IkBa Degradation
Our data showed that the whole spv locus contributed to IkBa degradation. However, previous studies have identified that SpvC does not affect NF-kB pathway, while SpvD prevents NF-kB activation by interfering with nuclear translocation of p65 but not degradation of IkBa . Therefore, we further studied the potential role of spvA and spvB in this process. We constructed DspvA and DspvB mutant Salmonella strains and assessed their roles in disturbing NF-kB activity, with DspvD mutant Salmonella strain as a control. There was no change in p65 phosphorylation level or IkBa expression in cells infected with the DspvA strain compared with cells infected with the WT strain at 2 h post-infection. Interestingly, the protein level of p-P65 in DspvB-infected cells was significantly higher than that detected in WT-infected cells, and the level of IkBa was markedly lower in cells infected with the DspvB strain ( Figures  2A, B). Additionally, HeLa cells were transfected with plasmid expressing luciferase under the transcriptional control of NF-kB, and then infected with different Salmonella strains. NF-kB activity was measured at 2 h post-infection by luciferase reporter assay. The results showed that DspvB-infected cells exhibited an increased NF-kB activity, which is consistent with DspvD-infected cells, while there was no apparent difference in luciferase activity among DspvAand WT-infected cells ( Figure  2C). These observations suggested an important effect of SpvB on spv-mediated NF-kB inhibition. We further constructed DspvBD mutant Salmonella strain and the complemented Salmonella strains harboring spvB (DspvBD,pspvB) or spvD (DspvBD,pspvD). The data of luciferase assay showed that cells infected with DspvBD,pspvB or DspvBD,pspvD strain significantly reduced the NF-kB activity compared with those infected with DspvBD strain ( Figure 2D). In line with the previous study , SpvD inhibited NF-kB activation ( Figure 2D) but did not interfere with IkBa degradation ( Figure 2B). Together, these results demonstrated that SpvB is critical for spv-mediated NF-kB inhibition and IkBa degradation.

SpvB Inhibits IL-1b/LPS/TNF-a/PMA-Triggered Activation of the NF-kB Pathway
The NF-kB signaling pathway can be triggered by several cellular receptors during Salmonella infection, including IL-1R, TLRs, TNFR, and NLRs. To elucidate the underlying mechanisms by which SpvB inhibits NF-kB activity, the spvB gene was cloned into a mammalian expression vector pEGFP-N1 to generate HA-SpvB-EGFP fusion protein. After stimulation with IL-1b, LPS, TNF-a, or PMA, the NF-kB activity was measured in 293T cells with ectopic expression of SpvB using NF-kB-luciferase reporter assays. As shown in Figures 3A-D, the expression of SpvB efficiently blocked all four stimulants-induced NF-kB activation. In agreement with this observation, western blot analysis illustrated that the expression of HA-SpvB-EGFP, unlike the empty vector, decreased p65 phosphorylation and increased IkBa expression in the context of either LPS or TNF-a treatment ( Figures 3E, F). Given that interleukin 8 (IL-8) transcription is regulated by NF-kB, we further analyzed the activity of IL-8 in 293T cells stimulated with IL-1b, LPS, TNF-a, or PMA using an IL-8 reporter assay to confirm SpvB-mediated inhibition of NF-kB activity. The results showed that IL-8 reporter activity in response to each of the four stimulants in cells transfected with SpvB-EGFP was significantly attenuated compared with mock transfected cells either stimulated with LPS or TNF-a ( Figures 3G-J). Moreover, the mRNA level of IL-8 in cells expressing SpvB-EGFP was obviously lower than that detected in cells expressing EGFP ( Figures 3K, L). Taken together, these findings indicated that SpvB inhibits the activation of NF-kB pathway triggered by all four stimulants.

SpvB Prevents the Activation of NF-kB by Acting at IKKb
To better understand the SpvB inhibitory mechanism and where in the NF-kB signaling pathway SpvB might exert its function, we tested whether SpvB itself was able to repress NF-kB activation in a context different from Salmonella infection. We also examined the effect of SpvB on the activation of NF-kB by several essential components of the signaling pathway. Thus, 293T cells expressing NF-kB luciferase reporter gene together with MyD88, TRAF6, TRAF2, TAK1/TAB1, IKKa, IKKb, or P65 were transfected with SpvB-EGFP or the empty vector. As shown in Figures 4A-F, SpvB prevented the activation of NF-kB induced by expression of MyD88, TRAF6, TRAF2, TAK1/ TAB1, IKKa, or IKKb. In contrast, SpvB did not block the activation of NF-kB caused by expression of P65 subunit ( Figure  4G). These results suggested that SpvB might exert its function at the level of IKKb. We further studied the relationship between SpvB and IKKb reduction. Western blot analysis showed that SpvB efficiently reduced the expression of IKKb in context of either LPS or TNFa activation (Figures 5A, B). Luciferase activity assay and quantitative PCR also confirmed that, even without stimulation, SpvB suppressed the promoter activity and mRNA level of IKKb ( Figures 5C, D). We further investigated the effect of SpvB on IKKb by transiently co-expressing differentially epitope-tagged SpvB and IKKb. The results showed that expression of SpvB led to a drastic reduction in the level of either IKKb expression or IKKb phosphorylation ( Figure 5E). All these results indicated that SpvB downregulates NF-kB activity via acting on IKKb.

SpvB Inhibits IKKb Expression and Phosphorylation by Targeting KEAP1
We next focused on elucidating the mechanisms underlying SpvB-mediated downregulation of IKKb. We first investigated whether SpvB directly interacted with IKKa or IKKb. To this end, we transfected EGFP-tagged SpvB together with HA-tagged IKKa or Flag-tagged IKKb into 293T cells to analyze molecular interactions by co-immunoprecipitation (co-IP). The results exhibited that neither IKKa nor IKKb could bind to SpvB ( Figures 6A, B). In previous studies, we found that SpvB interferes with intracellular iron homeostasis via regulation of a transcription factor Nrf2 (Yang et al., 2019). It is known that the cytoplasmic protein KEAP1 interacts with Nrf2 and represses its function, which suggests that SpvB might positively modulate KEAP1 to downregulate IKKb and suppress NF-kB activity. To address this issue, 293T cells or HeLa cells were transfected with Values are expressed as mean ± SEM of three independent experiments. p values less than 0.05 were considered statistically significant and are presented as *P < 0.05, **P < 0.01, ***P < 0.001 and ****p < 0.0001.
HA-tagged SpvB or the empty vector, and western blot was employed to analyze the effect of SpvB expression on KEAP1. Data showed that the expression of SpvB significantly increased the level of KEAP1 (Figures 6C, D). Therefore, we speculated that SpvB might inhibit the expression and phosphorylation of IKKb by affecting KEAP1. To further characterize the effect of KEAP1 in this process, 293T cells or HeLa cells were transfected with HA-tagged SpvB or the empty vector, and then treated with KEAP1 siRNA or the negative control (NC siRNA). Western blot analysis illustrated that SpvB potently reduced the expression of IKKb following the treatment of NC siRNA, whereas this reduction was dissolved in the context of KEAP1 siRNA treatment ( Figures 6E, F). To determine the phosphorylation of IKKb, FLAG-tagged IKKb and HA-tagged SpvB or the empty vector were co-transfected into 293T cells, and the cells were treated with KEAP1 siRNA or NC siRNA. Western blot analysis showed that in NC siRNA treatment group, SpvB not only decreased the expression of IKKb but also restrained the phosphorylation of IKKb. Conversely, whether SpvB was expressed or not, there was no significant difference in the expression or phosphorylation of IKKb following the treatment by KEAP1 siRNA ( Figure 6G). All these observations indicated that SpvB inhibits IKKb expression and phosphorylation by targeting KEAP1. The reporter activity was subsequently measured 24 h after transfection. Statistical analysis was performed with IBM SPSS Statistics 22. Data were compared by independent Student's t test. Values are expressed as mean ± SEM of three independent experiments. p values less than 0.05 were considered statistically significant and are presented as *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant.

DISCUSSION
Bacterial pathogens have a broad arsenal of genes that are tightly regulated and coordinated to facilitate adaptation to the host environment (Ilyas et al., 2017). S. Typhimurium, a facultative intracellular Gram-negative pathogen causing both gastrointestinal and systemic diseases in humans and other mammals, primarily depends on two T3SSs encoded within SPI-1 and SPI-2 to deliver effectors into host cells to alter cell physiology for bacterial invasion and colonization (Jennings et al., 2017;Lou et al., 2019). Emerging evidence indicates that Salmonella effector proteins are utilized by bacteria to modulate host inflammatory response and prolong intracellular bacterial survival. A few effectors have been shown to have an impact on the activity of key signaling pathways such as NF-kB, signal transducer and activator of transcription 3 (STAT3), and MAPK, which result in reduced subsequent production of pro- inflammatory cytokines and/or suppressed host inflammation. For example, SseK1 and SseK3 inhibit NF-kB signaling and necroptotic cell death in Salmonella-infected macrophages (Gunster et al., 2017), while PipA, GtgA, and GogA are proteases that target NF-kB transcription factors to preserve host homeostasis (Sun et al., 2016). AvrA inhibits MAPK pathway in epithelial cells via a JNKdependent manner (Collier-Hyams et al., 2002;Jones et al., 2008), whereas SteE drives M2 macrophage polarization via GSK3 and STAT3 (Panagi et al., 2020). Nonetheless, the function of most Salmonella effectors is still unclear.
Previous studies have reported that S. Typhimurium carries spv genes, highly conserved in the pSLT virulence plasmid and closely related to bacterial pathogenicity, have been implicated in suppressing host innate immune response against bacterial infection (Wu et al., 2016). However, the mechanisms through which spv affects host innate immune response remain elusive. Since NF-kB signaling pathway is one of the central host defense responses to limit Salmonella infection, we hypothesized that spv may restrain host inflammation and promote Salmonella infection by efficiently attenuating NF-kB activation. Consistent with this hypothesis, we found that spv contributed to the inhibition of the phosphorylation of p65 and the degradation of IkBa, indicating that Spv could negatively regulate NF-kB signaling during Salmonella infection. The spv locus contains five genes, designated as spvRABCD. The SpvB, SpvC, and SpvD proteins are thought to be delivered into the host cells mainly through the SPI-2 T3SS and have been proven as virulence effectors in a variety of cell types. It has been reported that SpvC has phosphothreonine lyase activity on host MAPK but no effect on NF-kB signaling pathway (Mazurkiewicz et al., 2008). SpvD was found to inhibit NF-kB activation by prevention of nuclear transport of p65 but not degradation of IkBa . In this study, we found that spv inhibited IkBa degradation, which suggests that spv-mediated NF-kB inhibition might depend on SpvA or SpvB instead of SpvC or SpvD. Therefore, we generated deletion mutants for spvA or spvB to identify their effect on the activation of NF-kB and constructed spvD mutant strain as a control.
Our data showed that spvB, rather than spvA, was critical for the inhibition of NF-kB activation. Furthermore, a double mutant strain lacking both spvB and spvD exhibited significantly stronger activation of the NF-kB reporter than each of the single mutants complemented with either spvB or spvD, suggesting that these effectors are not functionally redundant. SpvB has been characterized as an ADP-ribosylase, which can prevent actin polymerization, thereby leading to loss of the actin cytoskeleton (Guiney and Fierer, 2011). SpvD is a cysteine hydrolase with a serovar-specific polymorphism . Both effectors are likely to have distinct biochemical activities and physiological effects during Salmonella infection. Nathalie R et al. have demonstrated that SpvD disrupts normal recycling of importin-a from the nucleus, leading to a defect in nuclear translocation of p65 and inhibition of activation of NF-kB-regulated promoters, while it does not lead to the degradation of IkBa . In this context, we further investigated the mechanism by which SpvB inhibits the IkBa degradation and NF-kB activation.
NF-kB is a transcription factor that is sequestered in the cytosol by IkBa in resting cells, and multiple exogenous and endogenous stimuli are needed to induce its translocation to the nucleus. In canonical NF-kB signaling pathway, cellular receptors including TNFR, IL-1R, TLRs, and NLRs sense different stimuli, and then utilize various adaptors and signaling molecules to transmit signals to initiate the phosphorylation of IKKs, degradation of IkBa, nuclear transfer of p65, and final activation of NF-kB signaling pathway (Bhoj and Chen, 2009). To characterize the SpvB-dependent dampening of NF-kB activation, we employed NF-kBluciferase reporter assays to measure the levels of NF-kB activity in SpvB-expressing 293T cells that were stimulated with TNF-a, IL-1b, LPS, or PMA. Interestingly, we found that SpvB blocked NF-kB activation induced by all these stimuli. Consistent with this, SpvB restrained the activated IL-8 expression without specificity. Given that the inhibition of NF-kB signaling mediated by the ectopic expression of SpvB is independent of different stimuli, we speculated that SpvB might act on the components of their common downstream pathway (Ruchaud-Sparagano et al., 2011).
We subsequently investigated which of the components function as a potential SpvB target. Previous studies have shown that MyD88 and TRAF6 were downstream of the IL-1b receptor, and TRAF2 was the TNF-a receptor. Here we found that SpvB inhibition of NF-kB reporter activity was driven by expression of MyD88, TRAF6, and TRAF2. Importantly, we observed that SpvB downregulated NF-kB activity stimulated by TAK1/TAB1, IKKa, or IKKb downstream of TRAF, but not by P65 expression. These findings suggested that SpvB might affect IKKb activity. The following results showed that SpvB not only inhibited the transcription and expression of IKKb but also suppressed the phosphorylation of IKKb. All these results indicated that SpvB directly or indirectly targets IKKb to exert the inhibitory effect on NF-kB activity. To elucidate the underlying modulatory mechanism, we further performed immunoprecipitation assays to determine whether IKK complex can be physically associated with SpvB. However, SpvB failed to bind with either IKKa or IKKb.
Our previous study reported that SpvB facilitates the pathogenicity of Salmonella within cells via disrupting host iron metabolism by targeting the transcription factor Nrf2 (Yang et al., 2019). In mammals, Nrf2 interacts with the E3 ligase adaptor KEAP1, the major endogenous negative regulator of Nrf2, to form a defense system aimed to preserve cellular homeostasis (Bellezza et al., 2018). In fact, KEAP1 also functions as an E3 ubiquitin ligase to dysregulate IKKb degradation and phosphorylation (Lee et al., 2009). In view of the Nrf2 degradation enhanced by SpvB, we detected the expression of KEAP1 in cells transiently transfected with HA-tagged SpvB, and our data showed that SpvB significantly increased the expression of KEAP1. We further investigated whether the Salmonella effector SpvB inhibits the expression of IKKb by modulation of KEAP1. Cells expressing SpvB were treated with KEAP1 siRNA or NC siRNA. Western blot analysis illustrated that SpvB reduced the expression of IKKb following the treatment with NC siRNA. However, there was no significant difference in IKKb level between SpvB-and vector-transfected cells following KEAP1 siRNA treatment. All these findings indicated that the Salmonella effector protein SpvB can prevent IKKb expression and NF-kB activation by targeting KEAP1. Moreover, our data confirmed that SpvB restrains the phosphorylation of IKKb through KEAP1. Wei et al. recently reported that S53 is a potential phosphorylation site of KEAP1 and its phosphorylation is critical to the activity of KEAP1 (Wei et al., 2019). It is possible that SpvB upregulates the expression of cytosolic KEAP1, and the latter competes with IKKb for phosphate groups in order to maintain its own activity, which leads to the inhibition of NF-kB signaling pathway.
Together, our work revealed a novel mechanism for a Salmonella effector to inhibit NF-kB activity as it was demonstrated that Salmonella effector SpvB has a potent and specific ability to prevent activation of NF-kB via suppressing IKKb activity by targeting KEAP1. Even though inflammation is ultimately induced by Salmonella infection, SpvB may allow Salmonella to delay host immune response to establish infection and disseminate to other tissues. These findings revealed a remarkable adaptation of a bacterial pathogen to promote its own survival and virulence.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
SY, QD, and YL designed and conducted the experiments. SY, QD, and YL contributed to development of methodology. SY, QD, LS, YZ, KD, and YL contributed to analysis and interpretation of data. SY, QD, and LS prepared figures and wrote the manuscript. SW, RH, and YL supervised the project and edited the manuscript. All authors contributed to the article and approved the submitted version.