Functional Diversification of the Nleg Effector Family in Enterohemorrhagic Escherichia Coli

Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli

Dylan Valleaua, Dustin J. Littleb, Dominika Borekc,d, Tatiana Skarinaa, Andrew T. Quailea, Rosa Di Leoa, Scott Houlistone,f, Alexander Lemake,f, Cheryl H. Arrowsmithe,f, Brian K. Coombesb, and Alexei Savchenkoa,g,1

aDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada; bDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON L8S 4K1, Canada; cDepartment of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390; dDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390; ePrincess Margaret Cancer Centre, University of Toronto, Toronto, ON M5G 2M9, Canada; fDepartment of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada; and gDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB T2N 4N1, Canada

Edited by Ralph R. Isberg, Howard Hughes Medical Institute and Tufts University School of Medicine, Boston, MA, and approved August 15, 2018 (receivedfor review November 6, 2017) The pathogenic strategy of Escherichia coli and many other gram- chains. Depending on the length and nature of the polyubiquitin negative pathogens relies on the translocation of a specific set of chain, it posttranslationally regulates the target protein’s locali- proteins, called effectors, into the eukaryotic host cell during in- zation, activation, or degradation. Ubiquitination is a multistep fection. These effectors act in concert to modulate host cell pro- process which begins with the ubiquitin-activating enzyme (E1) cesses in favor of the invading pathogen. Injected by the type III using ATP to “charge” ubiquitin, covalently binding the ubiquitin secretion system (T3SS), the effector arsenal of enterohemorrhagic C terminus by a thioester linkage. The ubiquitin-conjugating E. coli (EHEC) O157:H7 features at least eight individual NleG ef- enzymes (E2s) are then involved in conjugation of “charged” fectors, which are also found across diverse attaching and effacing ubiquitin and interaction with the ubiquitin protein ligases (E3s), pathogens. NleG effectors share a conserved C-terminal U-box E3 which regulate the specificity and nature of ubiquitin attachment ubiquitin ligase domain that engages with host ubiquitination to the target protein (5, 6). There are two main classes of E3 machinery. However, their specific functions and ubiquitination ligase mechanisms: catalytic, such as HECT-type E3s which bind targets have remained uncharacterized. Here, we identify host ubiquitin covalently before ubiquitin-target transfer, and non- BIOCHEMISTRY proteins targeted for ubiquitination-mediated degradation by catalytic, where the homologous really interesting new gene two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 (RING) and U-box (RING domain homolog) families bind the localizes to the host cell nucleus and targets the MED15 subunit E2-ubiquitin conjugate and transfer ubiquitin directly from the of the Mediator complex, while NleG2-3 resides in the host cytosol E2 to the target protein (7, 8). and triggers degradation of Hexokinase-2 and SNAP29. Our struc- E. coli effectors have been shown to disrupt, hijack, or impair tural studies of NleG5-1 reveal a distinct N-terminal α/β domain ubiquitin signaling pathways through a variety of mechanisms that is responsible for interacting with host protein targets. The (5). The most common ubiquitination-manipulating T3SS ef- core of this domain is conserved across the NleG family, suggest- fectors are those that mimic the function of eukaryotic E3 li- ing this domain is present in functionally distinct NleG effectors, gases. In pathogenic E. coli, the effector NleL is a catalytic E3 which evolved diversified surface residues to interact with specific host proteins. This is a demonstration of the functional diversification and the range of host proteins targeted by the most ex- Significance panded effector family in the pathogenic arsenal of E. coli. Pathogenic Escherichia coli strains represent a persistent health ubiquitination | effectors | Escherichia coli | pathogenesis risk worldwide, with the enterohemorrhagic E. coli (EHEC) strain O157:H7, in particular, responsible for many deadly nteropathogenic (EPEC) and enterohemorrhagic (EHEC) outbreaks. During infection of the gastrointestinal tract, EHEC “ ” EEscherichia coli are responsible for gastrointestinal infections injects pathogenic proteins called effectors into cells of the worldwide. EHEC is responsible for the majority of severe in- human intestinal lining to subvert normal host processes in fections, in particular the O157:H7 serotype, which together with benefit of the pathogen. In this work, we investigate the other non-O157 Shiga toxin-containing E. coli, has been associ- largest family of EHEC effectors, the NleG family, revealing ated with outbreaks and severe disease in humans, and the po- them to have a distinct N-terminal domain that binds to specific tentially fatal hemolytic uremic syndrome (HUS) (1–3). To human protein targets and causes their degradation via their colonize the intestinal epithelium, EHEC and EPEC depend on conserved C-terminal E3 ubiquitin ligase domain during EHEC a type III secretion system (T3SS) that facilitates the injection of infection of human cells. This provides the first insight into the a specific set of effector proteins into host cells. These T3SS- functional diversity among NleG effectors and their roles in translocated effector proteins cause host cell modifications that EHEC pathogenesis. lead to attaching and effacing lesions, immune evasion, and Author contributions: D.V., D.J.L., C.H.A., B.K.C., and A.S. designed research; D.V., D.J.L., nutrient acquisition. Although specific effector repertoires vary T.S., A.T.Q., R.D.L., and S.H. performed research; R.D.L. contributed new reagents/analytic between pathogenic E. coli strains, certain “core” effectors are tools; D.V., D.J.L., D.B., A.T.Q., A.L., B.K.C., and A.S. analyzed data; and D.V., D.J.L., and highly conserved throughout EHEC and EPEC. Despite signif- A.S. wrote the paper. icant progress in understanding the role of T3SS effectors in The authors declare no conflict of interest. EPEC and EHEC’s infection strategy, the molecular target of This article is a PNAS Direct Submission. many effectors and the host cell consequence of these interac- Published under the PNAS license. tions remain unknown (1, 2). Data deposition: The solution NMR and crystal structures for NleG5-1 have been depos- One of the key targets of bacterial effector proteins is the ited in the Protein Data Bank, www.pdb.org (PDB ID codes 6B3N and 5VGC). eukaryotic ubiquitination system (4, 5). Ubiquitination is a major 1To whom correspondence should be addressed. Email: [email protected]. eukaryote-specific posttranslational regulation mechanism which This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. involves the transfer of ubiquitin to lysine residues in target 1073/pnas.1718350115/-/DCSupplemental. proteins, typically followed by the formation of polyubiquitin

www.pnas.org/cgi/doi/10.1073/pnas.1718350115 PNAS Latest Articles | 1of6 Downloaded by guest on September 29, 2021 ligase that has functional similarities to HECT E3 ligases and is and NleG8-1 and acquired (1H-15N)-heteronuclear single quan- involved in pedestal formation (9), while the NleG effector family tum coherence (HSQC) spectra to evaluate whether they adopted shares a conserved C-terminal U-box domain that mediates in- a stable tertiary structure. These NleG effectors were chosen be- teraction with the human UBE2D ubiquitin E2-conjugating en- cause they are secreted by EHEC (10) and none share greater zyme family (4). NleG effectors are part of the core effector than 30% pairwise amino acid identity to one another. The HSQC repertoire present in EPEC and EHEC, while EHEC O157:H7 spectrum of the N-terminal region of NleG5-1 (residues 1–116) contains an expanded array of 14 nleG genes, at least 8 of which showed good peak dispersion and signal-to-noise, indicating this are expressed and secreted by the T3SS, making NleGs the largest fragment adopts a stable structure suitable for determination by family of EHEC effectors (10). Notably, an analysis of non-O157 NMR spectroscopy (SI Appendix, Fig. S2). We therefore pro- Shiga-toxin containing E. coli strains found that certain combi- ceeded with the solution structure determination of the NleG5-1 nations of NleG effectors are more prevalent in strains associated N-terminal domain by NMR. with outbreaks and severe disease (3). Other T3SS-dependent Standard 2D and 3D spectra were acquired with 15N/13C-labeled pathogenic bacteria such as Salmonella enterica and the murine N-terminal NleG5-1 and the solution structure of NleG5-1 for pathogen Citrobacter rodentium also contain homologs of NleG residues 1–100 was determined (PDB ID code 6B3N). The struc- effectors (10), suggesting that NleG family members play an im- ture of NleG5-1[1–100] revealed a two-layer α/β-sandwich fold, portant role in disease potential among gram-negative pathogens. β α α ∼ with an antiparallel -sheet packed against the 1and 2 helices E. coli NleG effectors are 200 amino acids in size, consisting of (Fig. 1A). Consistent with our interpretation of conserved hydro- a highly variable ∼100 residue N-terminal region and a conserved phobic residues in the N-terminal domain, mapping these residues C-terminal U-box domain. Although the ubiquitin E3 ligase ac- onto the structure of NleG5-1[1–100] confirmed that most are part tivity of the C-terminal U-box domain has been established (4), of the hydrophobic core (Fig. 1B). Structural homology searches the role of the N-terminal domain of NleG effectors and their general role during infection has not been addressed. Here we using the DALI (11) and PDBeFold (12) servers confirmed only general similarity between the N-terminal NleG5-1 domain and show that NleGs target distinct host proteins for degradation α β during infection, identifying a human target for the EHEC ef- several layered / domains (SI Appendix, Tables S1 and S2). fectors NleG5-1 and NleG2-3 and revealing the dependence of However, this analysis did not suggest possible functions that could these interactions on the NleG N-terminal target-binding domain. be inferred from structural similarity. – Furthermore, we present the solution NMR structure of the Although the structure of NleG5-1[1 100] along with the NleG5-1 N-terminal domain and the full-length crystal structure NleG2-3 U-box domain, reported previously by Wu et al. (4), of NleG5-1 that establishes a two-domain architecture for NleG provide structural details for understanding their function in- effectors. These data reveal that NleG effectors provide a versatile dividually, how these domains are linked and their function in and structurally unique scaffold for host–pathogen interactions, context of full-length NleG remained elusive. To understand the whereby the C-terminal domain contains conserved features re- architecture of NleGs, we determined the crystal structure of full-length quired for eukaryotic E3 ligase activity, while the N-terminal do- NleG5-1 to 2.6 Å by molecular replacement (PDB ID code 5VGC). main provides a modifiable platform to allow targeting of different Each NleG5-1 molecule featured distinct N- and C-terminal do- host proteins for degradation. mains connected by two consecutive α-helices, designated α3and Results Sequence Variability in NleG Effectors Suggests Divergent Targeting. NleG effectors were originally divided into 12 distinct clades based on sequence diversity (10). The 14 NleG effectors identified in EHEC O157:H7 represent 8 of these clades, sharing between 23% and 99% of primary sequence identity in pairwise comparisons (excluding the truncated predicted pseudogenes nleG2-1 and nleG3) (4, 10). We hypothesized that sequence variability among NleG effectors reflects a diversification of host target-binding specificity and ubiquitination. We built a phylo- genetic tree for the NleG family that includes several newly identified NleG effector sequences (SI Appendix, Fig. S1 A and B). The group represented by NleG2 from EHEC O157:H7 appears most prevalent, with at least one representative encoded in all EPEC or EHEC strains included in this analysis. In contrast, other NleG clades, such as the subfamily represented by NleG5-1 and NleG5-2 from EHEC O157:H7 are only present in a subset of EHEC strains. This comparative analysis of NleG sequences revealed a conserved set of residues in the N-terminal region, including Arg28, Ile38, Gly42, Val45, Ile47, Phe56, Leu65, Ile70, Ile80, Leu84, Asn85, and Gly87 (numbering based on NleG5-1) (SI Appendix, Fig. S1A). We interpreted the prevalence of conserved hydrophobic residues within this N-terminal region as an indication of Fig. 1. The NleG5-1 NMR and crystal structures reveal a conserved N- a structurally maintained hydrophobic core. Since the NleG N- terminal fold. (A) Bundle of the 20 lowest-energy NMR models represent- – terminal region lacked a known sequence motif or similarity with ing the solution structure of NleG5-1 for residues 1 100 (PDB ID code 6B3N). (B) The most conserved residues in the NleG5-1 N-terminal domain form the known functional domains, we proceeded with structural char- core of the fold. Depiction of the most conserved residues between NleGs acterization of the NleG N-terminal region. made using the ConSurf server (40) and the multiple sequence alignment shown in SI Appendix, Fig. S1.(C) The crystal structure of full-length NleG5-1 The NleG5-1 Structure Defines the Conserved Architecture of NleG (PDB ID code 5VGC) with the N-terminal domain colored in blue as in A and Effectors. To characterize the NleG N-terminal domain, we the U-box domain in purple. Due to slight secondary structure annotation expressed and purified N-terminal fragments of NleG2-3, NleG5-1, differences the first β-strand in the NMR structure is annotated as β0.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1718350115 Valleau et al. Downloaded by guest on September 29, 2021 α4, which are tethered by an unstructured linker (Fig. 1C). The The human Mediator complex is composed of ∼26 subunits and NMR-derived NleG5-1[1–100] N-terminal domain and the C- plays a critical role in transcriptional regulation for eukaryotes (15). terminal U-box domain of NleG2-3 (4) superimposed with the Hexokinase-2 (HK2) is an enzyme involved in glycolysis and reg- NleG5-1 full-length crystal structure with an rmsd of 2.14 (over 89 ulation of intrinsic apoptosis (16, 17). SNAP29 is a SNARE protein Cα atoms) and 1.35 Å (over 91 Cα atoms), respectively (SI Ap- involved in vesicle fusion and transport, as well as phagocytosis pendix,Fig.S3A). To interpret this full-length NleG structure in (18). To determine which of these proteins were involved in direct the context of host target ubiquitination, we modeled NleG5-1 interactions with NleG5-1 and NleG2-3 we used a yeast two-hybrid against the structure of an activated eukaryotic E3–E2∼Ub com- (Y2H) approach (19). To control for the E3 ligase activity of the NleGs, which could lead to ubiquitination and degradation of their plex, RNF4–UbcH5a∼Ub (13). Superimposition of the NleG5-1 interacting partner, the NleG variants NleG5-1V144K and NleG2- U-box domain to the RING domain of RNF4 revealed no major 3I121K were tested alongside the wild-type NleGs as they have clashes between NleG5-1 and the E2-conjugating enzyme previously shown to be deficient in ubiquitination due to disruption UbcH5a, or the conjugated ubiquitin (SI Appendix,Fig.S3B). of the hydrophobic E2-binding surface (4). Coexpression of wild- Thus, our structural analysis of NleG5-1 has established the two- type NleG2-3 with HK2 or SNAP29 did not result in yeast growth domain architecture shared by NleG effectors and is consistent on selective media (Fig. 2C). However, the coexpression of NleG2- with eukaryotic ubiquitination processes. 3I121K with HK2, but not with SNAP29, consistently showed yeast growth, implying that NleG2-3 directly interacts with HK2, con- Different NleG Effectors Target Unique Host Proteins. To test the sistent with HK2 representing the most highly enriched NleG2-3 functional diversification among NleG effectors, we searched for target identified in affinity purification (AP)-MS experiments. human protein targets of the EHEC O157:H7 effectors NleG5-1 This observation suggested that the absence of growth for strains and NleG2-3, which share only ∼20% sequence identity in their coexpressing wild-type NleG2-3 with HK2 is a false negative re- N-terminal domain. For this, affinity-purified NleGs immobi- sult, due to degradation of HK2 via NleG-triggered ubiquitination. lized on magnetic beads were used to capture potential in- The Y2H screens for interaction between NleG5-1 and indi- teraction partners from human U937 cell lysate, which were then vidual Mediator complex subunits were complicated by autoac- identified using mass spectrometry (Fig. 2 A and B). For NleG5-1, tivation of yeast growth for DNA-binding (DB) domain fusions V144K we identified 23 subunits of human Mediator complex and several of NleG5-1 (SI Appendix, Fig. S4A) and of the Mediator additional Mediator complex-affiliated proteins as possible in- subunits MED1, MED4, MED15, MED26, and MED31 (Fig. teraction targets (Fig. 2A) (14). In contrast, NleG2-3 coprecipi- 2D). Coexpression of the activating domain (AD) fusion of BIOCHEMISTRY tated with only three human proteins, Hexokinase-2 (HK2), NleG5-1 with the rest of the tested Mediator subunits did not reveal any specific interactions. However, when plated on se- Synaptosomal-associated protein 29 (SNAP29), and Hexokinase-1 lective media containing 3-amino-1,2,4-triazole (3AT), which (HK1), with HK2 accounting for the majority of the identified increases the stringency of the Y2H readout, coexpression of peptides (Fig. 2B). wild-type NleG5-1 with MED15 resulted in decreased yeast growth compared with NleG5-1V144K. We interpreted this as a positive interaction between NleG5-1 and MED15 and the A subsequent ubiquitination-mediated degradation of MED15, analogous to the results observed with coexpression of wild-type MED15 MED14 MED23 TRIM11 MED16 MED1 MED24 MED17 MED8 MED4 LONP2 MED10 RASAL2 MED27 MED11 MED25 MED21 CAMK2G MED26 UBR5 MED22 MED6 MED9 POLR2A MED18 RPS13 DECR1 MED30 MED19 MED20 MED28

NleG5-1 NleG2-3 and HK2 (Fig. 2C). This hypothesis was examined

Pepde count further by monitoring the growth of yeast coexpressing MED15 V144K 78 0 with wild-type NleG5-1 or NleG5-1 in the presence of in- B C creasing concentrations of 3AT. Under these conditions, the

HK2 SNAP29 HK1 NleG2-3-AD Vector HK2 SNAP29 Vector HK2 SNAP29 Vector HK2 SNAP29 MED15-triggered autoactivation of yeast growth was decreased EV-AD NleG2-3 Target-DB only by the presence of wild-type NleG5-1, while growth with WT V144K Pepde count the NleG5-1 mutant was amplified (SI Appendix,Fig. 160 0 I121K S4B). Considering that MED15 was the most represented NleG2-3-DB human protein coprecipitated with NleG5-1 from human cell EV-DB Target-AD lysate (Fig. 2A), these results strongly suggest that the WT MED15 subunit of the Mediator complex is a direct target I121K of NleG5-1. -Leu-Trp -His -His+3-AT D To determine whether the N-terminal domain of NleGs mediate host target interactions, we tested the ability of the individual Vector MED1 MED4 MED6 MED7 MED8 MED14 MED15 MED16 MED17 MED18 MED19 MED20 MED21 MED22 MED23 MED24 MED25 MED26 MED27 MED28 MED29 MED30 MED31 TRIM11 CDK8 MED15_c2 N- and C-terminal domains of NleG2-3 and NleG5-1 to bind their

EV-AD -His+3AT -His -Leu-Trp WT respective targets using Y2H. In this assay, yeast growth indicating V144K direct binding was observed between the N-terminal domains of EV-AD NleG2-3 with HK2 and NleG5-1 with MED15 (for details see SI WT Appendix, Supplementary Results and Fig. S5 A and B). Consider- V144K NleG5-1-AD ing the NleG2-3 and NleG5-1 N-terminal domains mediate EV-AD WT interactions with specific human protein targets, we then hy- V144K pothesized that sequence variability in their N-terminal surface- exposed residues mediates the specificity of these interactions. A Fig. 2. Human protein targets of NleG5-1 and NleG2-3 are MED15 and HK2, model of the N-terminal domain of NleG2-3 was constructed respectively. AP-MS experiments with U937 cell lysate reveals the human based on the crystal structure of NleG5-1 using MODELER (20) protein target candidates for NleG5-1 (A) and NleG2-3 (B). AP-MS results are and used to select a panel of NleG2-3 N-terminal surface-exposed displayed as the average number of peptides identified for the indicated human proteins in at least four MS runs (two MS runs for each replicate AP residues that are not conserved with NleG5-1, which were then experiment). To identify the direct interactors from the AP-MS candidate probed for their role in HK2 interaction. These NleG2-3 residues pools, Y2H experiments were performed with human protein candidates were individually substituted with alanine and tested for their in- I121K and NleG2-3 (C) or NleG5-1 (D), confirming interactions between MED15 and teraction with HK2 by Y2H in the NleG2-3 ubiquitination- NleG5-1 and between HK2 and NleG2-3. deficient variant. The substitutions D30A, T32A, G35A, T37A,

Valleau et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 29, 2021 V41A, Y42A, S44A, L63A, and L64A resulted in a detectable A C decrease of Y2H growth, particularly when tested on the medium WT I121K WT V144K

containing 3AT (SI Appendix, Fig. S5 C and D). Of these, Y42A, WT I121K WT V144A V144K L63A, and L64A mutations had the most severe effect on Y2H --- signal. Since these residues are conserved in a subset of NleG Vector NleG2-3 NleG2-3 NleG5-1 NleG5-1 Vector + ------NleG2-3 NleG2-3 NleG5-1 NleG5-1 NleG5-1 EPEC wt effectors, but are not found in NleG5-1, we postulated that these Lysate + - + - + - + - + - + - EPEC ΔescN - + ------residues may be part of a common target interaction interface in Flag-IP - + - + - + - + - + - + EPEC ΔnleG -- +++ +++ several NleGs, while the rest of the identified NleG2-3 residues MED15 MED15 are responsible for specificity of NleG2-3 toward HK2. The large HK2 MED6 HK2 number of NleG2-3 N-terminal residues that appeared to be in- SNAP29 long exp. volved in interaction with HK2 is indicative of a broad NleG2- Flag-NleG HK2 3–HK2 interface. Intriguingly, the mutation H10A in NleG2-3 short exp. GAPDH SNAP29 resulted in increased Y2H growth on selective medium, suggesting that this substitution may strengthen the NleG2-3 interactions Flag-NleG with HK2. Tir Our results identify distinct host targets for two EHEC NleG GAPDH effectors, revealing the functional diversification in this family Actin DAPI NleG-FLAG Merge and identify a number of N-terminal residues specific to NleG2-3 B that are critical for HK2 interaction, supporting the hypothesis pGEN222 that sequence plasticity of this domain is responsible for host vector control target specificity of NleG effectors.

NleG Effectors Bind and Induce Degradation of Their Targets in WT Human Cells. To determine whether NleG2-3 and NleG5-1 af- NleG2-3 fected endogenous levels of HK2 and MED15, HEK293T cells were transiently transfected with constructs expressing wild-type and ubiquitination-deficient NleG5-1 or NleG2-3 and the levels of I121K MED15 and HK2 were probed by immunoblotting. Cells expressing NleG2-3 wild-type NleG5-1 or NleG2-3, but not their ubiquitination-inactive variants, showed significantly decreased levels of MED15 and HK2, respectively (Fig. 3A). In addition, the NleG5-1V144K and NleG2- NleG5-1WT 3I121K mutants consistently coimmunoprecipitated with MED15 and HK2, respectively, further confirming specific interactions between NleG effectors and these human proteins (Fig. 3A). Transfection of HEK293T cells with a construct expressing the partially inactive NleG5-1V144K NleG5-1V144A variant also led to reduction of MED15 levels, although to a lesser degree than in the case of the wild-type NleG5-1. These studies confirmed that NleG5-1 and NleG2-3 specifically Fig. 3. NleG5-1 and NleG2-3 bind and degrade host targets in human cells. target the human proteins MED15 and HK2 and induce their (A) NleG2-3 and NleG5-1 bind and degrade their host targets following ectopic degradation in human cells. expression in HEK293T cells using the vector pcDNA3.1/nFLAG-DEST and their To further address individual roles of the NleG N- and C-terminal subsequent immunoprecipitation. (B) The indicated FLAG NleG2-3 and NleG5-1 domains in interaction with host proteins, we constructed a chimeric constructs were expressed in EPEC O126:H7 ΔnleG and used to infect HeLa NleG effector composed of the N-terminal binding domain of cells, which were then detected by anti-FLAG immunofluorescence (green) and – costained for nucleus (DAPI, blue) and F-actin (phalloidin, red). (C)NleG2-3, NleG5-1 (residues 1 112) and the U-box domain of NleG2-3 (res- Δ – NleG5-1, and their indicated mutants were expressed in EPEC O126:H7 nleG idues 90 191), which we called NleG5-1chi. This chimeric effector and used to infect HeLa cells in culture along with wild-type and secretion- was transiently expressed in HEK293T cells and the level of MED15 deficient (ΔescN) EPEC and probed for target degradation. Immunoblotting of was measured by immunoblotting (SI Appendix,Fig.S5E). While the targets reveals specific degradation by both NleG2-3 and NleG5-1 during NleG5-1chi did not degrade MED15 as efficiently as wild-type infection. GAPDH is a loading control in A and C. NleG5-1, the NleG5-1chi/I121K coimmunoprecipitated with MED15 but not with HK2, confirming the ability of this chimeric effector to specifically interact with only the identified target of NleG5-1. levels of HK2 and MED15. Treatment of cells with MG132 for Furthermore, the N-terminal fragment of NleG5-1 spanning resi- 2 h resulted in restoration of HK2 levels in cells carrying NleG2- WT dues 1–112 transiently expressed in HEK293T cells also coimmu- 3 , although the levels of HK2 slowly lowered after this point, noprecipitated with MED15 and not with HK2. In the reciprocal potentially showing the rate of HK2 turnover (SI Appendix, Fig. experiment, we constructed a chimeric effector composed of the S6A). Similarly, a 2-h treatment with MG132 restored the levels WT N-terminal domain of NleG2-3 (residues 1–88) and the C-terminal of MED15 in cells expressing NleG5-1 (SI Appendix, Fig. U-box of NleG5-1 (residues 105–213), called NleG2-3chi, although S6B), together supporting our hypothesis that NleG-mediated we were not able to obtain substantial expression of NleG2-3chi or ubiquitination directs their specific host protein targets for NleG2-3chi/V144K. However, the N-terminal fragment of NleG2-3 degradation by the proteasome. To confirm that NleGs are individually expressed in HEK293T cells coimmunoprecipitated directly ubiquitinating their targets, we transiently expressed with HK2 and not with MED15 (SI Appendix,Fig.S5E). Combined, NleG5-1WT or NleG5-1V144K in combination with HA-tagged these results show that the N-terminal domain in NleG2-3 and ubiquitin in HEK293T cells and treated these cells with MG132 NleG5-1 are sufficient for specific interactions with human HK2 for 6 h. Next, MED15 was immunoprecipitated under denaturing and MED15, respectively. conditions (21) and probed for ubiquitinated MED15 species by To test whether ubiquitination of the identified targets by immunoblotting using anti-HA antibodies. The amount of ubiq- NleG effectors triggers their degradation by the proteasome, we uitinated MED15 was noticeably higher in cells expressing the transiently expressed NleG2-3 or NleG5-1 in HEK293T cells and NleG5-1WT relative to the cells expressing NleG5-1V144K variant tested the effect of the proteasome inhibitor MG132 on the and the vector control (SI Appendix,Fig.S6C), thus confirming

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1718350115 Valleau et al. Downloaded by guest on September 29, 2021 that NleG5-1 directly ubiquitinates MED15 to trigger its degra- targets via variation in the N-terminal domain. This hypothesis is dation by the proteasome. supported by site-directed mutagenesis studies of the NleG2-3 N-terminal domain surface based on a model established using NleG5-1 and NleG2-3 Induce Specific Host Target Degradation During the NleG5-1 structures, with a number of surface-exposed resi- EPEC Infection. The repertoire of NleG effectors varies between dues revealed to be crucial for interaction between NleG2-3 and pathogenic E. coli, ranging from 14 members in EHEC O157:H7 HK2, all of which are residues that are not conserved with to a single NleG (also known as NleI) in the EPEC strain E2348/ NleG5-1. A number of these residues are conserved between 69 (22, 23). We decided to take advantage of the low represen- members of the NleG2 family, which share 60% or greater tation of NleGs in EPEC to test the individual function of NleG5- overall identity in their primary sequence, suggesting that these 1 and NleG2-3 during infection. An EPEC ΔnleG strain was effectors may share a common target recognition interface. constructed and transformed with plasmids expressing FLAG- The two-domain architecture established for NleG effectors by tagged NleG2-3, NleG5-1, or their ubiquitination-inactive vari- our structural analysis is reminiscent of other characterized ef- ants. The secretion of NleG5-1 and NleG2-3 by the EPEC ΔnleG fector E3 protein ubiquitin ligases (26–28). However, the NleG5-1 strain was confirmed by stimulating T3SS-dependent secretion in N-terminal domain fold bears no resemblance to domains found defined media (SI Appendix,Fig.S7A and B) (24). Next, to test in other E3 ubiquitin ligases or other characterized proteins in whether the identified interactions of NleG2-3 and NleG5-1 with general. The expansion of the NleG family in pathogenic E. coli host proteins were in line with their cellular localization during strains such as EHEC O157:H7 through apparent diversification infection, we infected HeLa cells with EPEC expressing FLAG- of the N-terminal region suggests that this domain is a versatile tagged NleG2-3, NleG5-1, or empty vector. Consistent with its interface for host protein recognition. Comparing against an interaction with the nuclear Mediator complex, NleG5-1 pre- activated E3–E2∼ubiquitin complex, RNF4–UbcH5a∼Ub (13), dominantly localized to the nucleus, while NleG2-3 displayed shows the positioning of the NleG5-1 N-terminal domain is con- nonspecific localization and was generally present in the cytosol, in sistent with the N-terminal domain’s role of bringing the substrate line with the fact that HK2 is cytosolic and commonly localized to into contact with the eukaryotic ubiquitination machinery. How- the surface of mitochondria (Fig. 3B) (14, 16, 25). ever, the true conformation adopted by NleG5-1 during target To determine whether NleG2-3 and NleG5-1 triggered degra- ubiquitination remains to be determined through further structural dation of their identified targets in the context of infection, we studies. infected HeLa cells with EPEC strains expressing NleG2-3, We identified distinct human proteins targeted for degrada-

NleG5-1, or empty vector, and monitored the levels of HK2 and tion by two EHEC NleGs with divergent N-terminal domains, BIOCHEMISTRY MED15. The infection of HeLa cells by EPEC carrying wild-type NleG2-3 and NleG5-1. HK2, a target of NleG2-3, is a major NleG2-3, but not the inactive NleG2-3I121K variant, resulted in a metabolic enzyme that converts glucose to glucose-6-phosphate, decrease of HK2 levels (Fig. 3C). Unexpectedly, the level of the initial step of glucose metabolism. In addition, HK2 carries SNAP29 was also reduced during infection with EPEC expressing apoptosis-preventing cellular functions, whereby consistent NleG2-3. Similarly, infection by EPEC expressing NleG5-1WT led binding of HK2 to mitochondria is crucial to inhibit cytochrome to complete degradation of MED15 (Fig. 3C). Additional probing C release and subsequent apoptosis (16, 17, 29). Both the met- for the levels of another Mediator subunit, MED6, showed no abolic and apoptotic roles of HK2 are implicated in E. coli in- change between infections with EPEC expressing NleG5-1WT or fection. HK2 is up-regulated in host cells in the presence of NleG5-1V144K, indicating that NleG5-1–mediated degradation of uropathogenic E. coli, possibly to support the energy demands of MED15 does not lead to degradation of the entire Mediator fighting infection (30), while other effectors have been shown to complex. We did not observe any detectable changes in the level manipulate mitochondrial apoptosis, in particular EspF, which of MED15 for EPEC carrying NleG2-3, nor for HK2 or SNAP29 induces cytochrome C release from mitochondria and causes following infection by EPEC expressing NleG5-1. Next, we fol- apoptosis (31). Interestingly, we found that expression of NleG2- lowed the infection of HeLa cells with EPEC expressing FLAG- 3 in host cells also leads to degradation of SNAP29, a SNARE tagged NleG2-3, NleG5-1, or a vector control with an anti-FLAG protein involved in vesicle fusion and transport (18). Although immunoprecipitation and immunoblotting for HK2, MED15, and SNAP29 coprecipitated with purified NleG2-3 in AP experi- SNAP29. We detected HK2 and MED15 coimmunoprecipitating ments we were not able to demonstrate a direct interaction be- with NleG2-3 and NleG5-1, respectively, consistent with our other tween these two proteins using Y2H or immunoprecipitation. It results, suggesting that these are the direct targets of NleG2-3 and is therefore tempting to suggest that NleG2-3 triggering the NleG5-1 during infection. This is in line with our hypothesis that degradation of SNAP29 is indicative of an interaction between NleG2-3 and NleG5-1 bind and ubiquitinate HK2 and MED15 SNAP29 and HK2, the primary identified target of NleG2-3, during infection, respectively (SI Appendix,Fig.S7C). Taken to- or the presence of additional targets for this effector, although gether, our results demonstrate the EHEC effectors NleG2-3 and the functional implications of this interaction remain to be NleG5-1 are functionally diverse E3 ubiquitin ligases that are able determined. to trigger the ubiquitination-mediated degradation of specific NleG5-1 targets the Mediator complex member MED15, human proteins during infection. which functions as an end point for many signaling pathways, with a number of transcription factors (TFs) mediating tran- Discussion scription of their target genes through interaction with specific NleG effectors represent an expanded family that is part of the Mediator subunits (15, 32). Although NleG5-1 does not possess core arsenal of type III-secreted effectors in EHEC and EPEC. an identifiable nuclear localization signal, it is able to localize to Our previous work established these effectors as E3 ubiquitin the nucleus and target MED15. This may be due to the passive ligases with conserved C-terminal U-box domains (4). Phyloge- diffusion of NleG5-1 into the nucleus as its size of ∼24 kDa is netic analysis of the NleG family of effectors revealed conserved well under the presumed nuclear pore diffusion limit of at least residues in the N-terminal domain, which, as revealed by our 60 kDa (33). MED15 is specifically involved in integration of structural analysis, form part of the hydrophobic core in the various transcriptional signals, including TGF-β and SREBP1 NleG5-1 N-terminal domain. We further demonstrated that the signaling (15, 32, 34). Intriguingly, TGF-β is implicated in E. coli N-terminal domain of NleG2-3 and NleG5-1 is required and infection, as TGF-β treatment of epithelial cells has been shown sufficient for binding of specific host targets. This suggests that, to inhibit the ability of EHEC to disrupt tight junctions, and while diverse in sequence, NleGs share a core N-terminal elevated TGF-β levels are associated with reduced incidence of structural fold that has evolved to interact with distinct host hemolytic uremic syndrome induced by EHEC O157:H7 (35,

Valleau et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 29, 2021 36). The potential consequence of impaired SREBP1 signaling, EHEC NleG effectors and contributes to the diversity of host which is involved in responding to a sensor of intracellular cell processes targeted during bacterial infection. cholesterol and regulating of lipid homeostasis (37, 38) in the context of E. coli infection has not been addressed so far. Materials and Methods The infection strategy of attaching and effacing pathogens is Please refer to SI Appendix, Supplementary Materials and Methods for full dependent on a specific arsenal of virulence factors that ma- extensive details on cloning, protein expression, AP-MS, Y2H, structural nipulate and control host cells. Thus, it is plausible that the determination, cell culture, transfection experiments, EPEC infection ex- dramatic expansion of the NleG effectors in EHEC species is a periments, immunofluorescence, generation of EPEC mutants, and T3SS direct contributor to the high virulence of these bacteria in hu- translocation assays. mans or efficient asymptomatic colonization of cattle, their ACKNOWLEDGMENTS. We thank Zdzislaw Wawrzak for assistance in X-ray natural reservoir. Similarly, the human-specific bacterial patho- data collection for the crystal structure of NleG5-1 and Joan and Ron gen Shigella also has a large arsenal of sequence-related yet Conaway for their gift of HeLa cell lines expressing FLAG-tagged MED26 and functionally diverse E3 ubiquitin ligase effectors, the IpaHs (26, CDK8. The structural information was obtained as part of the Center for 27, 39). Functionally similar to NleGs but structurally distinct, Structural Genomics of Infectious Diseases (https://csgid.org/). This project the IpaH effectors contain a conserved C-terminal catalytic E3 has been funded in whole or in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health ligase domain and a variable N-terminal leucine rich repeat and Human Services, under Contract nos. HHSN272201200026C and (LRR) domain that enables them to target a variety of discrete HHSN272201700060C. The C.H.A. NMR facility is generously supported with human proteins for degradation (39). This is indicative of the operating funds provided by the Princess Margaret Cancer Centre. The func- expanded arsenal of E3 ubiquitin ligase effectors, representing a tional data were obtained using a Canadian Institutes of Health Research (CIHR) operating grant to A.S. (principal investigator) and B.K.C. (coprincipal versatile evolutionary mechanism that allows bacterial pathogens investigator). B.K.C. is supported by a Canada Research Chair in Infectious to efficiently adapt to a specific host niche. Our current work Disease Pathogenesis. D.J.L. has been supported in part by Michael G. provides insight into the broad range of host proteins targeted by DeGroote and CIHR fellowships.

1. Kaper JB, Nataro JP, Mobley HL (2004) Pathogenic Escherichia coli. Nat Rev Microbiol 23. Ogura Y, et al. (2009) Comparative genomics reveal the mechanism of the parallel 2:123–140. evolution of O157 and non-O157 enterohemorrhagic Escherichia coli. Proc Natl Acad 2. Caprioli A, Morabito S, Brugère H, Oswald E (2005) Enterohaemorrhagic Escherichia Sci USA 106:17939–17944. – coli: Emerging issues on virulence and modes of transmission. Vet Res 36:289 311. 24. Deng W, et al. (2005) Regulation of type III secretion hierarchy of translocators and 3. Coombes BK, et al. (2008) Molecular analysis as an aid to assess the public health risk effectors in attaching and effacing bacterial pathogens. Infect Immun 73:2135–2146. of non-O157 Shiga toxin-producing Escherichia coli strains. Appl Environ Microbiol 74: 25. Schindler A, Foley E (2013) Hexokinase 1 blocks apoptotic signals at the mitochondria. 2153–2160. Cell Signal 25:2685–2692. 4. Wu B, et al. (2010) NleG type 3 effectors from enterohaemorrhagic Escherichia coli are 26. Singer AU, et al. (2008) Structure of the Shigella T3SS effector IpaH defines a new U-Box E3 ubiquitin ligases. PLoS Pathog 6:e1000960. class of E3 ubiquitin ligases. Nat Struct Mol Biol 15:1293–1301. 5. Ashida H, Kim M, Sasakawa C (2014) Exploitation of the host ubiquitin system by 27. Zhu Y, et al. (2008) Structure of a Shigella effector reveals a new class of ubiquitin human bacterial pathogens. Nat Rev Microbiol 12:399–413. – 6. Komander D (2009) The emerging complexity of protein ubiquitination. Biochem Soc ligases. Nat Struct Mol Biol 15:1302 1308. Trans 37:937–953. 28. Quaile AT, et al. (2015) Molecular characterization of LubX: Functional divergence of – 7. Metzger MB, Hristova VA, Weissman AM (2012) HECT and RING finger families of E3 the U-Box fold by Legionella pneumophila. Structure 23:1459 1469. ubiquitin ligases at a glance. J Cell Sci 125:531–537. 29. Roberts DJ, Miyamoto S (2015) Hexokinase II integrates energy metabolism and cel- 8. Berndsen CE, Wolberger C (2014) New insights into ubiquitin E3 ligase mechanism. lular protection: Akting on mitochondria and TORCing to autophagy. Cell Death Nat Struct Mol Biol 21:301–307. Differ 22:248–257. 9. Piscatelli H, et al. (2011) The EHEC type III effector NleL is an E3 ubiquitin ligase that 30. Reigstad CS, Hultgren SJ, Gordon JI (2007) Functional genomic studies of uropatho- modulates pedestal formation. PLoS One 6:e19331. genic Escherichia coli and host urothelial cells when intracellular bacterial commu- 10. Tobe T, et al. (2006) An extensive repertoire of type III secretion effectors in Escherichia nities are assembled. J Biol Chem 282:21259–21267. coli O157 and the role of lambdoid phages in their dissemination. Proc Natl Acad Sci 31. Nougayrède JP, Donnenberg MS (2004) Enteropathogenic Escherichia coli EspF is – USA 103:14941 14946. targeted to mitochondria and is required to initiate the mitochondrial death path- 11. Holm L, Laakso LM (2016) Dali server update. Nucleic Acids Res 44:W351–W355. way. Cell Microbiol 6:1097–1111. 12. Krissinel E, Henrick K (2004) Secondary-structure matching (SSM), a new tool for fast 32. Malik S, Roeder RG (2010) The metazoan mediator co-activator complex as an in- protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr tegrative hub for transcriptional regulation. Nat Rev Genet 11:761–772. 60:2256–2268. 33. Wang R, Brattain MG (2007) The maximal size of protein to diffuse through the 13. Plechanovová A, Jaffray EG, Tatham MH, Naismith JH, Hay RT (2012) Structure of a – RING E3 ligase and ubiquitin-loaded E2 primed for catalysis. Nature 489:115–120. nuclear pore is larger than 60kDa. FEBS Lett 581:3164 3170. 14. Conaway RC, Conaway JW (2013) The mediator complex and transcription elonga- 34. Kato Y, Habas R, Katsuyama Y, Näär AM, He X (2002) A component of the ARC/ – tion. Biochim Biophys Acta 1829:69–75. mediator complex required for TGF beta/nodal signalling. Nature 418:641 646. 15. Allen BL, Taatjes DJ (2015) The mediator complex: A central integrator of transcrip- 35. Proulx F, Litalien C, Turgeon JP, Mariscalco MM, Seidman E (2000) Circulating levels of tion. Nat Rev Mol Cell Biol 16:155–166. transforming growth factor-beta1 and lymphokines among children with hemolytic 16. Pastorino JG, Shulga N, Hoek JB (2002) Mitochondrial binding of hexokinase II inhibits uremic syndrome. Am J Kidney Dis 35:29–34. Bax-induced cytochrome c release and apoptosis. J Biol Chem 277:7610–7618. 36. Howe KL, Reardon C, Wang A, Nazli A, McKay DM (2005) Transforming growth fac- 17. Mathupala SP, Ko YH, Pedersen PL (2006) Hexokinase II: Cancer’s double-edged sword tor-beta regulation of epithelial tight junction proteins enhances barrier function and acting as both facilitator and gatekeeper of malignancy when bound to mitochon- blocks enterohemorrhagic Escherichia coli O157:H7-induced increased permeability. – dria. Oncogene 25:4777 4786. Am J Pathol 167:1587–1597. 18. Wesolowski J, Caldwell V, Paumet F (2012) A novel function for SNAP29 (synapto- 37. Yang F, et al. (2006) An ARC/mediator subunit required for SREBP control of cho- somal-associated protein of 29 kDa) in mast cell phagocytosis. PLoS One 7:e49886. lesterol and lipid homeostasis. Nature 442:700–704. 19. Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. 38. Taubert S, Van Gilst MR, Hansen M, Yamamoto KR (2006) A mediator subunit, Nature 340:245–246. MDT-15, integrates regulation of fatty acid metabolism by NHR-49-dependent and 20. Webb B, Sali A (2016) Comparative protein structure modeling using MODELLER. Curr – Protoc Bioinformatics 54:5.6.1–5.6.37. -independent pathways in C. elegans. Genes Dev 20:1137 1149. 21. Tansey WP (2007) Denaturing protein immunoprecipitation from mammalian cells. 39. Ashida H, Sasakawa C (2016) Shigella IpaH family effectors as a versatile model for CSH Protoc 2007:pdb.prot4619. studying pathogenic bacteria. Front Cell Infect Microbiol 5:100. 22. Iguchi A, et al. (2009) Complete genome sequence and comparative genome analysis 40. Ashkenazy H, et al. (2016) ConSurf 2016: An improved methodology to estimate of enteropathogenic Escherichia coli O127:H6 strain E2348/69. J Bacteriol 191: and visualize evolutionary conservation in macromolecules. Nucleic Acids Res 44: 347–354. W344–W350.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1718350115 Valleau et al. Downloaded by guest on September 29, 2021