The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation

ABSTRACT A central signal that marshals host defense against many infections is the lymphocyte-derived cytokine interferon-gamma (IFNγ). The IFNγ receptor is expressed on most human cells and its activation leads to the expression of antimicrobial proteins that execute diverse cell-autonomous immune programs. One such immune program consists of the sequential detection, ubiquitylation, and destruction of intracellular pathogens. Recently, the IFNγ-inducible ubiquitin E3 ligase RNF213 was identified as a pivotal mediator of such a defense axis. RNF213 provides host protection against viral, bacterial, and protozoan pathogens. To establish infections, potentially susceptible intracellular pathogens must have evolved mechanisms that subdue RNF213-controlled cell-autonomous immunity. In support of this hypothesis, we demonstrate here that a causative agent of bacillary dysentery, Shigella flexneri, uses the type III secretion system (T3SS) effector IpaH1.4 to induce the degradation of RNF213. S. flexneri mutants lacking IpaH1.4 expression are bound and ubiquitylated by RNF213 in the cytosol of IFNγ-primed host cells. Linear (M1-) and lysine-linked ubiquitin is conjugated to bacteria by RNF213 independent of the linear ubiquitin chain assembly complex (LUBAC). We find that ubiquitylation of S. flexneri is insufficient to kill intracellular bacteria, suggesting that S. flexneri employs additional virulence factors to escape from host defenses that operate downstream from RNF213-driven ubiquitylation. In brief, this study identified the bacterial IpaH1.4 protein as a direct inhibitor of mammalian RNF213 and highlights evasion of RNF213-driven immunity as a characteristic of the human-tropic pathogen Shigella .

Once bound to Gram-negative bacteria such as Salmonella, RNF213 directly ubiquitylates the bacterial surface molecule, lipopolysaccharide (LPS).These ubiquitylated cytosolic bacteria can be captured by ubiquitin-binding proteins and delivered into autolysosomes for degradation (3).
While the predominantly vacuolar pathogen Salmonella is susceptible to RNF213mediated cytosolic host defense (3), the cytosol-adapted Gram-negative pathogen Burkholderia thailandensis is resistant.B. thailandensis escapes RNF213-driven host defense in part through the secretion of the virulence factor TssM, a ubiquitin esterase that removes ubiquitin from LPS on the bacterial surface (9).However, whether and how other cytosolic bacterial pathogens escape from RNF213-driven ubiquitylation is unknown.
Shigella spp.are the etiological agent of bacillary dysentery and a leading cause of diarrheal deaths, malnutrition, and growth retardation in children (10)(11)(12).Shigella spp.invade the intestinal epithelium, enter the host cell cytosol of infected human colonic epithelial cells, and trigger an acute inflammatory response that involves the secretion of IFN by lymphocytes (13)(14)(15)(16).IFN signaling in epithelial cells induces the expression of human guanylate binding protein 1 (GBP1), a defense protein that blocks bacterial intracellular motility and cell-to-cell spread (17)(18)(19)(20).To counter this potent cellautonomous immune program, Shigella flexneri employs its type III secretion system (T3SS) to deliver the GBP1 inhibitor IpaH9.8 into the host cell cytosol.Loss of IpaH9.8 results in diminished S. flexneri virulence and a potent antibacterial host response (19)(20)(21)(22).
IpaH9.8 belongs to a family of T3SS effectors that contain a leucine-rich repeat (LRR) domain involved in substrate recognition and a novel E3 ligase (NEL) domain that transfers K48-linked ubiquitin to its bound substrate (23).K48-linked ubiquitylation tags the substrate for proteasomal degradation.These IpaH proteins belong to a 'second wave' of T3SS effectors that are under the control of the transcriptional regulator MxiE.
Second wave T3SS effectors have been shown to promote host cell survival, to dampen inflammation, and to counteract cell-autonomous immune programs (24), yet their full repertoire of targets remains to be determined.
In the present work we found that the MxiE-regulated T3SS effector IpaH1.4 facilities the proteasomal degradation of RNF213.Remarkably, RNF213 functions independent of the linear ubiquitin chain assembly complex (LUBAC) to promote the linear and lysine-linked ubiquitylation of ipaH1.4-deficientS. flexneri mutants.We observed that ubiquitylated S. flexneri continued to replicate inside the host cell cytosol, indicating that S. flexneri employs additional unknown virulence mechanisms to interfere with its degradation.Thus, our study demonstrates that the human enteric pathogen S. flexneri directly interferes with RNF213 expression and also limits host defense downstream of RNF213-driven ubiquitylation.

Deletion of mxiE renders S. flexneri susceptible to linear ubiquitylation
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 (3,25).
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 (9).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 Shigella flexneri (serotype 2a, strain 257T) by immunofluorescence.We found that wildtype (WT) S. flexneri remained largely devoid of M1-linked ubiquitin in both alveolar A549 and colonic HT29 epithelial cell lines under both unprimed and IFN-primed conditions (Fig. 1A -C).In contrast to WT S. flexneri, a mxiE mutant strain that has diminished expression of 'second-wave' T3SS effector proteins (26,27), was robustly stained with anti-M1 antibodies in IFN-primed host cells (Fig. 1A -C).These observations indicate that S. flexneri uses MxiE-dependent effectors to evade an IFN-activated ubiquitylation pathway that targets cytosolic bacteria.

Linear and lysine-linked ubiquitin is attached to S. flexneri mxiE
The ubiquitin protein contains seven lysine residues.Any of these lysine residues or the N-terminal methionine can form isopeptide bonds with the C-terminal glycine residue of ubiquitin to form chains.To determine which ubiquitin linkage types are present on the surface of S. flexneri mxiE bacteria, we generated A549 cells that overexpressed internally tagged ubiquitin (INT-Ub) variants, which contain only a single lysine residue or lack lysines altogether.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 mxiE, albeit with diminished efficiency compared to WT INT-Ub (Fig. 2A).
These data confirmed that M1-linked ubiquitin is bound to S. flexneri mxiE 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 (Fig. 2A).Linkage-specific antibodies against K27 and K63 confirmed that these two linkage types are present on ΔmxiE bacteria in IFN-primed A549 cells (Fig. 2B-C).Staining with an antibody that detects most ubiquitin linkage types (FK2) showed near complete overlap with anti-M1 staining (Fig. 2D-E).Because the percentage of FK2-positve but M1-negatve bacteria (Fig. 2E).
was much smaller than the percentage of bacteria that were K27-or K63-positive (Fig. 2B-C), these data suggest that most S. flexneri ΔmxiE bacteria are decorated with both linear and lysine-linked ubiquitin.
Consequently, we hypothesized that LUBAC was essential for the ubiquitylation of ΔmxiE.
Similarly, the internally tagged 7KR-Ub variant decorated S. flexneri ΔmxiE independent of the LUBAC component HOIP (Fig. 3C).Collectively, these data demonstrate that LUBAC is dispensable for linear ubiquitylation of ΔmxiE bacteria in the host cell cytosol of IFN-primed A549 cells.
The ubiquitin E3 ligase RNF213 was recently shown to ubiquitylate pathogencontaining vacuoles (2,4) as well as Gram-positive and Gram-negative bacteria inside the host cell cytosol (3,6,9).Like HOIL-1, RNF213 is an IFN-inducible protein (Fig. 3A and (2,4)).Using immunofluorescence microscopy, we detected RNF213 on the surface of ΔmxiE but not WT bacteria in IFN-primed A549 as well colonic HT29 cells (Fig. 3D-G).The vast majority of ubiquitylated S.flexneri ΔmxiE mutants (~93%) co-stained with anti-RNF213 (Fig. 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 CRISPRgenerated A549 RNF213 KO cells (Fig. 3A) with S. flexneri ΔmxiE and stained these IFN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 (Fig. 3B and 3H-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 (Fig. 3C).Together, these data demonstrate that human RNF213 is essential for the linear and lysine-linked ubiquitylation of MxiE-deficient S. flexneri mutants.

S. flexneri virulence factors IpaH1.4 and IpaH2.5 are sufficient to induce RNF213 degradation
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 (20,22,28,29,(31)(32)(33)(34)(35).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 (Fig. 4A).
To directly test whether S. flexneri promote 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 (Fig. 4B).We next tested whether 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 were sufficient to promote RNF213 degradation in HEK293T cells (Fig. 4C).Of note, substratebinding 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 (28).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 (29).
Catalytically inactive forms of IpaH1.4 or IpaH2.5, in which Cys368 is mutated to a serine (C/S), failed to degrade RNF213 (Fig. 4D).As an additional control we included overexpression of IpaH9.8, an inhibitor of GBPs, which also did not impact RNF213 expression (Fig. 4D), as anticipated.Functional and physical interaction of the effector IpaH2.5 with murine Rnf213 in intestinal tissue was further demonstrated using a UBAIT (Ubiquitin-Activated Interaction Traps) approach (Fig. S1).Collectively, these data demonstrate that the virulence factors IpaH1.4 and IpaH2.5 promote the degradation of mouse as well as human RNF213.

Loss of IpaH1.4 is sufficient to render S. flexneri susceptible to RNF213-driven ubiquitylation in human and murine host cells
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 (Fig. 5A-B) or in serotype 5a strain M90T (Fig. S2) 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 ipaH1.4-ipaH2.5double mutant was indistinguishable from the ipaH1.4single deletion mutant (Fig. S2).These data suggested that endogenous IpaH2.5 is nonfunctional, which agrees with a previous characterization of ipaH2.5 as a minimally expressed pseudogene (36).
While infection of A549s with the ipaH1.4mutant had no appreciable impact on RNF213 expression, S. flexneri mxiE infections still led to a moderate reduction in RNF213 expression compared to uninfected (UI) cells (Fig. 5A).These data agree with previously published data that showed diminished but not abolished expression of IpaH proteins in mxiE mutants (27,37).As expected, a control infection with ipaH9.8reduced RNF213 expression in the same way as WT S. flexneri infections did (Fig. 5A).
Although S. flexneri is a human-adapted pathogen, some IFN-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) (22,38).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 IFN-primed WT and Rnf213 KO mouse embryonic fibroblasts (MEFs, Fig. S3A) with S. flexneri WT and ΔipaH1.4 mutant.We observed that ΔipaH1.4bacteria became decorated with mouse Rnf213 (Fig. S3B) and linear ubiquitin chains (Fig. S3C).Linear ubiquitin was present on ΔipaH1.4 S. flexneri in IFN-primed WT but not in Rnf213 KO MEFs (Fig. S3C).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 (39).Previous work showed that linear ubiquitylation of S. enterica leads to bacterial clearance by xenophagy (3).In contrast to the xenophagic killing of ubiquitylated S. enterica, we found that the ubiquitylated S. flexneri ΔIpaH1.4 and ΔmxiE mutants continued to replicate inside of IFN-primed A549 cells (Fig. 5H), reminiscent of the continued growth of ubiquitylated B. thailandensis tssM mutants inside the host cell cytosol of human IFN-primed cells (9).Thus, our data indicate that S. flexneri, like B. thailandensis, can block ubiquitindependent xenophagy both at the stage of ubiquitylation and at another so far undetermined downstream step of this cell-autonomous host defense pathway.

DISCUSSION
Xenophagy is a cell-autonomous defense program in which intracellular pathogens are captured and delivered into degradative lysosomes.Ubiquitylation of intracellular pathogens is the most common initiating step for xenophagy (39).Like other steps of the xenophagy cascade, pathogen ubiquitylation is augmented in cells primed with the lymphocyte-derived cytokine IFN (2,4,7,40,41).An antimicrobial E3 ubiquitin ligase, RNF213, is itself an IFN-inducible protein (2)(3)(4)(5)(6)(7).Here, we report that the enteric human pathogen S. flexneri employs the T3SS effector IpaH1.4 to induce the proteasomal degradation of RNF213.Accordingly, loss of IpaH1.4 results in RNF213-depdendent ubiquitylation of S. flexneri.However, IpaH1.4-deficient S. flexneri mutants, despite their ubiquitylation, continue to replicate inside the cytosol of IFN-primed human cells.These observations suggest that S. flexneri not only blocks RNF213-driven ubiquitylation but also the downstream degradation of ubiquitylated bacteria.
Accompanying previous reports on Chlamydia and Burkholderia (4, 9), our study on Shigella provides evidence that host-adapted intracellular escape from RNF213-driven host defense.Each of these pathogens evolved a mechanistically distinct strategy to escape ubiquitylation by RNF213.The soil-dwelling accidental pathogen B. thailandensis is recognized and ubiquitylated by RNF213 but instantly removes ubiquitin from its surface through the activity of its secreted ubiquitin esterase TssM (9).The human pathogen Chlamydia trachomatis decorates its surrounding vacuole with the secreted T3SS effector GarD, which renders the Chlamydia-containing vacuole invisible to RNF213 (4).In contrast to TssM and GarD, the Shigella effector IpaH1.4 is a direct inhibitor of RNF213.
IpaH1.4 and IpaH2.5 have nearly 99% sequence identity in their substrate-binding LRR and overexpression of either paralog is sufficient to trigger RNF213 degradation.However, only IpaH1.4 appears to be essential for S. flexneri-triggered RNF213 degradation -as shown in the present work -or for HOIP degradation (28).While these observations agree with a previous report demonstrating low expression of IpaH2.5 under standard culture conditions (36), it remains to be seen whether IpaH2.5 is expressed and possibly redundant with IpaH1.4 for virulence during in vivo infections.
The mechanism by which IpaH1.4 and its paralog IpaH2.5 promote RNF213 degradation can be gleaned from previous studies characterizing IpaH1.4 and IpaH2.5 as ubiquitin E3 ligases that directly conjugate K48-linked ubiquitin to the LUBAC subunit HOIP leading to HOIP degradation by the proteasome (28).We propose that IpaH1.Different ubiquitin linkage types and their incorporation into heterotypic conjugates create a rich diversity of ubiquitin modifications with unique functional outputs, which is commonly referred to as the 'ubiquitin code.' (42,43).We found that at least three linkage types are attached to S. flexneri ipaH1.4 in an RNF213-dependent manner: K27, K63, and linear (M1).Linear ubiquitylation is a head-to-tail inter-ubiquitin linkage through the amino group of the N-terminal methionine (M1) and the C-terminal carboxyl group (44).
The LUBAC complex is the only recognized ubiquitin E3 ligase able to conjugate M1linked ubiquitin chains (45,46).However, we observed that LUBAC is dispensable for M1-linked ubiquitylation of cytosolic S. flexneri ipaH1.4.We found that lysine-less internally tagged ubiquitin or an M1-specific antibody bound to S. flexneri ipaH1.4 in cells lacking LUBAC (HOIL-1 KO or HOIP KO ) but failed to bind bacteria in RNF213-deficient cells.These data corroborate our previous observations, which showed that IFN-induced linear ubiquitylation of Chlamydia-and Toxoplasma-containing vacuoles also occurs independent of LUBAC in human cells (2,4).While it seems likely that RNF213 itself is the enzyme catalyzing linear ubiquitin conjugation, additional biochemical studies are needed to validate this hypothesis.
It was previously reported that RNF213 ubiquitylates LPS on the surface of S. enterica prompting the recruitment of LUBAC, which catalyzes the conjugation of linear ubiquitin to the surface of S. enterica (3).Whereas LUBAC was found to be essential for M1-linked ubiquitylation of cytosolic S. enterica (25), we find that LUBAC is dispensable for M1-linked ubiquitylation of S. flexneri ipaH1.4bacteria.While differences between S. enterica and S. flexneri, for example in the composition of their bacterial cell walls, could possibly contribute to these discrepant results, it is also important to note that the previous S. enterica infection studies were conducted in naïve cells (3,25), whereas our studies were performed under IFN priming conditions.We therefore propose that a boost in the expression of RNF213 and other IFN-stimulated genes (ISGs) renders LUBAC obsolete for M1-linked ubiquitylation of Gram-negative bacteria.Future studies are needed to determine how RNF213 co-operates with LUBAC and other host factors to ubiquitin-tag intracellular pathogens.
Shigella is a human adapted pathogen that secretes T3SS effectors to directly interfere with type I and III IFN signaling in epithelial cells (47).Additionally, Shigella employs virulence factors that inhibits ISGs which act downstream from IFN signaling.
RNF213.Because ubiquitylated S. flexneri ipaH1.4 and mxiE mutants continue to replicate inside the cytosol of IFN-primed host cells, we propose that S. flexneri also uses a mxiE-independent T3SS effector to interfere with host defenses occurring downstream from RNF213.Collectively, these data indicate that Shigella is equipped with redundant counter-immune mechanisms.This type of bacterial effector redundancy is not unique to Shigella.It was recently shown that the rodent pathogen Citrobacter rodentium can tolerate various combinations of individual T3SS effector deletions in vivo, an observation that led to the 'T3SS effector networks' hypothesis (48,49).Further characterization of the Shigella T3SS effector network that renders bacteria resistant to IFN-inducible cell-autonomous immune program will provide a deeper understanding of the pathogenesis of this important human enteric pathogen.Given the rise of extensively drug-resistant Shigella strains (50)(51)(52)(53), such an understanding may prove to be essential for the development of critically needed novel therapeutic approaches to treat Shigellosis.

Knock out cell lines and mammalian expression systems
The production and characterization of A549 knockout cell lines (pooled RNF213KO, HOIP KO clone and HOIL-1 KO clone) was previously reported (2,4).Various GFP-tagged IpaH expression constructs including the IpaH1.4 and IpaH2.5 C/S mutants were previously described (28) according to 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.

Production of Rnf213 knockout mouse and derived cells
RNF213 knockout mice were generated using a modified iGONAD methodology (57-59).
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 set 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 bp, 284 bp, 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.

Lentivirus production
Plasmids were transfected into HEK293T cells using the TransIT 293 transfection reagent (Mirus).Wells were transfected with 1ug plasmid, 750 ng pGAGpol, 250 ng VSVG.6 hours post transfection, media was aspirated and replaced with fresh DMEM.24 hours post transfection, media was aspirated and replaced with pseudoparticle DMEM (3%fbs, 1% MEM non-essential aminoacids, 20mM HEPES).Supernatant was collected 48 and 72 hours after transfection.Supernatants containing lentivirus were filtered using 0.45um filters (corning) and later mixed with 10ug/ml polybrene (Millipore Sigma).Virus was frozen at -80 until use.For cell transduction, A549 cells were diluted to a concentration of 3.33 x 10 4 cells/ml and mixed with 10 ug/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 1µg/ml of puromycin and expanded.

IpaH mini-screen
HEK293T that express PiggyBac RNF213 were seeded at 10 5 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 HEK293T using the TransIT 293 transfection reagent (Mirus).24 hours after transfection, cells were lysed in RIPA buffer and prepared for immunoblotting.

Infection procedures
For immunofluorescence staining A549, HT29, and MEF cells were seeded at 1x10 5 cells per well on cover slips in 24-well plates.HT29 cells were plated at 2x10 5 cells per well.
For Western blotting cells were plated at 2x10 5

Immunofluorescence microscopy
Cells were fixed in 4% PFA in PBS and then permeabilized either with 0.1%triton in PBS for 15 minutes or with ice-cold methanol for 1 minute 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 minutes 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 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.

Bacterial luminescence assay
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 x10 4 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 50µg /ml gentamicin was added to each well.Bacterial bioluminescence was measured on a multimode microplate reader (Biotek Synergy H1, Agilent) every 30 minutes, starting at 1 hpi and finishing at 8 hpi.

Western blotting
After infection, cells were washed two times with cold HBSS and lysed in RIPA buffer (Sigma) in the presence of 4U/ml DNAse I (NEB) and protease inhibitor cocktail (Sigma).
Lysates were incubated at 4°C for 30 min and centrifuged at 20,000g for 12 minutes at 4°C.Protein concentrations were measured using the BCA kit (Thermofisher) and normalized.Normalized samples were mixed with Laemmli buffer that contained 5% βmercaptoethanol and heated at 95°C for 10 minutes.Sample were heated at 56 °C for 10 minutes 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 (Biorad) 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 hour 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 hour at RT.
UBAIT assays were performed essentially as described before (32).Briefly, unknown substrates were captured from colon and cecum lysates from 6-8 weeks old wild type 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 (HCD) for ions with charges 2-7.Dynamic exclusion was set for 25 seconds 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) was 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 (FDR) cutoff was 1% for all peptides.The abundance of identified peptides found for Strep-IpaH2.5UBAIT were 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 2-fold higher than UBAIT controls.

Statistical analysis
Graphpad Prism 10.2.2 was used to generate graphs and to perform statistical analyzes.
Experiments were conducted in three to five biological replicates.Data in graphs are displayed as mean ± standard error of the mean (SEM).Statistical analyses included student t-test one-way and two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests.Significance was considered when p values were <0.05

Data storage
Mass spectrometry data (Ubait) will be deposited in a publishably accessible Repository at the University of Texas Southwestern at the time at which the final version of the manuscript is published.The numerical values of all other quantified data depicted in data panels in this manuscript will be made openly available in the Digital Repositories at Duke at the time at which the final version of the manuscript is published.Digital object identifier will be provided.
4 and IpaH2.5 similarly bind to and directly ubiquitylate RNF213 to facilitate RNF213's proteasomal degradation.Such direct interactions are supported by our data showing that transient IpaH2.5-RNF213interactions can be trapped via formation of covalent bonds between these proteins in a UBAIT assay.Future studies are needed to further probe for direct RNF213-IpaH1.4/IpaH2.5interactions and to characterize the molecular structure of the protein binding interfaces.The deletion of ipaH1.4leads to S. flexneri ubiquitylation in IFN-primed host cells.
. Internally Strep-tagged ubiquitin (INT-Ub) 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 (55).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 (56) 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 table 1 in supplemental material.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), cells per well in 24-well plates.Cells were either left untreated or treated overnight with human or mouse cytokine IFN at a final concentration of 100 U/ml, as indicated.The day before 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 1h 30 minutes.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 to 100, unless otherwise specified.Cells were centrifuged at 1000 g for 10 minutes 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 minutes (A549 and HT29 cells) or 60 minutes (MEFs) post infection.Following these washes gentamicin at a final concentration of 50 ug/ml (for A549 and MEFs) or 100 ug/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.

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 Salmonella 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), 1mM DTT).Homogenates were then centrifuged at 13,000g at 4˚C for 10 minutes and supernatants were added to UBAIT reactions.The supernatant were incubated with 25 ug of purified Strep-IpaH2.5-3xFLAG-Ubor Strep-IpaH3-3xFLAG-Ub and rotated at 4˚C.After 1 hour, His-UbE1 (100nM), His-UbcH5b (2000nM), energy regeneration buffer (Boston Biochem), MgCl (1mM) and 1 x ubiquitination buffer were added to a final volume of 500 µl.The reaction was run at 30˚C for 10 minutes and then 300 µl TBS buffer (25 mM Tris-HCL pH7.4,150 mM NaCl, 1mM DTT) and 30 µl of washed Strep-Tactin Superflow beads (Iba LifeSciences) were added and rotated for 2 hours at 4˚C.The beads were washed 4 times in TBS-T (TBS+ 0.5% Triton-X100) and 2 times in TBS.Strep-IpaH2.5-3xFLAG-Ubwas 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 (5mM final concentration) were added, and samples were heated at 95˚C for 5 minutes.Next, 1.2 ml TBS was added, followed by 20 µl of M2-FLAG beads (Sigma).The mixture was rotated for 2 hours at 4˚C.M2-FLAG beads were washed 4 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 minutes at 95˚C.Samples were electrophoresed on a gradient precast SDS-PAGE gel (Biorad) and Coomassie stained.A lane of gel above the band with unmodified Strep-IpaH2.5-3xFLAG-Ubor 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% (v/v) 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 0-28% buffer B over 90 minutes.Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) 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

Fig. 1 .Fig. 2 .Fig. 3 Fig. 4 .Fig. 5 .
Fig. 1.Mutation of the bacterial transcription factor MxiE results in linear ubiquitylation of S. flexneri in IFNγ-primed epithelial cells.(A -C) Cells were primed with 100 U/ml IFNγ or left unprimed and then infected with WT or ΔmxiE S. flexneri at an MOI of 50 -100.Cells were fixed 3 -4 h post infection (hpi) and stained for linear ubiquitin (M1-Ub) with anti-M1 antibody.Percentage of M1-Ub-positive bacteria was quantified in infected A549 epithelial cells at 4 hpi (B) and HT29 epithelial cells at 3 hpi (C).Graphs show the average of three independent experiments and depict means ± SEM.Two-way ANOVA with Tukey's multiple comparison tests were performed; all statistically significant comparisons are shown.***p<0.001;**** p<0.0001; ns, not significant Fig.5