Previous studies identified a cell-autonomous host defense program in which mammalian cells attach linear ubiquitin (M1) to the surface of the Gram-negative pathogen Salmonella enterica upon bacterial spillage into the host cell cytosol (Otten et al., 2021; Noad et al., 2017). Recently, it was reported that the cytosol-adapted pathogen B. thailandensis secretes a ubiquitin esterase that renders the bacteria resistant to this type of cytosolic ubiquitylation (Szczesna et al., 2024). We therefore hypothesized that resistance to host-driven linear ubiquitylation is a common feature of bacterial pathogens that are adapted to a cytosolic milieu. To test this concept, we monitored ubiquitylation of the professional cytosolic pathogen S. flexneri (serotype 2a, strain 257T) by immunofluorescence. We found that wild-type (WT) S. flexneri remained largely devoid of M1-linked ubiquitin in both alveolar A549 and colonic HT29 epithelial cell lines under both unprimed and IFNγ-primed conditions (Figure 1A–C). In contrast to WT S. flexneri, a ΔmxiE mutant strain that has diminished expression of ‘second-wave’ T3SS effector proteins (Kane et al., 2002; Mavris et al., 2002), was robustly stained with anti-M1 antibodies in IFNγ-primed host cells (Figure 1A–C). These observations indicate that S. flexneri uses MxiE-dependent effectors to evade an IFNγ-activated ubiquitylation pathway that targets cytosolic bacteria.

The ubiquitin protein contains seven lysine residues. Any of these lysine residues or the N-terminal methionine of the acceptor ubiquitin can form isopeptide bonds with the C-terminal glycine residue of the donor ubiquitin to form chains. To determine which ubiquitin linkage types are present on the surface of S. flexneriΔmxiE bacteria, we generated A549 cells that overexpressed internally tagged ubiquitin (INT-Ub) variants, which contain only a single lysine residue or lack lysines altogether. As a ubiquitin acceptor, the lysine-less (7KR) variant can only be assembled into ubiquitin chains via its N-terminal methionine. We found that 7KR INT-Ub robustly co-localized with S. flexneri ΔmxiE, albeit with diminished efficiency compared to WT INT-Ub (Figure 2A).

In addition to its incorporation into linear ubiquitin chains, 7KR ubiquitin can also function as donor ubiquitin and be attached as mono-ubiquitin to a substrate or to an existing ubiquitin chain as a chain terminator. To distinguish between 7KR INT-Ub signals originating from linear versus mono-ubiquitylation, we generated an N-terminally HA-tagged 7KR INT-Ub variant. The N-terminal tag prevents linear ubiquitylation but still allows 7KR INT-Ub to be attached as mono-ubiquitin via its C-terminal carboxyl group. We found that the addition of this N-terminal HA-tag significantly reduced but not completely abolished the number of ΔmxiE bacteria decorated with 7KR INT-Ub (Figure 2 –figure supplement 1). These data indicate that much of the association of 7KR INT-Ub with cytosolic ΔmxiE bacteria is due to the formation of M1-linked linear ubiquitin chains.

Collectively, our data confirmed that M1-linked ubiquitin is bound to S. flexneri ΔmxiE but also indicated that other additional ubiquitin linkage types are attached to mxiE-deficient bacteria. The functional importance of these lysine-linked ubiquitin chains was demonstrated by improved bacterial targeting of INT-Ub variants that can form additional K27-linkages or both K27- and K63-linkages (Figure 2A). Linkage-specific antibodies against K27 and K63 confirmed that these two linkage types are present on ΔmxiE bacteria in IFNγ-primed A549 cells (Figure 2B, C). Staining with an antibody that detects most ubiquitin linkage types (FK2) showed near complete overlap with anti-M1 staining (Figure 2D, E). Because the percentage of FK2-positive but M1-negative bacteria (Figure 2E) was much smaller than the percentage of bacteria that were K27- or K63-positive (Figure 2B, C), these data suggest that most S. flexneri ΔmxiE bacteria are decorated with both linear and lysine-linked ubiquitin.

Previous work showed that the multimeric E3 ligase LUBAC can deposit M1-linked ubiquitin on the surface of cytosolic S. enterica Typhimurium (Noad et al., 2017). Moreover, S. flexneri secretes the MxiE-regulated effector IpaH1.4 which specifically recognizes and marks the LUBAC components HOIP and HOIL-1 for proteasomal degradation (de Jong et al., 2016; Liu et al., 2022; Hiragi et al., 2023). Consequently, we hypothesized that LUBAC was essential for the ubiquitylation of ΔmxiE. To test this hypothesis, we infected HOIP- and HOIL-1-deficient A549 cells (HOIP^KO, HOIL-1^KO, Figure 3A) with the ΔmxiE mutant under IFNγ-primed conditions. Unexpectedly, deletion of either HOIP or HOIL-1, each an essential component of the LUBAC complex, did not diminish linear ubiquitylation of ΔmxiE when compared to WT cells (Figure 3B). Similarly, the internally tagged 7KR-Ub variant decorated S. flexneri ΔmxiE independent of the LUBAC component HOIP (Figure 3C). Collectively, these data demonstrate that LUBAC is dispensable for linear ubiquitylation of ΔmxiE bacteria in the host cell cytosol of IFNγ-primed A549 cells.

The ubiquitin E3 ligase RNF213 was recently shown to ubiquitylate pathogen-containing vacuoles (Hernandez et al., 2022; Walsh et al., 2022) as well as Gram-positive and Gram-negative bacteria inside the host cell cytosol (Otten et al., 2021; Thery et al., 2021; Szczesna et al., 2024). Like HOIL-1, RNF213 is an IFNγ-inducible protein (Figure 3A and Hernandez et al., 2022; Walsh et al., 2022). Using immunofluorescence microscopy, we detected RNF213 on the surface of ΔmxiE but not WT bacteria in IFNγ-primed A549 as well as colonic HT29 cells (Figure 3D–G). The vast majority of ubiquitylated S. flexneri ΔmxiE mutants (~93%) co-stained with anti-RNF213 (Figure 3G), indicative of a functional role for RNF213 in the ubiquitylation of ΔmxiE bacteria. To directly probe for RNF213 function, we infected a pool of CRISPR-generated A549 RNF213^KO cells (Figure 3A) with S. flexneri ΔmxiE and stained these IFNγ-primed cells for M1-, K27-, and K63-linked ubiquitin. We found that in the absence of human RNF213, antibodies specific for M1-, K27-, and K63-linked ubiquitin no longer stained the surface of S. flexneri ΔmxiE (Figure 3B, H–I). In agreement with these immunofluorescence data, both WT and lysine-less ubiquitin (7KR-Ub) localized to ΔmxiE bacteria in WT but not in RNF213^KO cells (Figure 3C). Together, these data demonstrate that human RNF213 is essential for the linear and lysine-linked ubiquitylation of MxiE-deficient S. flexneri mutants.

The transcription factor MxiE induces the expression of several secreted effector proteins of the IpaH family that act as bacterial ubiquitin E3 ligases and tag host proteins for proteasomal degradation (Wandel et al., 2017; Li et al., 2017; de Jong et al., 2016; Liu et al., 2022; Luchetti et al., 2021; Hansen et al., 2021; Otsubo et al., 2019; Rohde et al., 2007; Ashida et al., 2010). Since our data revealed that the S. flexneri virulence factor MxiE blocks bacterial ubiquitylation by RNF213, we hypothesized that S. flexneri infections induce the proteasomal degradation of RNF213 in a MxiE-dependent manner. To test this hypothesis, we measured RNF213 protein levels by immunoblotting in cells that had been infected with WT or ΔmxiE S. flexneri. Our data revealed that RNF213 protein levels are drastically reduced upon infection with WT S. flexneri and that the deletion of mxiE partially restored RNF213 expression in infected cells (Figure 4A). Similarly, and as reported previously (Noad et al., 2017; de Jong et al., 2016; Liu et al., 2022), HOIP protein expression is diminished in cells infected with WT S. flexneri but much less so in the case of infections with ΔmxiE S. flexneri (Figure 4A).

To directly test whether S. flexneri promotes proteasomal degradation of RNF213, we conducted infections in the presence of the proteasomal inhibitor MG132. As expected, MG132 largely restored RNF213 total protein levels in S. flexneri-infected cells (Figure 4B). We next tested whether a specific IpaH effector facilitates the degradation of RNF213. To this end, we transiently expressed GFP-fusion proteins of individual IpaH family members in HEK 293T cells that ectopically expressed RNF213 and measured their effects on RNF213 protein levels by immunoblotting. Our mini-screen revealed that the ectopic expression of the two closely related paralogs IpaH1.4 and IpaH2.5 was sufficient to promote RNF213 degradation in HEK 293T cells (Figure 4C). Of note, the substrate-binding LRR domains of IpaH2.5 and IpaH1.4 are 98.9% identical in DNA sequence and were previously shown to have comparable substrate specificity and functional redundancy (de Jong et al., 2016). Both IpaH1.4 and 2.5 share a highly conserved NEL E3 ubiquitin ligase domain that contains an essential catalytic cysteine residue at position 368 (Liu et al., 2022). Catalytically inactive forms of IpaH1.4 or IpaH2.5, in which Cys368 is mutated to a serine (C/S), failed to degrade RNF213 (Figure 4D). As an additional control, we included overexpression of IpaH9.8, an inhibitor of GBPs, which also did not impact RNF213 expression (Figure 4D), as anticipated.

To investigate potential physical interactions between IpaH1.4 and IpaH2.5, we reanalyzed a previously generated dataset that employed a method known as ubiquitin-activated interaction traps (UBAITs) (Hansen et al., 2021). As depicted in Figure 4—figure supplement 1A, the human ubiquitin gene was fused to the 3′ end of IpaH2.5, producing a C-terminal IpaH2.5-ubiquitin fusion protein. When incubated with ATP, a ubiquitin-activating enzyme E1, and a ubiquitin-conjugating enzyme E2, the IpaH2.5-ubiquitin ‘bait’ protein is capable of binding to and ubiquitylating target substrates. This ubiquitylation creates an iso-peptide bond between the IpaH2.5 bait and its substrate, thereby enabling purification via a Strep affinity tag incorporated into the fusion construct (Hansen et al., 2021). IpaH2.5-ubiquitin bait and IpaH3-ubiquitin control proteins were incubated with lysates from murine intestinal tissue. To detect interaction partners in a physiologically relevant setting, we used intestinal lysates derived from mice infected with Salmonella, which in contrast to Shigella causes pronounced inflammation in WT mice and therefore better simulates human Shigellosis in an animal model. Using UBAIT, we identified HOIP (Rnf31) as a likely IpaH2.5 binding partner (Figure 4—figure supplement 1B), thus confirming previous observations (de Jong et al., 2016) and validating the effectiveness of our approach. Strikingly, we identified mouse Rnf213 as the most abundant interaction partner of the IpaH2.5-ubiquitin bait protein (Figure 4—figure supplement 1B). Collectively, our data and concurrent reports showing direct interactions between IpaH1.4 and human RNF213 (Naydenova et al., 2025; Zhou et al., 2025) indicate that the virulence factors IpaH1.4 and IpaH2.5 directly bind and degrade mouse as well as human RNF213.

Given that ectopic expression of either IpaH1.4 or IpaH2.5 was sufficient to degrade RNF213, we expected these two endogenous effectors to be functionally redundant. However, we found that genetic deletion of ipaH1.4 in either S. flexneri serotype 2a strain 257T (Figure 5A, B) or in serotype 5a strain M90T (Figure 5—figure supplement 1A) was sufficient to restore RNF213 protein expression and RNF213 recruitment to cytosolic bacteria. A deletion mutant of ipaH2.5 was as resistant to RNF213 targeting as co-isogenic WT bacteria, and a ΔipaH1.4-ipaH2.5 double mutant was indistinguishable from the ΔipaH1.4 single deletion mutant (Figure 5—figure supplement 1A). These data suggested that endogenous IpaH2.5 is non-functional, which agrees with a previous characterization of ipaH2.5 as a minimally expressed pseudogene (Silué et al., 2020).

Previous work showed that IpaH1.4 binds to and degrades the LUBAC subunit HOIP (Noad et al., 2017; de Jong et al., 2016; Liu et al., 2022). To determine whether the degradation of RNF213 could be indirect and HOIP-dependent, or vice versa, we monitored RNF213 expression in HOIP^KO cells and HOIP expression in RNF213^KO cells. We found that expression of RNF213 remained at WT levels in the absence of HOIP and that the degree of IpaH1.4-dependent RNF213 degradation was comparable in WT and HOIP^KO cells (Figure 5A). Similarly, infections with WT but not ΔipaH1.4 mutant S. flexneri resulted in a substantial reduction of HOIP protein expression in RNF213^KO cells (Figure 5—figure supplement 1B). These observations show that IpaH1.4 targets RNF213 and HOIP for degradation independent from each other.

The sustained RNF213 expression in cells infected with S. flexneriΔipaH1.4 (Figure 5A) correlated with staining of cytosolic ΔipaH1.4 bacteria with RNF213 (Figure 5B, C). As expected, the complementation of the ΔipaH1.4 mutant with WT IpaH1.4 efficiently prevented RNF213 recruitment to cytosolic bacteria, whereas complementation with catalytically inactive IpaH1.4 did not (Figure 5C, D). Through the use of linkage-specific antibodies, we also observed M1-, K27-, and K63-linked ubiquitin on cytosolic ΔipaH1.4 bacteria in IFNγ-primed WT A549 cells (Figure 5E; Figure 5—figure supplement 1C–E). In contrast to IFNγ-primed WT A549 cells, IFNγ-primed RNF213^KO A549 cells lacked linear ubiquitylation of ΔipaH1.4 (Figure 5E). This ubiquitylation of S. flexneriΔipaH1.4 was independent of LUBAC, as it occurred in HOIL-1^KO, HOIP^KO, and WT A549 cells at comparable frequencies (Figure 5—figure supplement 1F).

Although S. flexneri is a human-adapted pathogen, some IFNγ-inducible host defense programs targeting S. flexneri and the corresponding bacterial evasion mechanisms transcend host barriers. For example, the Shigella virulence factor IpaH9.8 inhibits both human and murine guanylate binding proteins (GBPs) (Li et al., 2017; Ji et al., 2019). We therefore asked whether RNF213 ubiquitylates S. flexneri ΔipaH1.4 in at least one other mammalian species, the mouse. To do so, we infected unprimed and IFNγ-primed WT and Rnf213^KO/KO mouse embryonic fibroblasts (MEFs) (Figure 5—figure supplement 2A) with S. flexneri WT and ΔipaH1.4 mutant. We observed that ΔipaH1.4 bacteria became decorated with mouse Rnf213 (Figure 5—figure supplement 2B) and linear ubiquitin chains (Figure 5—figure supplement 2C). The IFNγ-dependent increase in the percentage of M1-ubiquitin-decorated ΔipaH1.4 S. flexneri bacteria was reversed in Rnf213^KO/KO MEFs (Figure 5—figure supplement 1C). Collectively, these data depict a conserved role for the mammalian ubiquitin E3 ligase RNF213 in cytosolic immune surveillance and demonstrate that IpaH1.4 can evade both mouse and human RNF213-driven linear ubiquitylation.

Ubiquitylated microbial cargo can be delivered into microbicidal lysosomes in an autophagy-like process commonly referred to as xenophagy (Tripathi-Giesgen et al., 2021). Previous work showed that linear ubiquitylation of S. enterica leads to bacterial clearance by xenophagy (Otten et al., 2021). In contrast to the xenophagic killing of ubiquitylated S. enterica, we found that ubiquitylated S. flexneri ΔIpaH1.4 and ΔmxiE mutant bacteria continued to replicate inside IFNγ-primed A549 cells (Figure 5H), reminiscent of the continued growth of ubiquitylated B. thailandensis tssM mutants inside the host cell cytosol of human IFNγ-primed cells (Szczesna et al., 2024). Thus, our data indicate that S. flexneri, like B. thailandensis, can block ubiquitin-dependent xenophagy both at the stage of ubiquitylation and at another so far undetermined downstream step of this cell-autonomous host defense pathway.

A549 (ATCC #CCL-185), HT-29 (ATCC #HTB-38), and MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 1% MEM non-essential amino acids (Gibco), 55 µM 2-Mercaptoethanol (Gibco) and 10% heat-inactivated fetal bovine serum (FBS). MEFs were generated from E12 to E14 embryos as previously described (Coers et al., 2008). Cells were grown at 37°C in 5% C0₂. Cell lines were authenticated using GenePrint10 (Promega) by the Duke University DNA Analysis Facility. All cell lines were routinely tested for mycoplasma.

The production and characterization of A549 knockout cell lines (pooled RNF213KO, HOIP^KO clone, and HOIL-1^KO clone) was previously reported (Hernandez et al., 2022; Walsh et al., 2022). Various GFP-tagged IpaH expression constructs including the IpaH1.4 and IpaH2.5 C/S mutants were previously described (de Jong et al., 2016). Internally Strep-tagged ubiquitin (INT-Ub) and Internally Strep-tagged and N-Terminally HA-tagged ubiquitin (NTerm-HA-Ub_ INT-Strep) open reading frames were cloned via Gateway cloning into the lentiviral expression plasmid pLEX307, a gift from Dr. David Root (Addgene plasmid # 41392; http://n2t.net/addgene:41392; RRID:Addgene_41392). DNA of full-length human RNF213 with N-terminal mCherry was a gift from Daisuke Morito (Sugihara et al., 2019). RNF213 was amplified in segments using PCR and cloned using Golden Gate assembly with the enzyme kit BsmBI-v2 (New England Biolabs). Endogenous BsmBI sites were removed from RNF213 by introducing silent mutations in primers used to add enzyme overhangs for Golden Gate assembly. RNF213 was first cloned into a modified Gateway donor vector pDONR221 (Thermo Fisher Scientific) containing a golden gate cloning site in between ATTP sites to make pENTR-mCherry-RNF213. mCherry-RNF213 was transferred into piggybac vector PB-TA-ERN using LR clonase enzyme (Thermo Fisher Scientific) for dox-inducible expression of mCherry-RNF213. PB-TA-ERN (Kim et al., 2016) was a gift from Knut Woltjen (Addgene plasmid #80474; http://n2t.net/addgene:80474; RRID:Addgene_80474). Whole plasmid sequencing of PB-TA-ERN-mCherry-RNF213 and the INT-Ub vectors was performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation. The INT-Ub protein sequences are provided in Supplementary file 1. For mCherry-RNF213 expression in human cells, 293T cells (ATCC CRL-3216) were transfected with PB-TA-ERN and piggybac transposase (pRP[Exp]-EGFP-EF1A>hyPBase, VectorBuilder) using lipofectamine 2000 transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. Cells were grown for approximately 8 days to allow for loss of the transiently transfected vectors, while mCherry-RNF213 that had integrated into the 293T genome was being maintained.

RNF213 knockout mice were generated using a modified iGONAD methodology (Skavicus and Heaton, 2023; Gurumurthy et al., 2019; Ohtsuka et al., 2018). Briefly, 1 day time-mated, vaginal-plug-positive 8- to 24-week-old C57BL/6J female mice were put under isoflurane anesthesia. A dorsal surgical incision was made, and the ovary/oviduct was exposed. The oviduct was injected with a 0.5-μl Cas9/CRISPR mix which included 6.1 μM Cas9 protein (IDT, Cat. #1081058), 30 μM sgRNAs (IDT, Alt-R CRISPR–Cas9 sgRNA), and 0.02% Fast Green FCF (VWR, Cat. #AAA16520-14) in Opti-MEM (Thermo Fisher Scientific, Cat. #11058021). The 30 μM sgRNA concentration included the following two sgRNAs targeting exon 28 of Rnf213: 5′-TTAAATACTGGTAAGGTCGT(TGG)-3′ and 5′-AGTCGGAGTAGCAAAATCCC(TGG)-3′. After the Cas9/sgRNA mix injection, the oviducts were covered with tissue and then electroporated using a CUY21EDIT II (BEX Co, Ltd) electroporator with the settings: Square mA mode, Pd V: 60 V, Pd A: 100 mA, Pd on: 5 ms, Pd off: 50 ms, Pd N: 3, and Decay: 10%. This procedure was done on both sets of oviducts in the mouse. After surgery, female mice were housed together and at 19–21 days post-surgery monitored for pup birth. Pups were genotyped, and three founder mice with out-of-frame 101, 284, or 407 bp deletions within exon 28, respectively, were selected and bred to homozygosity. Loss of Rnf213 protein expression in cells derived from these knockout mice was validated by western blotting.

Plasmids were transfected into HEK 293T cells using the TransIT 293 transfection reagent (Mirus). Wells were transfected with 1 µg plasmid, 750 ng pGAGpol, 250 ng VSVG. Six hours post-transfection, media was aspirated and replaced with fresh DMEM. Twenty-four hours post-transfection, media was aspirated and replaced with pseudoparticle DMEM (3% FBS, 1% MEM non-essential amino acids, 20 mM HEPES). Supernatant was collected 48 and 72 hr after transfection. Supernatants containing lentivirus were filtered using 0.45 µm filters (Corning) and later mixed with 10 µg/ml polybrene (Millipore Sigma). Virus was frozen at –80 until use. For cell transduction, A549 cells were diluted to a concentration of 3.33 × 10⁴ cells/ml and mixed with 10 µg/ml polybrene. 500 µl of lentivirus was added to each well of a 6-well plate, and 1.5 ml of diluted cells were added on top. Cells were selected in media with 1 µg/ml of puromycin and expanded.

HEK 293T that express PiggyBac RNF213 were seeded at 10⁵ cells/well. Sixteen hours before transfection, aTc was added to cells to induce expression of RNF213. The next morning, 2.5 µg of IpaH effector-encoding plasmids were used to transfect HEK 293T using the TransIT 293 transfection reagent (Mirus). Twenty-four hours after transfection, cells were lysed in RIPA buffer and prepared for immunoblotting, as described (Piro et al., 2017).

All S. flexneri strains were grown on Tryptic Soy Broth (TSB, Millipore Sigma) agar plates in the presence of 0.01% of Congo Red (Millipore Sigma) and red colonies were cultured in TSB broth culture. S. flexneri 2457T wild-type and mutant strains (ΔmxiE and ΔipaH1.4) were previously described (Piro et al., 2017). S. flexneri M90T strains (wild-type, ΔipaH2.5, ΔipaH1.4, and ΔipaH1.4-ipaH2.5) were also previously reported (de Jong et al., 2016). For immunofluorescence studies, we used fluorescent S. flexneri strains carrying the pGFPmut2 (Cormack et al., 1996) or the pJUMP45-2A (sfGFP) (Valenzuela-Ortega and French, 2021) plasmids. Bacteria were transformed with the ilux pGEX(-) plasmid (Addgene plasmid #107879) (Gregor et al., 2018) to generate bioluminescent bacteria, as previously reported (Dickinson et al., 2023). Where specified, S. flexneri strains were used that harbor the pilT plasmid which encodes an adhesin (Clerc and Sansonetti, 1987) and enhances bacterial adhesion to host cells. To complement the ΔipaH1.4 strain, full-length IpaH1.4 and its catalytically inactive variant C368S were cloned into the bacterial expression vector pDSW206 using Takara In-Fusion Snap Assembly Master Mix (Cat# 638948) following the manufacturer’s instructions. Bacterial transformation was performed as previously described (Piro et al., 2017). Antibiotics were used as needed at the following concentration: carbenicillin (50 µg/ml), kanamycin (50 µg/ml), spectinomycin (50 µg/ml), and chloramphenicol (100 µg/ml).

For immunofluorescence staining, A549, HT29, and MEF cells were seeded at 1 × 10⁵ cells per well on cover slips in 24-well plates. HT29 cells were plated at 2 × 10⁵ cells per well. For Western blotting, cells were plated at 2 × 10⁵ cells per well in 24-well plates. Cells were either left untreated or treated overnight with human or mouse cytokine IFNγ at a final concentration of 100 U/ml, as indicated. The day before infection, S. flexneri was inoculated into TSB with antibiotics as needed and grown overnight at 37°C with aeration. The next day, bacteria were diluted (1:20) in TSB with antibiotics and grown for 1 hr 30 min. To induce the expression of WT and C368S mutant IpaH1.4 in complemented ΔipaH1.4 bacteria, 1 mM IPTG (Invitrogen 15529-019) was added 1 hr after subculturing. Subsequently, the optical density of the broth culture was measured and pelleted bacteria were washed in sterile PBS and resuspended in DMEM. Infections were carried out at an approximate multiplicity of infection (MOI) of 50–100, unless otherwise specified. Cells were centrifuged at 1000 × g for 10 min at the time of infection and infected cells were incubated at 37°C with 5% CO₂. Cells were washed with warm HBSS two times after 90 min (A549 and HT29 cells) or 60 min (MEFs) post infection. Following these washes, gentamicin at a final concentration of 50 µg/ml (for A549 and MEFs) or 100 µg/ml (for HT29) was added to the media. For fluorescence microscopy, infected cells were fixed with 4% PFA in phosphate-buffered saline (PBS) at 4 hpi (A549 cells), 3 hpi (HT29), or 2 hpi (MEFs). All S. flexneri strains carried the plasmid pGFPmut2 to visualize bacteria by fluorescence microscopy. For western blotting, bioluminescence experiments, and MEF infections, S. flexneri strains expressed PilT for improved adhesion and infection rates.

Cells were fixed in 4% PFA in PBS and then permeabilized either with 0.1% Triton in PBS for 15 min or with ice-cold methanol for 1 min followed by 0.05% saponin in blocking buffer. Blocking buffer was composed of 5% BSA, 2.2% glycine ± 0.05% saponin or 10% goat serum and 0.05% saponin in PBS. Following permeabilization, coverslips were blocked for 30 min at room temperature (RT). Next, coverslips were incubated with the respective primary antibody diluted in blocking buffer overnight at 4°C. The following primary antibodies were used: rabbit monoclonal anti-RNF213 (Sigma HPA026790; 1:1000), rabbit monoclonal anti-linear (M1) ubiquitin; clone 1E3 (Sigma ZRB2114; 1:250), mouse polyclonal anti-ubiquitin FK2 (Cayman Chemical 14220; 1:100), rabbit monoclonal anti-K63 ubiquitin, clone Apu3 (Sigma 05-1308; 1:100), rabbit monoclonal anti-K27 (Abcam ab181537; 1:100), mouse monoclonal anti-Strep (Genscript A01732; 1:200), custom rabbit anti-mRnf213 antibody from an animal immunized with peptides RKSNEGGNTQPEDQRKPGEGR (aa296–316) and KDTVEYEFIYEQAQKKGE (aa429–446) (Life Technologies, 1:100). Coverslips were then washed three times with 0.05% Triton in PBS or 0.05% saponin and incubated with an Alexa Fluor-conjugated secondary antibody (1:1000, Invitrogen) and nuclear dye Hoechst 33258 (2 µg/ml; Invitrogen) for 1 hr at RT. Next, coverslips were washed three times with 0.05% Triton or 0.05% saponin in PBS and mounted on glass slides using a mix of mowiol 4-88 combined with para-phenylenediamine antifading agent (9:1). Coverslips were imaged at a Zeiss Axio Imager microscope with Apotome 3 using a 63×/1.4 NA Oil immersion lens. Two to eleven fields of view were captured per coverslip to account for at least 100 bacteria, and z slices with an interval of 0.5 µm were taken per field of view. Images were processed on the software program Fiji. Bacteria that had protein signal surrounding at least 50% of its visible surface were considered as targeted.

Infections were conducted with bacterial strains carrying the pilT plasmid to promote cell adhesion and the ilux plasmid as a bioluminescence reporter. Host cells were seeded in 96-well plates at a density of 2.5 × 10⁴ cells per well. An approximate MOI of 5 in an infection volume of 100 µl per well was used. Twenty-five minutes post-infection, wells were washed once with 200 µl of warm HBSS and then fresh DMEM containing 50 µg /ml gentamicin was added to each well. Bacterial bioluminescence was measured on a multimode microplate reader (Biotek Synergy H1, Agilent) every 30 min, starting at 1 hpi and finishing at 8 hpi.

After infection, cells were washed two times with cold HBSS and lysed in RIPA buffer (Sigma) in the presence of 4 U/ml DNAse I (NEB) and protease inhibitor cocktail (Sigma). Lysates were incubated at 4°C for 30 min and centrifuged at 20,000 × g for 12 min at 4°C. Protein concentrations were measured using the BCA kit (Thermo Fisher) and normalized. Normalized samples were mixed with Laemmli buffer that contained 5% β-mercaptoethanol and heated at 95°C for 10 min. Samples were heated at 56°C for 10 min when samples were prepared for RNF213 immunoblotting. Ten to thirty µg of protein sample were resolved on a 4–20% Mini-Protean TGX stain-free gels (Bio-Rad) and then blotted onto a PVDF membrane using a tank transfer system overnight at 4°C. After the transfer, PVDF membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween20 (Sigma-Aldrich) (TBS-T) for 1 hr at RT. Subsequently, membranes were incubated with primary antibody diluted in blocking solution overnight at 4°C. After primary antibody incubation, blots were washed three times with TBST and incubated with the respective secondary antibody in blocking solution for 1 hr at RT. Afterwards, membranes were washed with TBS-T and developed with Clarity ECL (Cytiva). Blots were imaged on an Azure 500 visualization system. Primary antibodies used in this study include rabbit monoclonal anti-RNF213 (Sigma HPA026790; 1:2000), rabbit polyclonal anti-GAPDH (Abcam ab9845; 1:10000); mouse monoclonal anti HOIL-1, clone E3E (Sigma MABC576; 1:500); custom rabbit anti-mRnf213 (Life Technologies, 1:500); rabbit polyclonal anti-HOIP (Abcam ab46322; 1:500); and monoclonal Anti-β-Actin (Sigma A2228; 1:6000). Images were processed in Fiji.

UBAIT assays were performed essentially as described before (Hansen et al., 2021). Briefly, unknown substrates were captured from colon and cecum lysates from 6- to 8-week-old wild-type C57BL/6 (Charles River) mice. All mouse infection experiments conducted were in accordance with the policies of the Institutional Animal Care and Use Committee at UT Southwestern. Mice were orally infected by gavage with 1x10⁹ S. enterica Typhimurium S1344 WT, and after 4 days mice were euthanized, and colon and cecum were removed, washed, and homogenized in Lysis Buffer (1x ubiquitination buffer (Boston Biochem), 1x protease inhibitor (Sigma), 1 mM DTT). Homogenates were then centrifuged at 13,000 × g at 4°C for 10 min and supernatants were added to UBAIT reactions. The supernatant was incubated with 25 µg of purified Strep-IpaH2.5-3xFLAG-Ub or Strep-IpaH3-3xFLAG-Ub and rotated at 4°C. After 1 hr, His-UbE1 (100 nM), His-UbcH5b (2000 nM), energy regeneration buffer (Boston Biochem), MgCl (1 mM), and 1x ubiquitination buffer were added to a final volume of 500 µl. The reaction was run at 30°C for 10 min and then 300 µl TBS buffer (25 mM Tris-HCL pH 7.4, 150 mM NaCl, 1 mM DTT) and 30 µl of washed Strep-Tactin Superflow beads (Iba LifeSciences) were added and rotated for 2 hr at 4°C. The beads were washed four times in TBS-T (TBS + 0.5% Triton-X100) and two times in TBS. Strep-IpaH2.5-3xFLAG-Ub was eluted from the beads in 150 µl Strep-tag elution buffer (Iba LifeSciences). Eluted sample and beads were centrifuged, and supernatant moved to a new tube. SDS (0.25% final concentration) and DTT (5 mM final concentration) were added, and samples were heated at 95°C for 5 min. Next, 1.2 ml TBS was added, followed by 20 µl of M2-FLAG beads (Sigma). The mixture was rotated for 2 hr at 4°C. M2-FLAG beads were washed four times in TBS-T followed by 2 times in TBS. Finally, 35 µl of hot (95°C) SDS–PAGE Loading Buffer was added and the beads were heated for an additional 5 min at 95°C. Samples were electrophoresed on a gradient precast SDS–PAGE gel (Bio-Rad) and Coomassie stained. A lane of gel above the band with unmodified Strep-IpaH2.5-3xFLAG-Ub or Strep-IpaH3-3xFLAG-Ub was excised, and proteins were digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). Following solid-phase extraction cleanup with an Oasis HLB µelution plate (Waters), the resulting peptides were reconstituted in 10 µl of 2% (vol/vol) acetonitrile (ACN) and 0.1% trifluoroacetic acid in water. Two µl of each sample were injected onto an Orbitrap Fusion Lumos (Thermo) mass spectrometer, coupled to an Ultimate 3000 RSLC-Nano liquid chromatography systems (Thermo) at the Mass Spectrometry Core at University of Texas Southwestern. Samples were injected onto a 75 μm i.d., 75 cm long, (Lumos) EasySpray column (Thermo), and eluted with a gradient from 0 to 28% buffer B over 90 min. Buffer A contained 2% (vol/vol) ACN and 0.1% formic acid in water, and buffer B contained 80% (vol/vol) ACN, 10% (vol/vol) trifluoroethanol, and 0.1% formic acid in water. The Orbitrap Fusion Lumos mass spectrometer operated in positive ion mode with a source voltage of 2.0–2.4 kV and an ion transfer tube temperature of 275°C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the Orbitrap for each full spectrum acquired using higher-energy collisional dissociation for ions with charges 2–7. Dynamic exclusion was set for 25 s after an ion was selected for fragmentation. Raw MS data files were analyzed using Proteome Discoverer v.2.4 (Thermo), with peptide identification performed using Sequest HT searching against the human reviewed protein database from UniProt. Fragment and precursor tolerances of 10 ppm and 0.6 Da (Lumos) were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys was set as a fixed modification and oxidation of Met was set as a variable modification. The false-discovery rate cutoff was 1% for all peptides. The abundance of identified peptides found for Strep-IpaH2.5 UBAIT was compared to those found for control IpaH3 UBAIT that was included in the same experiment. Hits for IpaH2.5 were included when abundance levels were at least twofold higher than UBAIT controls.

Graphpad Prism 10.2.2 was used to generate graphs and to perform statistical analyses. Experiments were conducted in three to five biological replicates. Data in graphs are displayed as mean ± SEM. Statistical analyses included Student t-test one- and two-way ANOVA followed by Tukey’s multiple comparison tests. Significance was considered when p values were <0.05.

A large UBAIT mass spectrometry dataset broadly probing for IpaH protein family interaction partners was originally generated and published in de Jong et al., 2016. The numerical values of all other quantified data depicted in data panels in this manuscript are openly available in the Digital Repositories at Duke under the digital object identifier https://doi.org/10.7924/r4vq36v59.