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Shigella flexneri suppresses NF-kB activation by inhibiting linear ubiquitin chain ligation

The Linear Ubiquitin chain Assembly Complex (LUBAC) is a multimeric E3 ligase that catalyzes M1- or linear ubiquitination of activated immune receptor signaling complexes (RSCs). While mutations that disrupt linear ubiquitin assembly lead to complex disease pathologies including immunodeficiency and autoinflammation in both humans and mice, microbial toxins that target LUBAC function have not yet been discovered. Here, we report the identification of two homologous Shigella flexneri Type III Secretion System (T3SS) effector E3 ligases IpaH1.4 and IpaH2.5 that directly interact with LUBAC subunit HOIL-1L (RBCK1) and conjugate K48-linked ubiquitin chains to the catalytic RING-between-RING domain of HOIP (RNF31). Proteasomal degradation of HOIP leads to irreversible inactivation of linear ubiquitination and blunting of NF-κB nuclear translocation in response to TNF, IL-1β, and pathogen associated molecular patterns (PAMPs). Loss of function studies in mammalian cells in combination with bacterial genetics explains how Shigella evades a broad spectrum of immune surveillance systems by cooperative inhibition of receptor ubiquitination, and reveals the critical importance of LUBAC in host defense against pathogens.

IpaH1.4 belongs to a large family of bacterial E3 ligases characterized by a highly variable NH 2 -terminal Leucine Rich Repeat (LRR) domain and a nearly identical COOH-terminal catalytic domain ( Supplementary Fig. 1a) 21,22 . LUBAC signaling was inhibited by both IpaH1.4 and IpaH2.5, which share 98% sequence homology 14 , but not other family members including IpaH4.5, IpaH7.8 and IpaH9.8 that share less than 80% homology ( Supplementary Fig. 1b). The E3 ligase activity of IpaH1.4/2.5 21,22 was necessary for inhibiting LUBAC stimulated NF-κB activation ( Supplementary Fig. 1c). Evaluation of experiments in Figure 1b also revealed a consistent downregulation of LUBAC-mediated NF-κB by Shigella kinase OspG. However, we found the kinase activity was unnecessary for LUBAC inhibition (Supplementary Fig. 1c) and an unbiased yeast two-hybrid screen failed to identify OspG substrates beyond its established activators Ub and Ube2L3 (UbcH7) E2conjugating enzyme ( Supplementary Fig. 2a) 16,23,24 . It is therefore likely that OspG inhibits LUBAC through non-specific sequestration of Ube2L3~Ub machinery required for linear ubiquitination under these experimental conditions 25,26 . Taken together, we confirmed previous work on Shigella effector proteins OspI and OspZ that target K63-linked Ub chains and demonstrate that Shigella may require additional effector proteins, and particularly IpaH1.4/2.5, to target M1-linked Ub chains to effectively suppress host defense mechanisms.
We performed Shigella infection experiments to test the ability of IpaH1.4/2.5 to degrade endogenous HOIP in a human intestinal epithelial cell line (CaCo-2). An infection rate of ~50% could be achieved without appreciable cell death ( Supplementary Fig. 3), which set the limitation on the total HOIP degradation that could be monitored in bulk biochemical experiments. As shown in Figure 2e, WT Shigella reduced endogenous HOIP by more than 50% (consistent with the rate of bacterial infection), but not HOIL-1L or β-actin control. HOIP degradation was not observed during infection with the ShigellaΔipaH1.4/2.5 strain and analysis of single mutations revealed IpaH1.4 is the essential bacterial E3 ligase necessary for targeting LUBAC in this cellular model of infection (Fig. 2e). The varying promoter sequences upstream of ipaH1.4 and ipaH2.5 genes (52.0% identity), but nearly identical coding sequences (98.9% identity) suggest that although ipaH1.4 and ipaH2.5 encode identical proteins, expression may vary depending on conditions encountered by Shigella during infection ( Supplementary Fig. 4).
We were unable to complement the ShigellaΔipaH1.4 strain with an arabinose inducible copy of ipaH1.4 or ipaH2.5 encoded on a plasmid. To then determine if IpaH1.4/2.5 targets LUBAC function at protein levels secreted by bacterial pathogens, we engineered an attenuated Listeria monocytogenes (Lm) strain to secrete IpaH2.5 during intracellular invasion ( Supplementary Fig. 5a). Both Lm and Shigella have very similar intracellular lifestyles, yet Lm does not encode IpaH homolog or induce HOIP degradation. We observed dose and time dependent degradation of endogenous HOIP in U2OS cells infected with de Jong et al.

IpaH1.4/2.5 is the major E3 ligase regulator of cytokine RSCs
S. flexneri encodes 12 IpaH effector proteins including IpaH4.5, IpaH9.8 and IpaH0722 (IpaH5), which have been reported to target NF-κB subunit p65, NEMO and TRAF2, respectively, for degradation 13,15,30,31 . To directly test the contributions of chromosomal and plasmid encoded IpaH-type E3 ligases on innate immune evasion, a cellular degradation assay was performed with all major components of TNF and IL-1 RSCs. To first demonstrate the specificity of this assay, Shigella and Salmonella E3 ligases were expressed together with PKN1, a known Salmonella SspH1 substrate 28,32 . As expected, SspH1 induced the cellular degradation of PKN1, whereas other IpaH family members had no effect ( Supplementary Fig. 8a, b). Contrary to previous studies 13,15,31 , the reported targets of de Jong et al.
IpaH4.5, IpaH9.8 and IpaH0722 (IpaH5) including p65, NEMO and TRAF2 were stable in the presence of Shigella IpaH proteins. In contrast, HOIP was readily degraded by IpaH1.4/2.5 (Fig. 4a). These results were confirmed by monitoring expression levels of endogenous HOIP, NEMO, p65 or TRAF2 in the presence of overexpressed Shigella IpaH proteins ( Supplementary Fig. 9). We noted that during Shigella infection of CaCo-2 or U2OS cells only a fraction of cells were infected ( Supplementary Fig. 3). Accordingly, HOIP levels were 53.8% (+/− 6.7) in Shigella infected CaCo-2 cells compared to uninfected control cells, while in the same samples endogenous NEMO was upregulated significantly and p65 or TRAF2 levels were not significantly altered (Fig. 4b). In addition, no other signaling components tested were degraded in the presence of IpaH proteins ( Fig. 4 and Supplementary Fig. 9). These data reveal the central importance of IpaH1.4/2.5 among IpaH family members for blocking cytokine RSCs.

IpaH1.4 and OspI cooperate to limit RSC signaling during Shigella infection
Our studies now indicate that IpaH1.4/2.5 is a highly specific inhibitor of M1-Ub chain assembly. In addition, the S. flexneri M90T strain used in this study does not encode a functional OspZ 18 , suggesting that OspI is the central effector targeting K63-Ub chains. Thus, we explored the question whether inhibition of M1-and K63-Ub chains by IpaH1.4/2.5 and OspI, respectively, are sufficient to suppress multiple RSCs during Shigella infection. To first determine if HOIP ubiquitination and degradation suppresses immune system activation during Shigella infection, we quantified endogenous NF-κB (p65) nuclear translocation at single cell resolution (Fig. 5a). Less than half (44.3+/−7.9%) of WT Shigella infected U2OS cells exhibited nuclear NF-κB accumulation (Fig. 5b). In contrast, NF-κB translocated to the nucleus in 94.3% (+/− 3.6) of U2OS cells infected with ShigellaΔipaH1.4/2.5, indicating that secretion of IpaH1.4/2.5 suppresses the host immune responses to PAMPs ( Fig. 5a and b). Comparison between ShigellaΔipaH1.4 and ShigellaΔipaH2.5 mutant strains showed that IpaH1.4 is the essential T3SS effector protein required for blocking NF-κB activation ( Fig. 5a and b), which is consistent with the role of IpaH1.4 in HOIP degradation during Shigella infection (Fig. 2e). Importantly, NF-κB activation by ShigellaΔipaH1.4 was independent of the total bacterial load in infected cells and it was not due to alterations in bacterial intracellular growth characteristics ( Fig. 5c and Supplementary Fig. 10). Interestingly, p65 readily accumulated in the nucleus of U2OS cells infected with ShigellaΔospI and ShigellaΔipaH1.4ΔospI strains indicating that similar to IpaH1.4, OspI secretion is required for immune suppression ( Fig. 5a and b). These data indicate that IpaH1.4 and OspI perform non-redundant functions to inhibit PAMP-mediated NF-κB activation.

Cooperative termination of M1-and K63-Ub chain assembly limits the IL-1β response to pathogens
LUBAC post-translationally modifies numerous cytokine RSCs 2,3,33-36 , suggesting that HOIP degradation may limit a broad spectrum of immune responses to Shigella infection. We found that WT Shigella, but not ShigellaΔmxiD (which lacks a functional T3SS), suppressed NF-κB nuclear translocation in response to IL-1β and TNF ( Supplementary Fig.  11). In contrast, NF-κB accumulated in the nucleus of TNF and IL-1β stimulated U2OS and CaCo-2 cells infected with Shigella strains lacking either ipaH1.4 or ospI genes, suggesting de Jong et al. Page 6 that these effectors limit immune responses to these cytokines (Fig 6a, Supplementary Fig.  11, 12, 13). However, because NF-κB activation can occur by PAMPs under these conditions (see Figure 5a and b), we sought to more directly define the effects of M1-and K63-Ub chain elimination on cytokine signaling in genetically modified human cells.
We noted that cytokine RSCs exhibit different sensitivities to loss of M1-Ub chains as nuclear NF-κB was eliminated in TNF, but not IL-1β treated HOIP −/− cells (generated by CRIPSR/Cas9; Supplementary Fig. 14 and 15, Fig. 6b). Similar results were found in TAK1 −/− cells that are unable to signal through K63-Ub chains ( Fig. 6b and Supplementary  Fig. 15). Thus, elimination of signaling through TNF-RSCs would require inhibition of either M1-or K63-Ub chain assembly, whereas IL-1β RSC inhibition would likely require loss of both.
To then deconstruct potential cooperative regulation of the IL-1β RSC by multiple T3SS effector proteins, we evaluated NF-κB nuclear translocation in HOIP −/− and TAK1 −/− cells infected with Shigella. By genetically eliminating signaling through either M1-linked  (Fig. 6d). The observed suppressive effect of OspI in TAK1 −/− cells is likely due to the partial dependence of linear Ub chain generation on K63-linked Ub chains ( Fig. 6e) 2,20 . Taken together, these data demonstrate that Shigella T3SS effectormediated inhibitions of K63-linked and M1-linked ubiquitination is a critical step in suppressing signal transduction through diverse PRR and cytokine receptors (Fig. 6e).

Discussion
Here, we demonstrate that Shigella flexneri suppresses immune signal transduction by specifically recognizing the LUBAC machinery and targeting its enzymatic center HOIP for proteasomal degradation.  13,15,17,18,31 . However, except for OspI and OspZ we have not been able to reproduce these results. Unexpectedly, we found that the effectors IpaH4.5, IpaH9.8 and IpaH0722 had no effect on cytokine RSC signaling as suggested by previous reports 13,15,31 . These findings were confirmed in NF-κB activation assays (

Yeast 2-Hybrid assay
Shigella flexneri M90T genes encoding OspG, IpaH2.5 and IpaH4.5 were cloned into vector pLexNA. After introduction of constructed plasmids into Saccharomyces cerevisiae L40, expression of OspG, IpaH2.5 and IpaH4.5 was confirmed by Western blotting using anti-LexA antibodies (Millipore). In this Y2H screen LexA-effector expressed in the yeast Saccharomyces cerevisiae L40 served as the bait and a library of mouse embryo cDNA cloned in the vector pVP16 served as the prey as previously described 41,42 . The pLexNA plasmid provides L40 the ability to grow on Trp − minimal medium (SD -W), and the pVP16 plasmid on Leu − (SD -L) medium. An interaction between bait and prey allows LexA to be shuttled into the nucleus (via the NLS of VP16) resulting in expression of LacZ and HIS3 genes, and growth in Trp − , Leu − , His − (SD -WHULK) medium. Plasmids were isolated from yeast colonies growing on SD -WHULK (+ 3 mM 3-Amino-1,2,4-triazole) using phenol/chloroform and were electroporated into E. coli HB101 strain. The pVP16 and de Jong et al.
pLexNA plasmids were isolated from E. coli and inserts in pVP16 were sequenced using the pVP16 5' primer. For hits, pVP16 plasmids were isolated and reintroduced together with pLexNA plasmids containing effectors into the L40 yeast strain. Drops of tenfold dilution of colonies picked from SD -UWL plates were plated on SD -UWL and SD -WHULK plates.

GST pulldowns
HOIP UBA-RBR fragment was fused to the C-terminus of GST (in pGEX6P-1) and GST purified using glutathione coated beads. IpaH2.5 full length was fused to the C-terminus of His 6 -MBP-His 6 (in pET28b) and His-tag purified on Ni-NTA beads. GST beads were loaded with 20 ug GST or GST-HOIP UBA-RBR for 2h, washed 3x and then incubated for 2h with His 6 -MBP-HOIL-1L UBL (1-137) followed by 2h incubation with His 6 -MBP or His 6 -MBP-IpaH2.5. Then beads were washed 6x, and proteins were eluted from the beads in 50 mM reduced glutathione, pH8.0 and run on a SDS-PAGE gel for Coomassie staining or Western blotting.

In vitro Ubiquitination assays
Shigella flexneri genes encoding IpaH proteins were cloned into pGEX4T-1, expressed in E. coli BL21 and purified using glutathione coated beads. His 6 -UbcH5b and His 6 -UbE1 were expressed in E. coli BL21 and purified using Ni-NTA agarose beads (Qiagen). LUBAC

Mass Spectrometry
To determine which lysines in HOIP are ubiquitinated by IpaH2.5, an Ub reaction (500 μl volume) was performed with GST-IpaH2.5 or GST-IpaH2.5C368S, and after 90 mins at 37°C, Myc-HOIP was immunoprecipitated (IP) using anti-Myc antibodies (Covance). The IP fractions were then run on a SDS-PAGE gel (Biorad) and Coomassie stained bands were excised and proteins were digested in-gel with trypsin and run on a Q Exactive MS platform at the Mass Spectrometry Core at UTSW.

Infections of tissue culture cells and microscopy
For infections with Shigella flexneri M90T, strains were grown overnight at 30°C in Brain Heart Infusion (BHI) broth (BD Biosciences) followed by 1:50 dilution in BHI broth and additional 2h growth at 37°C to an OD 600nm of 0.4 to 0.6. Bacteria were then washed 1x in PBS and resuspended in 0.03% Congo Red (Sigma Aldrich) for 15 min at 37°C prior to infection of cells. Listeria monocytogenes were grown overnight at 30°C in BHI broth prior to infection of cells. After addition (at indicated MOI) of Shigella or Listeria strains to HeLa, U2OS or CaCo-2 cells (grown in DMEM + 10% FBS), infection was initiated through centrifugation of plates at 1000g for 10 mins, followed by incubation at 37°C, 5% CO 2 , for 90 mins. To kill extracellular bacteria, media was then replaced with DMEM + 50 μg/ml gentamycin. Cell lines used in this study HEK293T (CRL-3216), HeLa (CCL-2), CaCo-2 (HTB-37) and U2OS (HTB-96) were purchased from ATCC. Parental cell lines were tested and determined to be mycoplasma free.

Statistical analysis
A two-tailed unpaired Student's t-test was used to calculate statistical differences. Experiments were performed at least three times independently. A P-value of less than 0.05 was considered significant.

Construction of Shigella mutants
A Shigella flexneri M90T ΔipaH1.4, ΔipaH2.5 and ΔospI single and double mutants were constructed by λ red recombinase mediated replacement by homologous recombination of respective genes with a kanamycin resistance cassette followed by FLP recombinase catalyzed removal of the cassette 43  Briefly, a kanamycin resistance cassette (matching primer sequences are underlined) flanked with ~50 base pairs homologous to the gene of interest was amplified from plasmid DNA (pKD3). PCR products were electroporated into Shigella flexneri strain M90T carrying the red recombinase plasmid pKD46. Transformants were selected by growth on LB agar plates containing kanamycin (50μg/ml) and simultaneously cured of pKD46 by growth at 42°C overnight. The kanamycin resistance gene was eliminated through the introduction of the pCP20 helper plasmid that contains the FLP recombinase. Subsequent curing of pCP20 was carried out by growing strains at 42°C for 5 hours. Deletions were confirmed by PCR using primers flanking the deletion site, and sequencing of the PCR product.

Construction Listeria monocytogenes strains
Shigella effectors IpaH2.5 (wt and C368S) were cloned into pActAN100-pPL2 44 , in which effectors were Gibson assembled (New England Biolabs) together with a 3×Flag-tag and a de Jong et al. Page 12 529 nt Listeria monocytogenes actA fragment (247 nt of the actA promoter region and 282 nt of the actA ORF). Thus, the resulting construct ActA(1-94aa)-3×Flag-IpaH2.5(aa 1-563) contains the ActA Sec secretion signal from Listeria and is under transcriptional control of the actA promoter. The resulting plasmids were introduced into the (attenuated) Listeria 10403S ΔinlA strain through conjugation with E. coli SM10.

CRISPR/Cas9 genome editing
For CRISPR/Cas9 mediated knockout of MAP3K7/TAK1 or RNF31/HOIP from U2OS cells a previously described method was used 45,46 . The sequences TTGTCCGCTGCAACGCTCAT in the first exon of RNF31 gene and GATCGACTACAAGGAGATCG in the first exon of MAP3K7 gene were cloned into lentiCRISPRv2 vector, and introduced into U2OS cells through Lentiviral infection, to facilitate genomic targeting of these sites by Cas9. After obtaining a culture derived from a single cell, knockout of RNF31/HOIP or MAP3K7/TAK1 was confirmed by Western blotting and sequencing of the affected site.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. High confidence assignment of ubiquitination on Lys 735, 783 and 875 was based on sequence coverage from y and b ions and miscleavage by trypsin due to GlyGly residues on Lys. In addition these lysines were not ubiquitinated in IpaH2.5 C368S treated samples (see Supplementary Fig. 7