Gram-negative bacteria export virulence proteins (effectors) into host cells using a type three secretion system (T3SS) [1
]. Effectors bind and/or post-translationally modify host proteins, preventing the host from generating inflammatory responses to the pathogen. Enterohemorrhagic E. coli
(EHEC) is an attaching/effacing (A/E) pathogen that causes hemorrhagic colitis and pediatric renal failure in humans [2
]. The EHEC T3SS and some effectors are encoded on a pathogenicity island termed the locus of enterocyte effacement (LEE) [3
]. Most of the effectors encoded within the LEE are referred to as E. coli
secreted proteins (Esps), while effectors encoded outside the LEE are referred to as non-LEE encoded effectors (Nles). Salmonella enterica
serovars encode multiple T3SSs; T3SS-1 is encoded on the Salmonella
pathogenicity island (SPI-1) and delivers effectors across the host cell plasma membrane to trigger actin rearrangements that contribute to bacterial invasion [4
]. T3SS-2 is encoded on SPI-2 and translocates virulence proteins from the Salmonella
containing vacuole (SCV) into the host cell cytoplasm [5
The recognition of bacterial pathogens by host cells triggers multiple signaling pathways to induce host inflammatory responses [6
], many of which are regulated by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). The ribosomal protein S3 (RPS3) is a component of the eukaryotic 40S small ribosomal subunit and plays multifunctional roles in DNA repair and apoptosis [7
]. RPS3 possesses an endonuclease activity that mediates some DNA repair processes [9
]. Knockdown of RPS3 protects cells from genotoxic stress after hydrogen peroxide treatment [10
]. RPS3 can be phosphorylated by PKCδ, leading to its mobilization in the nucleus to repair damaged DNA [11
]. RPS3 is one of a large number of ribosomal proteins that have extraribosomal functions [12
]. Many of these functions are related to tumorigenesis [13
], immune signaling [14
], and cell development [15
RPS3 functions as a “specifier” component in NF-κB complexes [16
]. RPS3 guides NF-κB to specific κB sites by increasing the affinity of the NF-κB p65 subunit for target gene promoters [16
]. RPS3 associates with p65 in the inhibitory p65-p50-IκBα complex in the cytoplasm of resting cells [16
]. Activation of NF-κB is initiated by external stimuli that activate the IκB kinase (IKK) complex. Activated IKKβ phosphorylates IκBα, leading to its subsequent ubiquitination and degradation, which allows for p65 and p50 nuclear translocation [17
]. IKKβ also phosphorylates RPS3 on Ser209, enhancing its association with importin-α and mediating RPS3 nuclear translocation [18
Two EHEC effectors disrupt the activation of the innate immune system of intestinal epithelial cells by inhibiting RPS3 nuclear translocation. NleH1 binds to RPS3 [19
] and inhibits its phosphorylation by IKK-β [18
]. NleC proteolysis of p65 generates an N-terminal p65 fragment that competes for full-length p65 binding to RPS3, thus inhibiting RPS3 nuclear translocation [20
]. Thus, E. coli
has multiple mechanisms by which to block RPS3-mediated transcriptional activation. With this in mind, we considered whether other enteric pathogens also encode T3SS effectors that affect this important host regulatory pathway. Here we report that the Salmonella
Secreted Effector L (SseL), which has been previously shown to inhibit NF-κB signaling [21
] and function as a deubiquitinase [22
], also inhibits RPS3 nuclear translocation.
Here we further examined the mechanism of RPS3 nuclear translocation and gained novel insights into the importance of RPS3 ubiquitination in this process. We provide evidence that the Salmonella
effector SseL binds to RPS3 and functions as a DUB to inhibit its nuclear translocation. RPS3 was identified as a “specifier” component in NF-κB complexes [26
]. RPS3 guides NF-κB to specific κB sites by increasing the affinity of the NF-κB p65 subunit for target gene promoters [16
Evidence is emerging that some ribosomal proteins have extraribosomal functions that affect NF-κB signaling. The ribosomal protein rpL3 stabilizes IκBα to inhibit p65 nuclear translocation in p53-mutated cells, thus reducing IL-8 production [27
]. TNF-induced synthesis of H2
S sulfhydrates the C38 residue of the NF-κB p65 subunit and promotes p65 association with RPS3 to enhance the expression of cytoprotective genes [28
]. Human RPS3 contains 20 lysine residues that are potential ubiquitination sites [29
]. The unfolded protein response triggers ubiquitination of RPS3 K214 [32
]. Regulatory RPS3 ubiquitination catalyzed by ZNF598 plays a pivotal role in regulating mammalian ribosome-associated quality control pathways [24
]. RPS3 was also shown to interact with p53 [34
], but its mono-ubiquitination is independent of p53 [24
Non-ribosomal RPS3 is protected from ubiquitination and proteasome-dependent degradation by interacting with Heat shock protein 90 (Hsp90), which helps retain the function and biogenesis of the ribosome [35
]. There is little information available concerning the role of RPS3 ubiquitination in its nuclear translocation, although it is known that nuclear RPS3 can be ubiquitinated by ring finger protein 138 (RNF138) [8
]. This ubiquitination leads to RPS3 degradation and affects radioresistance [8
]. As no active E3 ligase that is specific to RPS3 is commercially available, we were unable to perform experiments to detect RPS3 ubiquitination and deubiquitination in vitro.
Two E. coli
effectors inhibit RPS3 nuclear translocation; NleH1 inhibits RPS3 phosphorylation by IKK-β an essential aspect of the RPS3 nuclear translocation process [19
]. NleC proteolysis of p65 generates an N-terminal p65 fragment that competes for full-length p65 binding to RPS3, thus also inhibiting RPS3 nuclear translocation [20
]. We examined whether Salmonella
effectors also target this pathway, and, by using nuclear fractionation and transfection experiments, we observed a significant reduction in nuclear RPS3 abundance by expressing SseL (Figure 1
). SseL interacted with RPS3 in mammalian cells and bound directly to RPS3 in vitro (Figure 2
). SseL deubiquitinated RPS3 in transfected mammalian cells (Figure 3
). However, we were unable to confirm this activity by performing in vitro deubiquitination assays, as no specific E3 ligase for RPS3 is available.
Ubiquitination plays an important role in regulating protein localization [36
], and there is a precedent for K63-linked polyubiquitination in regulating the relative cytoplasmic vs. nuclear abundance of proteins [38
]. In the context of SseL, deubiquitination of K63-RPS3 significantly reduced the extent of RPS3 nuclear translocation (Figure 4
). This phenotype was dependent upon SseL DUB activity, as the SseL C262A mutant had no impact on RPS3 nuclear translocation. Overall, we propose that, similarly to E. coli
, an important aspect of Salmonella
virulence may be that Salmonella
injected at least one effector through their conserved T3SS into host cells where they interfere with host cell signaling cascades, particularly the RPS3 signaling, to modulate host innate immune responses to the pathogen’s advantage.
4. Materials and Methods
Cloning, Chemicals, and Antibodies.
The strains and plasmids used in this study are listed in Table 1
. Chemicals were used according to manufacturers’ recommendations and were obtained from (Sigma Corporation: Kawasaki, Japan), except for the following: Polyjet DNA In Vitro Transfection Reagent (SignaGen Laboratories, Rockville, MD, USA), glutathione sepharose 4B (GE healthcare Life Sciences, Marlborough, MA, USA), nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen, Hilden, Germany), and TNF-α (Cell Signaling, Danvers, MA, USA). Antibodies were obtained from the following sources: anti-HA and anti-FLAG from Sigma; anti-RPS3 from Proteintech Group; anti-β–tubulin, anti-β–actin, and anti-His from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc. Dallas, TX, USA); anti-IκBα and anti-ubiquitin from Cell Signaling; anti-PARP from BD Transduction Laboratories.
Cell culture and transient DNA transfection. HEK293 cells were maintained at 37 °C, 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 U/mL). Cells were seeded in a 6-well plates 18–24 h prior to transfection. Media was replaced with 1 mL fresh complete DMEM per well 0.5–1 h before transfection. DNA was transfected into cells using Polyjet transfection reagent (SignaGen Laboratories). After 24 h of incubation at 37 °C, the cells were harvested.
Nuclear and cytosolic protein extracts were obtained as described previously [23
]. Briefly, HEK293 cells were transfected and 48 h later, TNF-α was added at 50 ng/mL for 30 min. Nuclear and cytosolic protein extracts were prepared using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher: Thermo Fisher Scientific, Waltham, MA, USA). Data were analyzed by Western blotting for nuclear RPS3. Poly (ADP-ribose) polymerase and β-tubulin or β–actin were used to normalize the protein concentrations of nuclear and cytoplasmic fractions, respectively.
Co-immunoprecipitation Assay. Transfected HEK293 cells were washed using pre-chilled PBS, scraped into ice-cold PBS, and centrifuged at 16,000× g for 5 min. Supernatants were removed, and cells were lysed in 50 mM Tris-HCl, pH 7.4, 0.15 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with 1× halt protease inhibitor cocktail (Thermo Fisher). Samples were incubated on ice for 30 min, with occasional shaking, and cell lysates were collected by centrifugation at 12,000× g for 10 min at 4 °C. Anti-FLAG M2 Gel (Sigma) was centrifuged at 7000× g for 30 s at 4 °C, supernatants were removed, and beads were washed twice using 50 mM Tris-HCl and 250 mM NaCl, pH 7.4 (TBS). Prepared beads were incubated with lysates for 45 min at 4 °C. The mixture was pelleted by centrifugation at 7000× g for 1 min at 4 °C and washed 3 times with pre-chilled TBS. The beads were resuspended in 2× SDS loading dye, incubated for 5 min at 95 °C, and analyzed using 10% SDS-PAGE.
Protein purification. RPS3 was cloned into pET28a, and SseL was cloned into pET42a. They were expressed in E. coli BL21(DE3) cells. Bacterial cultures were grown to A600 = 0.5, and isopropyl β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. After 3 h of additional growth, cells were pelleted using centrifugation and lysed in 50 mM sodium phosphate, pH 8.0, 0.5 mg/mL lysozyme. Lysates were incubated on ice for 30 min with occasional shaking, after which an equal volume of 50 mM sodium phosphate, pH 8.0, 1 M NaCl, 8 mM imidazole, 20% glycerol, and 1% sarkosyl was added, followed by further incubation on ice for 30 additional minutes. The bacterial lysate was sonicated and then clarified by centrifugation. The supernatant was incubated with nickel-nitrilotriacetic acid beads (Qiagen) with end-to-end rotation for 1 h at 4 °C, and slurries were loaded on a Poly-Prep Chromatography Column (BioRad: Bio-Rad Laboratories, Hercules, CA, USA) and washed twice with 5–7 bead volumes of 50 mM sodium phosphate, pH 8.0, 600 mM NaCl, 60 mM imidazole, and 10% glycerol. Proteins were eluted in 50 mM sodium phosphate, pH 8.0, 600 mM NaCl, 250 mM imidazole, and 20% glycerol. Proteins were analyzed using 10% SDS-PAGE.
Pulldown assays. GST-tagged bait proteins (10 µM) were immobilized on glutathione sepharose 4B beads (GE Healthcare) in 20 mM Tris-HCl, pH 7.9, 0.1 M NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 20% glycerol, and 0.1% Nonidet P-40, supplemented with 0.33 unit/µL of DNase I and RNase A. After overnight incubation at 4 °C, the beads were incubated with His-tagged proteins (10 µM) for 1 h at 4 °C. The beads were then washed 3–4 times with 20 mM Tris-HCl, pH 7.9, 1 M NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 20% glycerol, and 0.1% Nonidet P-40. Proteins were eluted with 10 mM reduced glutathione and analyzed using 10% SDS-PAGE.
Deubiquitination assays. HEK293 cells were transfected with FLAG-RPS3, Ubiquitin-HA, in the presence of SseL-HA or SseL(C262A). The cells were washed with pre-chilled 1× PBS, and cell pellets were lysed in 50 mM Tris-HCl, pH7.4, 0.15 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with 1× halt protease inhibitor cocktail (Thermo Fisher) on ice for 30 min and then mixed with anti-FLAG M2 affinity resin and rotated at 4 °C for 2 h. The resins were centrifuged at 7000× g for 30 s at 4 °C and then washed three times with pre-chilled TBS. The resins were resuspended in 2× SDS loading dye, boiled for 5 min at 95 °C, and immunoblotted with appropriate antibodies.
RT-PCR. Total RNA was isolated from cells using the RNeasy Plus Mini kit (QIAGEN). RNA was first reverse-transcribed using a first-strand cDNA synthesis kits (QIAGEN) and quantitative PCR was then carried out using a Rotor-Gene SYBR Green kit (QIAGEN). The comparative Ct method was used to calculate the relative abundance of IL-8 transcripts with normalization to beta-actin expression.
Statistical analyses. Protein abundance was quantified using Li-COR Image Studio software. RPS3 nuclear translocation and ubiquitination were analyzed statistically using the Kruskal–Wallis test or the Dunn’s multiple comparison test where appropriate. p-values < 0.05 were considered significant.