Abstract
Salmonella Typhimurium creates an intracellular niche for its replication by utilizing a large cohort of effectors, including several that function to interfere with host ubiquitin signaling. Although the mechanism of action of many such effectors has been elucidated, how the interplay between the host ubiquitin network and bacterial virulence factors dictates the outcome of infection largely remains undefined. Here we found that the SPI-2 effector SseK3 inhibits SNARE pairing to promote the formation of Salmonella-induced filament by Arg-GlcNAcylation of SNARE proteins, including SNAP25, VAMP8, and Syntaxin. Further study reveals that host cells counteract the activity of SseK3 by inducing the expression of the ubiquitin E3 ligase TRIM32, which catalyzes K48-linked ubiquitination on SseK3 and targets its membrane-associated portion for degradation. Hence, TRIM32 antagonizes SNAP25 Arg-GlcNAcylation induced by SseK3 to restrict SIF biogenesis and Salmonella replication. Our study reveals a mechanism by which host cells inhibit bacterial replication by eliminating specific virulence factor.
Introduction
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen capable of infecting multiple hosts to cause salmonellosis (Acheson and Hohmann, 2001). S. Typhimurium encodes two type III secretion systems (T3SSs), SPI-1 and SPI-2 that inject a large cohort of effector proteins into host cells(Dos Santos et al., 2020). Whereas the function of SPI-1 primarily is to promote invasion of non-phagocytic intestinal epithelial cells to establish a nascent phagosome, effectors translocated by SPI-2 function to remodel the phagosome into a niche permissive for bacteria replication called the Salmonella-containing vacuole (SCV)(Lou et al., 2019). A number of SPI-2 effectors are dedicated to subverting the endolysosomal system, including the formation of a complex and highly dynamic structure termed Salmonella-induced filaments (SIFs)(Jennings et al., 2017; Knuff and Finlay, 2017). Although many pathogens have the ability to create bacteria-containing vacuoles (BCVs) to support their intracellular replication, the formation of the SIF network is unique to S. Typhimurium (Santos and Enninga, 2016). The ability of this pathogen to form SIF strongly correlates with its ability to cause diseases in animal infection models (Stein et al., 1996). It had been hypothesized that SIF structures facilitate the bacterium to gain access to nutrients and/or to evade host immunity (Liss et al., 2017; Noster et al., 2019). Thus, understanding the mechanisms of SIF biogenesis and maintenance will not only lead to a better appreciation of the S. Typhimurium pathogenesis and host cell biology but also provide clues for its disruption as novel interference strategies against salmonellosis.
Ubiquitin signaling plays an essential role in immunity by regulating various cellular processes, particularly protein turnover and vesicle trafficking(Dikic, 2017). Consistent with the existence of multiple mechanisms designed to sense and eliminate invading S. Typhimurium, the bacterium has evolved several virulence factors to counteract such surveillance by directly interfering with ubiquitin signaling. At least four Salmonella effectors have been shown to function as E3 ubiquitin ligases to modulate host immunity(Herhaus and Dikic, 2018). Among them, SopA is a HECT-like E3 ligase (Zhang et al., 2006) that modifies and targets TRIM56 and TRIM65, two host E3 ligases for degradation(Fiskin et al., 2017; Kamanova et al., 2016). The two IpaH-type E3 enzymes SspH1 and SspH2 target protein kinase 1 and NO1, respectively (Bhavsar et al., 2013; Haraga and Miller, 2006; Keszei et al., 2014). SlrP promotes host cell death by targeting thioredoxin and the Hsp40/DnaJ chaperone family associated with the endoplasmic reticulum (Bernal-Bayard et al., 2010; Bernal-Bayard and Ramos-Morales, 2009). In addition, the two deubiquitinases, AvrA and SseL have been found to regulate host immunity, particularly the NF-κB pathway(Hermanns and Hofmann, 2019; Mesquita et al., 2013; Ye et al., 2007). The fate of S. Typhimurium that escape the phagosome to reach the cytosol differs greatly from those residing in the membrane-bound vacuoles, these bacteria are first ubiquitinated by the E3 ligase RNF213, which strikingly directly recognizes and modifies the lipid A moiety of bacterial lipopolysaccharide (LPS)(Otten et al., 2021). Several other E3 ligases coordinate to build the ubiquitin coat on the bacterial surface to initiate xenophagy(Herhaus and Dikic, 2018), a process that was recently found to be inhibited by the effector SopF that ADP-ribosylates the ATP6V0C subunit of the v-ATPase complex (Xu et al., 2019).
Another cohort of S. Typhimurium effectors contribute to the development of the SCV by modulating vesicle trafficking(Galán, 2021). Although the cellular function of these effectors has been extensively studied (Tuli and Sharma, 2019), how the host cell counteracts their activity largely remains elusive. In the present study, we identified the host E3 ligase TRIM32 as a regulator for the activity of the SPI-2 effector SseK3. We found that SseK3 catalyzes Arg-GlcNAcylation on SNAP25 and restricts SIF biogenesis mediated by the SseK3-SNARE axis and that infection by S. Typhimurium induces the expression of TRIM32, which antagonizes the activity of SseK3 by ubiquitination-mediated degradation.
Results
SseK3 promotes SIF formation during S. Typhimurium infection
Effectors translocated by the SPI-2 are required for the formation of SIFs (Knuff and Finlay, 2017). Among these, the three SseK proteins, SseK1, SseK2 and SseK3, are arginine GlcNAc transferases that are important for S. Typhimurium virulence(Meng et al., 2020). To test whether any of these effectors is involved in SIF biogenesis or maintenance, we examined the phenotypes by infecting a cell line derived from HeLa that expresses GFP-VAMP8 with several relevant S. Typhimurium strains, including the wild-type, ΔsseK1/2/3, and ΔssaV which is defective in the SPI-2. Samples were assessed for the integrity of SCV membrane and SIF formation by confocal microscopy. At 2 h post-invasion, the frequency of VAMP8-coated SCVs for strain ΔsseK1/2/3 was similar to that of the wild-type and the ΔssaV mutant, with rates at 81±2%, 83±6%, and 83.1±4%, respectively (Figure S1). Thus, SseKs do not contribute to the formation of nascent SCVs, which is consistent with the observation that Arg-GlcNAcylation induced by members of SseK family does not occur until after 6 h post-infection(Meng et al., 2020). At 10 h post-invasion, we observed a significant decrease in the frequency of SIFs in cells infected with the ΔsseK1/2/3 strain when compared to those infected with wild-type S. Typhimurium. Furthermore, expression of SseK3, but not its enzymatically inactive mutant SseK3D226A/D228A restored the development of SIF to wild-type levels (Figures 1A and 1B). These results indicate that SseK3 plays a role in promoting SIF formation during S. Typhimurium infection.
SseK3 attacks SNARE proteins by Arg-GlcNAcylation
We next investigated the mechanism underlying SseK3-induced SIF development. Our previous studies have shown that SseK3 catalyzes Arg-GlcNAcylation on death domain-containing receptor proteins and members of the Rab small GTPases(Meng et al., 2020; Pan et al., 2020). Yet, despite extensive efforts, we did not detect SseK3-induced modification of most of the Rabs involved in SIF formation, including Rab11, Rab9, and Rab7 in cells infected with S. Typhimurium (Meng et al., 2020). We thus attempted to identify SseK3 targets involving in SIF biogenesis by Arg-GlcNAc (Pan et al., 2014) followed by mass spectrometry analysis (Figure 1C). KEGG analysis of the putative SseK3 modified proteins revealed five significantly enriched pathways. Among them, nineteen proteins are involved in SNARE interactions in the vesicular transport pathway, including SNAP23, SNAP25, VAMP8, Vti1b, Syntaxin7, Syntaxin8, and Sec22b (Figure 1D, red dots). Further comparative analyses between SseK3 samples and controls led to the identification of several GTPase proteins that had been previously identified (green dots, e.g., Rab1 and Rab8), demonstrating the effectiveness of this strategy (Figure 1D). Complementation experiments with SseK1, SseK2 or SseK3 showed that SseK3 was the sole enzyme responsible for modifying SNARE proteins during infection (Figure 1E). Further experiments established that several SNARE proteins, including VAMP8, Syntaxin7, Syntaxin8, Vti1b, Sec22b, SNAP23, and SNAP25 are modified by SseK3 in cells infected with S. Typhimurium (Figure 1F). Among these, SNAP25 was most robustly modified by SseK3, suggesting that this protein is its preferred substrate (Figures 1F and S2). Taken together, these results indicate that SNARE proteins are new cellular targets of SseK3.
SseK3 induces GlcNAcylation of SNAP25 on Arg30 and Arg31, two residues important for its interaction with VAMPs
SNARE proteins share a common coiled-coil motif of 60–70 residues essential for membrane fusion (Figure 2A)(Harbury, 1998). By deletion mutagenesis, we found that the amino-terminal domain (1-90 aa, NTD) containing the coiled-coil motif of SNAP25 can be modified by SseK3 in cells infected with S. Typhimurium (Figure 2B). To precisely map the modification site(s), we affinity-purified Flag-SNAP25 from 293T cells co-transfected with either wild-type SseK3 or empty vector. By tandem MS (MS/MS) analysis, we detected one GluC-LysC peptide 28STRRMLQLVEE38 with a mass shift of 406 Da only in samples from cells that co-expressed SseK3. The 406 Da increase in mass corresponds to the attachment of two GlcNAc moieties. Quantitation analysis revealed that approximately 75% of the peptides were modified (Figure S3). MS/MS analyses assigned the modification sites to Arg30 and Arg31 (Figure 2C), which were further verified by mutagenesis analysis. Modification signals were no longer detected in samples expressing the SNAP25 R30K/R31K mutant (Figure 2D). Importantly, both Arg30 and Arg31 are located within the coiled-coil domain of the t-SNARE, which is involved in SNAP25 self-association and in interactions with its binding proteins (Figure 2E). To determine the impact of SseK3 on the binding of SNAP25 to its interacting partners, we used mass spectrometry to analyze proteins pulled down by SNAP25 under conditions with and without SseK3, which revealed that VAMP8 was not present in the pulldown products from samples that expressed SseK3 (Figure 2F). This phenomenon can be recapitulated by experiments in which such binding was detected by immunoblotting of samples that expressed enzymatically inactive SseK3 (Figure 2G).
SseK3 limits the size of SNAP25-decorated infection-associated macropinosomes (IAMs) during late stages of infection
SNAP25 is enriched on the fluid-filled infection-associated macropinosome (IAM) (Stévenin et al., 2021), and it plays a crucial role in homotypic fusion among IAMs and their heterotypic fusion with SCVs, an event that is required for the expansion of the bacterial phagosome shortly after phagocytosis (Stévenin et al., 2019). To explore the possibility that SseK3-induced GlcNAcylation may interfere with its ability to promote such fusion events and the expansion of the SCV, we measured the dimension of the SNAP25-containing vacuoles (SNAP25-CVs) that represent SCVs (with bacteria) and IAMs (without bacteria), respectively. Our results indicate that at 0.5 h and 2 h infection time, there was no significant difference between the diameter of SNAP25-CVs found in cells infected with wild-type bacteria and the ΔsseK1/2/3 mutant. Intriguingly, when the infection has proceeded for 6 h, the size of the SNAP25-CVs was significantly smaller in cells infected with the wild-type strain than that found in cells infected with the ΔsseK1/2/3 mutant (Figure 3A). Such difference disappeared when SseK3 was expressed in strain ΔsseK1/2/3 from a plasmid (Figures 3B and 3C). In agreement with these results, in cells transfected to express SseK3 prior to infection, the size of vacuoles formed by strain ΔsseK1/2/3 became smaller (Figures 3D and 3E). Furthermore, signals of protein Arg-GlcNAcylation induced by SseK3 in infected cells displayed clear co-localization with SNAP25 on bacterial vacuoles (Figure 3D). Thus, SseK3 limits the size of the SNAP25-containing IAM vacuole when the infection has proceeded for 6 h. Given the fact that SseK3 attenuates the interaction between SNAP25 and VAMPs (Figure 3F), such alternations in the size of the vacuoles may be caused by the inhibition of fusion among IAMs and between IAMs and SCVs. Because SNAREs are essential for SIF formation (Kehl et al., 2020), it is likely that SseK3 interferes with SNARE paring to impact SCV size and SIF formation at the late stage of infection (Figure 1A).
Expression level of the host ubiquitin E3 ligase TRIM32 is induced upon S. Typhimurium infection
To determine the mechanism the host may employ to counteract the activity S. Typhimurium effectors, we re-analyzed the available transcriptomic data of host cells infected with strain SL1344 (Avraham et al., 2015). These efforts led to the identification of trim32 that codes for an E3 ubiquitin ligase as one of the significantly induced genes in response to S. Typhimurium challenge (Figure 4A). Further analyses by quantitative real-time PCR (qRT-PCR) and immunoblotting confirmed that trim32 is induced at both mRNA and protein levels upon bacterial challenge (Figures 4B and 4C). Moreover, overexpression of TRIM32 led to a significant decrease in the frequency of the intracellular SIF structures found in cells infected with S. Typhimurium (Figures 4D and 4E).
TRIM32 interacts with and ubiquitinates SseK3
An earlier study has demonstrated interactions between TRIM32 and SseK3 (Yang et al., 2015). Yet, SseK3 does not detectably modify TRIM32 by Arg-GlcNAcylation, and the physiological significance of this interaction is unknown. TRIM32 consists of an amino-terminal RING domain, a type II B-box domain, a coiled-coil domain, and a carboxyl NHL domain. To determine which of these regions is important for its interaction with SseK3, we generated a series of TRIM32 deletion mutants and examined their ability to bind the effector. Results from these experiments indicate that the NHL domain is essential for SseK3 binding (Figures 5A and 5B).
TRIM32 is an E3 ubiquitin ligase that contains a RING finger domain. Therefore, we examined whether TRIM32 can catalyze ubiquitination on SseK3. Co-expression of TRIM32 with SseK3 and HA-ubiquitin led to ubiquitination of the effector. In contrast, no ubiquitination signal was detected in samples from cells transfected to express TRIM32ΔRING lacking the RING domain or the enzymatically inactive mutant TRIM32(C39S) in which the active cysteine was mutated to alanine (Kehl et al., 2020) (Figure 5C). In agreement with these results, knocking-out trim32 diminished SseK3 ubiquitination in a way that can be complemented with the wild-type gene but not the C39S mutant (Figure 5D). Furthermore, treatment of the cells with the proteasome inhibitor MG132 considerably enhanced SseK3 ubiquitination (Figure 5D). SseK3 is a Golgi-located protein(Meng et al., 2020), and we found that TRIM32-mediated ubiquitination occurred at the membrane components of SseK3 (Figure 5E). Finally, in biochemical assays, inclusion of TRIM32 in reactions containing huBE1 (E1), UBCH5c (E2), Ub, and ATP led to robust SseK3 ubiquitination. Such modification did not occur in reactions receiving TRIM32ΔRING (Figure 5F).Thus, TRIM32 ubiquitinates SseK3 by its NHL domain recognition.
TRIM32 catalyzes K48-linked ubiquitination on SseK3 and targets its membrane-associated portion for degradation
There are seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) in the ubiquitin molecule, each dictates the formation of a specific type of polyubiquitin chain that determines the biological consequence of the modification (Walczak et al., 2012). To further understand the implications of TRIM32-induced ubiquitination on the activity of Ssek3, we evaluated the chain type of the ubiquitin polymers on SseK3 using a series of lysine mutants of ubiquitin. Robust ubiquitination of SseK3 was observed in reactions receiving wild-type or K48-only ubiquitin (a mutant in which all Lys residues except for Lys48 have been replaced with Arg). Conversely, in reactions containing the K48R ubiquitin mutant, no ubiquitination was detected (Figure 6A). Polyubiquitination by K48 chain type typically results in protein degradation by the ubiquitin-proteasome system (UPS)(Clague and Urbé, 2010), we then investigated the stability of SseK3 in cellulo. Chase experiments with cycloheximide (CHX) showed that the levels of SseK3 associated with the membrane (M-SseK3) were markedly reduced in a manner that can be blocked by MG132 (Figure 6B). These results strongly suggest that SseK3 was degraded via the UPS after being ubiquitinated by TRIM32.
SseK3 has been shown to localize to the cis-Golgi apparatus(Meng et al., 2020). Importantly, RFP-TRIM32 also extensively targets to the Golgi apparatus when co-expressed with GFP-SseK3 in HeLa cells (Figure 6C). We therefore examined whether TRIM32 induces degradation of SseK3. Our results indicate TRIM32 indeed causes the reduction of SseK3 that is associated with membrane (Figure 6D). Consistently, knockout of trim32 led to elevated SseK3 in the membrane fraction, which can be reversed by expressing TRIM32 but not the ΔRING mutant. In contrast, the protein level of cytosol fraction of SseK3 (C-SseK3) was largely unaffected by TRIM32 (Figure 6D). Thus, TRIM32 induces K48-linked ubiquitination on SseK3, which results in its degradation, particularly for protein that is associated with the membrane.
TRIM32 antagonizes SNAP25 Arg-GlcNAcylation induced by SseK3 to restrict SIF biogenesis and Salmonella replication
The observation that TRIM32 ubiquitinates SseK3 and targets it for degradation suggests that this E3 ubiquitin ligase regulates the function of the effector. Indeed, we found that SseK3 injected into host cells by S. Typhimurium interacted with TRIM32 (Figure 7A). As expected, the level of GlcNAcylated SNAP25 decreased in samples expressing TRIM32 but not the mutant lacking the RING domain (Figure 7B). In line with these observations, overexpression of TRIM32 diminished the ability of SseK3 to promote SIF formation (Figures 7C and 7D). Considering that both SseK3 protein and SIF structure are required for Salmonella intracellular survival within macrophages(Meng et al., 2020; Singh et al., 2018), we next determined the effects of TRIM32 on bacterial replication in macrophage cells. We synthesized three pairs of siRNA and found that the 2nd pair had the best down-regulation effect (Figure 7E). As expected, trim32 knockdown efficiently facilitated bacterial replication of SseK3-expressing-Salmonella, but not S. Typhimurium lacking sseKs in RAW264.7 macrophage cells (Figure 7F). Together, these results indicate that TRIM32 functions to restrict the activity of SseK3 by targeting it for proteasome degradation, thus lowering Arg-GlcNAcylation on SNAP25, which ultimately causes a reduction in SIF formation and bacterial virulence (Figure 8).
Discussion
The outcome of bacterial infection is dictated by intimate interactions between host factors and virulence factors(Tripathi-Giesgen et al., 2021). Several members of the TRIM E3 ubiquitin ligase family have been found to be involved in regulating pathogen infection, including some that confer resistance to the virus by directly targeting viral proteins (Koepke et al., 2021; Lazzari and Meroni, 2016). TRIM56 and TRIM65 have been shown to be targeted by SopA, a HECT-like E3 ligase(Kamanova et al., 2016). Our discovery of TRIM32 as a host factor that functions to control S. Typhimurium virulence has expanded the role of these E3 ubiquitin ligases in defense against bacterial infection.
Importantly, an earlier study has shown that Trim32 plays a role in defending against S. Typhimurium infection because trim32-/- mice are more sensitive to inflammatory death caused by this bacterium (Yang et al., 2017). In this study, several lines of evidence show that TRIM32 is involved in the defense against S. Typhimurium by attacking the SPI-2 effector SseK3. First, the expression of TRIM32 and SseK3 are synchronously upregulated. SseK3 belongs to an SPI-2 effector and begins to catalyze Arg-GlcNAcylation after 6 h post-infection(Meng et al., 2020). Both trim32 mRNA and TRIM32 protein levels were significantly induced at this time (Figure 4). Second, TRIM32 interacts with SseK3 expressed by transfected or injected into host cells by S. Typhimurium via its carboxyl NHL repeats. Third, TRIM32 catalyzes K48-type polyubiquitin chains on SseK3 and targets its membrane-associated SseK3 for degradation. Fourth, overexpression of this E3 ligase led to less SIFs formation during S. Typhimurium infection, and trim32 knockdown facilitated Salmonella replication within macrophage cells.
Our findings have also provided novel insights into the role of effector-induced Arg-GlcNAcylation in S. Typhimurium infection. Additional proteins potentially modified by members of the SseK have been identified in cells ectopically expressing the effectors or in cells infected with S. Typhimurium strains expressing specific effectors (Meng et al., 2020; Newson et al., 2019). These results are consistent with observations made in an earlier RNAi screen which revealed that the canonical mammalian late endo-/lysosomal vesicle fusion machinery (SNARE and Rab GTPase) is involved in SIF biogenesis(Kehl et al., 2020). Our finding that multiple coiled-coil containing-domain proteins are Arg-GlcNAcylated in infected cells suggests that SseKs may act coordinately to target endomembrane components to facilitate S. Typhimurium replication. In particular, SseK3 appears to directly participate in this process by imposing Arg-GlcNAcylation on SNAP25, leading to inhibition of its paring with VAMP8 and the restriction of the expansion of SCVs. This activity of SseK3 also causes a reduction in the size of macropinosomes, but the physiological consequence of such reduction is not clear. A recent screen using proximity labeling (BioID) found that SPI-2 effectors SseG, SopD2, PipB2, and SifA potentially interact with proteins in the SNARE complex (D’Costa et al., 2019). Interestingly, these effectors have been suggested to be involved in SIF formation(Knuff and Finlay, 2017). It is likely that multiple effectors, including SseK3 coordinate to regulate the dynamics of SNARE pairing to promote SIF biogenesis during S. Typhimurium infection. Future research aiming at dissecting the potential interplay among these effectors and how each is temporally and spatially regulated to ensure successful infection will yield more insights into their roles in the intracellular lifecycle of the pathogen.
A majority of proteins anchored in membrane-containing organelles have been reported to be degraded via the cytosolic proteasome system(Guo, 2022; Ramachandran et al., 2018). The spatial separation between substrate selection and degradation requires either membrane-anchored substrates extraction from the membrane or recruitment of 26S proteasomes to the membrane. A well-studied example is the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Ubiquitinated proteins on ER are extracted from the membrane by Cdc48p/p97 complex and conveyed to the proteasome(Hwang and Qi, 2018). Besides, FKBP38, residing in the ER and mitochondrial membranes, functions to anchor the 26S proteasome to the organellar membrane(Nakagawa et al., 2007). Recently, proteasomes have been also reported to be constitutively associated with the Golgi membranes by PSMD6 and mediate the degradation of the Golgi protein GM130(Eisenberg-Lerner et al., 2020). SseK3 is a Golgi-located protein, and we here show that membrane counterparts of SseK3 are ubiquitinated and degraded in a proteasome-dependent manner. The mechanism is likely similar to GM130, and further investigations are needed.
Based on our results, we propose a model for the regulatory role of the TRIM32-SseK3-SNARE-SIF axis in S. Typhimurium infection. A few minutes after bacterial entry, the SCV increases in size through fusions with the macropinosomes. The t-SNARE protein SNAP25 localized on IAMs and v-SNARE VAMP8 are recruited to the SCV. The fusion between the SCV and IAMs allows the former to expand in size. As the infection has proceeded to late stages (>6h), SseK3 injected by the SPI-2 attacks SNAP25 by Arg-GlcNAcylation, leading to ablation of SNAP25-VAMP8 paring and the SNARE fusion events, which may contribute to SIF formation. Meanwhile, infected cells sense the presence of S. Typhimurium and induce the expression of trim32 by a yet unrecognized mechanism. Elevated TRIM32 captures membrane-associated SseK3 for ubiquitination and subsequent proteasome degradation, which restricts SseK3-SNARE-mediated SIF biogenesis and inhibits the intracellular replication of Salmonella (Figure 8).
Materials and Methods
Bacterial strains, cell culture and infection
S. Typhimurium strains (wild-type SL1344 and its mutant derivatives) used in this study were listed in SI Appendix, Table S1. pET28a vector-based complementation plasmids were introduced into S. Typhimurium by electroporation (2.5 kV, 200 Ω, 25 μF and 5 ms). Bacteria were cultured in LB Broth at 37 °C on a shaker (220 rpm/min). When necessary, cultures were supplemented with antibiotics at the following final concentrations: streptomycin, 100 μg mL-1; ampicillin, 100 μg mL-1; kanamycin, 50 μg mL-1.
The procedure for bacterial infection of mammalian cells was performed as previously described. Briefly, wild-type and mutant Salmonella were cultured overnight (approximately 16 h) at 37 °C on a shaker (220 rpm/min) and then were subcultured at 1:33 dilution in LB without antibiotics for 3 h. Bacteria were diluted in serum-free and antibiotics-free DMEM, and added to cells at a multiplicity of infection (MOI) of 100 for 30 min at 37 °C. 24-well plates were centrifuged at 700g for 5 min at room temperature to promote and synchronize infection. Extracellular bacteria were removed by extensive washing with PBS, and culture media was replaced with medium containing 100 μg mL-1 gentamicin. Cells were incubated at 37 °C, 5% CO2 for a further 1.5 h, and the culture medium was replaced with a medium containing 20 μg mL-1 gentamicin. Infected cells were incubated to the indicated time at 37 °C in a 5% CO2 incubator. Samples were processed further for immunoprecipitation or immunofluorescence.
To measure the S. Typhimurium replication fold, RAW264.7 cells were infected with indicated Salmonella strains at an MOI of 10. Infection was facilitated by centrifugation at 700 g for 5 min at room temperature. After 30 min incubation at 37 °C, cells were washed three times with PBS to remove extracellular bacteria and incubated with fresh DMEM containing 100 μg mL-1 gentamycin. At 2 h post-infection, the gentamicin concentration was reduced to 20 μg mL-1. At 2 h and 24 h post-infection, cells were lysed in cold PBS containing 1% Triton X-100, and colony-forming units were determined by serial-dilution plating on agar plates containing 100 μg mL-1 streptomycin and 50 μg mL-1 kanamycin. The replication fold was determined by dividing the number of intracellular bacteria at 24 h by the number at 2 h.
Plasmids, antibodies and reagents
Plasmids used in this study were listed in SI Appendix, Table S2. Genes coding for SseK1, SseK2, and SseK3 DNA were amplified from genomic DNA of S. Typhimurium strain SL1344 and were inserted into pCS2-EGFP, pCS2-RFP, pCS2-Flag, and pCS2-HA, respectively for transient expression in mammalian cells. For complementation in S. Typhimurium ΔsseK1/2/3 strain, DNA fragment containing genes encoding SseK1, SseK2, and SseK3, each together with their upstream promoter regions, was amplified from S. Typhimurium SL1344 genomic DNA and inserted into pET28A. cDNAs for SNAP23, SNAP25, VAMP8, Syntaxin7, Syntaxin8, Vti1b, Sec22b, Snapin, Rab1 and TRM32 were amplified from a cDNA library of HeLa cells. For mammalian expression, cDNAs were cloned into pCS2-EGFP and pCS2-Flag vectors. Truncation, deletion, and point-mutation mutants were constructed by the standard PCR cloning strategy. All plasmids were verified by sequencing analysis. Antibodies and reagents were listed in SI Appendix, Table S3.
Cell culture, transfection and stable cell-line construction
293T, HeLa cells were obtained from the American Type Culture Collection (ATCC) and were maintained in DMEM (HyClone) supplemented with 10% FBS (Gibco), 2 mM L-glutamine, 100U mL-1 penicillin, and 100 μg mL-1 streptomycin. Cells were cultivated in a humidified atmosphere containing 5% CO2 at 37 °C.
Transient transfection was performed using Vigofect (Vigorus) or Jetprime (Polyplus) reagents following the manufacturers’ instructions. For siRNA knockdown, 200 pmol of siRNAs were transfected into 2×106 RAW264.7 cells. Sense sequences for the siRNAs used are as follows: trim32 1# 5’-CCATCTGCATGGAGTCCTTTT -3’, trim32 2#: 5’-CCAAGTGTTCAACCGCAAATT-3’, trim32 3#: 5’-GCTATCAT CTGAGAAGATATT-3’, and negative control (NC): 5’-TTCTCCGAACGTGTCAC GT-3’.
To generate the cell line that stably expresses EGFP-VAMP8, pcDNA4-EGFP-VAMP8 was transfected into 293T cells. Cell emitting green fluorescence obtained by fluorescence-activated cell sorting were cultured in DMEM medium supplemented with 10% FBS, 1% v/v penicillin/streptomycin, and 50 μg mL-1 zeocin. Knockout cell lines were generated by the CRISPR-Cas9 method as previously described (ref). Briefly, the pHKO plasmid containing the guide RNA targeting Trim32 was co-transfected with packaging plasmid psPAX2 and envelope plasmid pMD2.G into Cas9-expressed 293T cell. The transfection cocktail was removed after 6 h and replaced by fresh medium. After 72 h, viral containing supernatant was collected and filtered with 0.45 μm membrane, and stored at 4 °C before transduction. 293T cells were transduced with the lentiviral particles. Three days later, GFP-positive cells were sorted into single clones in 96-well plates by flow cytometry and knockout lines were identified by PCR and by Western blot with antibodies specific for TRIM32. The sequence for the guide RNA used for trim32 knockout is 5′-CCAGTTTGTAGTAACCGATG-3′.
Immunoprecipitation
For immunoprecipitation, 293T cells at a confluency of 60-70% in 6-well plates were transfected with a total of 5 μg plasmids that code for the protein of interest. Twenty-four hours after transfection, cells were washed once with PBS and lysed in buffer A containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, and 1% Triton X-100, supplemented with a protease inhibitor mixture (Roche Molecular Biochemicals). Pre-cleared lysates were subjected to anti-Flag M2 or anti-GFP immunoprecipitation following the manufacturer’s instructions. The beads were washed four times with lysis buffer, and the immunoprecipitates were eluted by SDS sample buffer followed by standard immunoblotting analysis. All the immunoprecipitation assays were performed more than three times, and representative results were shown. For enrichment of the arginine-GlcNAcylated proteins from lysates of transfected cells, samples were washed three times in ice-cold PBS and lysed in buffer A containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, and 1% Triton X-100, supplemented with a protease inhibitor mixture (Roche Molecular Biochemicals). Pre-cleared lysates were subjected to immunoprecipitation with the anti-Arg-GlcNAc antibodies(Pan et al., 2014). The beads were washed four times with the lysis buffer, and the immunoprecipitates were dissolved by SDS sample buffer.
Immunofluorescence labeling and confocal microscopy
At the indicated time point post-transfection or bacterial infection, cells were fixed for 10 min with 4% PFA in PBS and permeabilized for 15 min with 0.2% Triton X-100 in PBS. After blockade of nonspecific binding by incubation of cells for 30 min with 2% bovine serum albumin (BSA) in PBS, coverslips were incubated with the appropriate primary antibodies and subsequently with fluorescein-labeled secondary antibodies (ThermoFisher). Confocal fluorescence images were acquired at the confocal microscope (Spinning Disc, Leica). All image data shown are representative of at least three randomly selected fields.
Expression and purification of recombinant proteins
Protein expression was induced in E. coli BL21(DE3) strain (Novagen) harboring the appropriate plasmid that directs the protein of interest at 22°C for 15 h with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) after the cultures have reached an OD600 of 0.8–1.0. Affinity purification of GST-SseK3 was performed using glutathione sepharose (GE Healthcare), and purification of 6×His-SUMO-hUBE1, 6×His-TRIM32, 6×His-TRIM32 (ΔRING), and 6×His-Flag-Ub was conducted using Ni-NTA agarose (Qiagen), following the manufacturer’s instructions. Proteins were concentrated in a buffer containing 20 mM HEPES pH 7.5, 150mM NaCl, and 5% glycerol. The protein concentration was determined by the Bradford method (ref).
In vitro ubiquitination assays
To assay ubiquitination of SseK3 in vitro, Ubiquitin (5 μg), E1 (200 ng), UBCH5a (300 ng), His-TRIM32 (0.8 μg) and GST-SseK3 (2 μg) were incubated with 2 mM ATP at 37°C for 2 h in ubiquitin assay buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 2 mM DTT). Reactions were stopped by adding 30 mM EDTA and 15 mM DTT. After GST pull-down, the sample was washed with 1 M urea for 60 min to exclude potential binding of unanchored polyubiquitin, then the sample was placed in an SDS-loading buffer and boiled at 95°C for 5 min. Samples were subsequently analyzed by SDS-PAGE followed by Western blotting.
Mass spectrometric analyses
For identification of the GlcNAcylated arginine and arginine-containing peptides, purified SNAP25 protein was subjected to digestion with GluC and LysC, and the resulting peptides were separated on an EASY-nLC 1200 system (Thermo Fisher Scientific). The nano liquid chromatography gradient was as follows: 0-8% B in 3 min, 8-28% B in 42 min, 28-38% B in 5 min, 38-100% B in 10 min (solvent A: 0.1% Formic acid in water, solvent B: 80% CH3CN in 0.1% formic acid). Peptides eluted from the capillary column were applied directly onto a Q Exactive Plus mass spectrometer by electrospray (Thermo Fisher Scientific) for mass spectrometry (MS) and MS/MS analyses. Searches were performed against the amino acid sequence of SNAP25 and were performed with cleavage specificity allowing four mis-cleavage events. Searches were performed with the variable modifications of oxidation of methionine, N-Acetyl-hexosamine addition to arginine (Arg-GlcNAc), and acetylation of protein N termini. For identification of the SNAP25-binding protein, immunoprecipitates were separated using SDS-PAGE, fixed, and visualized after silver staining as recommended by the manufacturer. An entire lane of bands was excised and subjected to in-gel trypsin digestion and MS/MS detections as described above. Identification of proteins was carried out using the Proteome Discoverer 2.2 program. Searches were performed against the Human proteomes depending on the samples with carbamidomethylation of cysteine set as a fixed modification. The precursor mass tolerance was set to 10 parts-per-million (ppm) and a fragment mass tolerance of 0.02 Da. A maximum false discovery rate (FDR) of 1.0% was set for protein and peptide identifications.
Quantification and statistical analysis
All results are presented as mean ± standard deviation containing a specified number of replicates. Data were analyzed using a Student’s t-test to compare two experimental groups. The comparison of multiple groups was conducted by using the one-way analysis of variance (ANOVA). A difference is considered significant as the following: *P <0.05, **P < 0.01.
Compliance and ethics
All authors declare no competing interests. This study does not involve human subjects and animals.
Author Contributions
S.L. and K.M. conceived the study. K.M. and J.Y. designed and performed the functional experiments. K.M. and J.X. conducted the mass spectrometry experiments. L.S. provided technical assistance in the analyses of the mass spectrometry data. J.L. and P.Z. provided assistance in preparing experiments materials. S.L. and K.M. analyzed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.
Supplementary Information for
SupplementaryTables
Acknowledgments
We thank members of the Li laboratory and the central laboratory of Taihe hospital for helpful discussions and technical assistance. We thank Prof. Hongbing Shu and Prof. Bo Zhong in Wuhan University (China) for providing the pRK-HA-Ub (K48 only), and pRK-HA-Ub (K63 only) plasmids. We thank Prof. Gang Cao in Huazhong Agricultural University (China) for providing the pHKO14-Cas9-Flag, and pHKO-GFP-sgRNA plasmids. This work was supported by the National Key Research and Development Programs of China 2021YFD1800404 and 2018YFA0508000, Huazhong Agricultural University Scientific & Technological Self-Innovation Foundation 2017RC003 to S.L., and Hubei Provincial Natural Science Foundation 2021CFB472 to K.M..
Footnotes
Email addresses of all the authors: Kun Meng: 15896536298{at}163.com, Jin Yang: 991529481{at}qq.com, Juan Xue: xuejuanjuan0505{at}163.com, Jun Lv: lvjunfisher{at}126.com, Ping Zhu: thzhuping01{at}163.com, Liuliu Shi: shi-liuliu{at}163.com