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Research Article

Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli

View ORCID ProfileDylan Valleau, Dustin J. Little, View ORCID ProfileDominika Borek, Tatiana Skarina, View ORCID ProfileAndrew T. Quaile, Rosa Di Leo, Scott Houliston, Alexander Lemak, Cheryl H. Arrowsmith, Brian K. Coombes, and Alexei Savchenko
PNAS October 2, 2018 115 (40) 10004-10009; first published September 14, 2018 https://doi.org/10.1073/pnas.1718350115
Dylan Valleau
aDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada;
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Dustin J. Little
bDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON L8S 4K1, Canada;
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Dominika Borek
cDepartment of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390;dDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
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Tatiana Skarina
aDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada;
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Andrew T. Quaile
aDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada;
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Rosa Di Leo
aDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada;
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Scott Houliston
ePrincess Margaret Cancer Centre, University of Toronto, Toronto, ON M5G 2M9, Canada;fDepartment of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada;
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Alexander Lemak
ePrincess Margaret Cancer Centre, University of Toronto, Toronto, ON M5G 2M9, Canada;fDepartment of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada;
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Cheryl H. Arrowsmith
ePrincess Margaret Cancer Centre, University of Toronto, Toronto, ON M5G 2M9, Canada;fDepartment of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada;
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Brian K. Coombes
bDepartment of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON L8S 4K1, Canada;
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Alexei Savchenko
aDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada;gDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB T2N 4N1, Canada
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  • For correspondence: alexei.savchenko@ucalgary.ca
  1. Edited by Ralph R. Isberg, Howard Hughes Medical Institute and Tufts University School of Medicine, Boston, MA, and approved August 15, 2018 (received for review November 6, 2017)

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Significance

Pathogenic Escherichia coli strains represent a persistent health risk worldwide, with the enterohemorrhagic E. coli (EHEC) strain O157:H7, in particular, responsible for many deadly outbreaks. During infection of the gastrointestinal tract, EHEC injects pathogenic proteins called “effectors” into cells of the human intestinal lining to subvert normal host processes in benefit of the pathogen. In this work, we investigate the largest family of EHEC effectors, the NleG family, revealing them to have a distinct N-terminal domain that binds to specific human protein targets and causes their degradation via their conserved C-terminal E3 ubiquitin ligase domain during EHEC infection of human cells. This provides the first insight into the functional diversity among NleG effectors and their roles in EHEC pathogenesis.

Abstract

The pathogenic strategy of Escherichia coli and many other gram-negative pathogens relies on the translocation of a specific set of proteins, called effectors, into the eukaryotic host cell during infection. These effectors act in concert to modulate host cell processes in favor of the invading pathogen. Injected by the type III secretion system (T3SS), the effector arsenal of enterohemorrhagic E. coli (EHEC) O157:H7 features at least eight individual NleG effectors, which are also found across diverse attaching and effacing pathogens. NleG effectors share a conserved C-terminal U-box E3 ubiquitin ligase domain that engages with host ubiquitination machinery. However, their specific functions and ubiquitination targets have remained uncharacterized. Here, we identify host proteins targeted for ubiquitination-mediated degradation by two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 localizes to the host cell nucleus and targets the MED15 subunit of the Mediator complex, while NleG2-3 resides in the host cytosol and triggers degradation of Hexokinase-2 and SNAP29. Our structural studies of NleG5-1 reveal a distinct N-terminal α/β domain that is responsible for interacting with host protein targets. The core of this domain is conserved across the NleG family, suggesting this domain is present in functionally distinct NleG effectors, 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 expanded effector family in the pathogenic arsenal of E. coli.

  • ubiquitination
  • effectors
  • Escherichia coli
  • pathogenesis

Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli are responsible for gastrointestinal infections worldwide. EHEC is responsible for the majority of severe infections, in particular the O157:H7 serotype, which together with other non-O157 Shiga toxin-containing E. coli, has been associated with outbreaks and severe disease in humans, and the potentially fatal hemolytic uremic syndrome (HUS) (1⇓–3). To colonize the intestinal epithelium, EHEC and EPEC depend on a type III secretion system (T3SS) that facilitates the injection of a specific set of effector proteins into host cells. These T3SS-translocated effector proteins cause host cell modifications that lead to attaching and effacing lesions, immune evasion, and nutrient acquisition. Although specific effector repertoires vary between pathogenic E. coli strains, certain “core” effectors are highly conserved throughout EHEC and EPEC. Despite significant progress in understanding the role of T3SS effectors in EPEC and EHEC’s infection strategy, the molecular target of many effectors and the host cell consequence of these interactions remain unknown (1, 2).

One of the key targets of bacterial effector proteins is the eukaryotic ubiquitination system (4, 5). Ubiquitination is a major eukaryote-specific posttranslational regulation mechanism which involves the transfer of ubiquitin to lysine residues in target proteins, typically followed by the formation of polyubiquitin chains. Depending on the length and nature of the polyubiquitin chain, it posttranslationally regulates the target protein’s localization, activation, or degradation. Ubiquitination is a multistep process which begins with the ubiquitin-activating enzyme (E1) using ATP to “charge” ubiquitin, covalently binding the ubiquitin C terminus by a thioester linkage. The ubiquitin-conjugating enzymes (E2s) are then involved in conjugation of “charged” ubiquitin and interaction with the ubiquitin protein ligases (E3s), which regulate the specificity and nature of ubiquitin attachment to the target protein (5, 6). There are two main classes of E3 ligase mechanisms: catalytic, such as HECT-type E3s which bind ubiquitin covalently before ubiquitin-target transfer, and noncatalytic, where the homologous really interesting new gene (RING) and U-box (RING domain homolog) families bind the E2-ubiquitin conjugate and transfer ubiquitin directly from the E2 to the target protein (7, 8).

E. coli effectors have been shown to disrupt, hijack, or impair ubiquitin signaling pathways through a variety of mechanisms (5). The most common ubiquitination-manipulating T3SS effectors are those that mimic the function of eukaryotic E3 ligases. In pathogenic E. coli, the effector NleL is a catalytic E3 ligase that has functional similarities to HECT E3 ligases and is involved in pedestal formation (9), while the NleG effector family shares a conserved C-terminal U-box domain that mediates interaction with the human UBE2D ubiquitin E2-conjugating enzyme family (4). NleG effectors are part of the core effector repertoire present in EPEC and EHEC, while EHEC O157:H7 contains an expanded array of 14 nleG genes, at least 8 of which are expressed and secreted by the T3SS, making NleGs the largest family of EHEC effectors (10). Notably, an analysis of non-O157 Shiga-toxin containing E. coli strains found that certain combinations of NleG effectors are more prevalent in strains associated with outbreaks and severe disease (3). Other T3SS-dependent pathogenic bacteria such as Salmonella enterica and the murine pathogen Citrobacter rodentium also contain homologs of NleG effectors (10), suggesting that NleG family members play an important role in disease potential among gram-negative pathogens.

E. coli NleG effectors are ∼200 amino acids in size, consisting of a highly variable ∼100 residue N-terminal region and a conserved C-terminal U-box domain. Although the ubiquitin E3 ligase activity of the C-terminal U-box domain has been established (4), the role of the N-terminal domain of NleG effectors and their general role during infection has not been addressed. Here we show that NleGs target distinct host proteins for degradation during infection, identifying a human target for the EHEC effectors NleG5-1 and NleG2-3 and revealing the dependence of these interactions on the NleG N-terminal target-binding domain. Furthermore, we present the solution NMR structure of the NleG5-1 N-terminal domain and the full-length crystal structure of NleG5-1 that establishes a two-domain architecture for NleG effectors. These data reveal that NleG effectors provide a versatile and structurally unique scaffold for host–pathogen interactions, whereby the C-terminal domain contains conserved features required for eukaryotic E3 ligase activity, while the N-terminal domain provides a modifiable platform to allow targeting of different host proteins for degradation.

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 phylogenetic 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 a structurally maintained hydrophobic core. Since the NleG N-terminal region lacked a known sequence motif or similarity with known functional domains, we proceeded with structural characterization of the NleG N-terminal region.

The NleG5-1 Structure Defines the Conserved Architecture of NleG Effectors.

To characterize the NleG N-terminal domain, we expressed and purified N-terminal fragments of NleG2-3, NleG5-1, and NleG8-1 and acquired (1H-15N)-heteronuclear single quantum coherence (HSQC) spectra to evaluate whether they adopted a stable tertiary structure. These NleG effectors were chosen because they are secreted by EHEC (10) and none share greater than 30% pairwise amino acid identity to one another. The HSQC spectrum of the N-terminal region of NleG5-1 (residues 1–116) showed good peak dispersion and signal-to-noise, indicating this fragment adopts a stable structure suitable for determination by NMR spectroscopy (SI Appendix, Fig. S2). We therefore proceeded with the solution structure determination of the NleG5-1 N-terminal domain by NMR.

Standard 2D and 3D spectra were acquired with 15N/13C-labeled N-terminal NleG5-1 and the solution structure of NleG5-1 for residues 1–100 was determined (PDB ID code 6B3N). The structure of NleG5-1[1–100] revealed a two-layer α/β-sandwich fold, with an antiparallel β-sheet packed against the α1 and α2 helices (Fig. 1A). Consistent with our interpretation of conserved hydrophobic residues in the N-terminal domain, mapping these residues onto the structure of NleG5-1[1–100] confirmed that most are part of the hydrophobic core (Fig. 1B). Structural homology searches using the DALI (11) and PDBeFold (12) servers confirmed only general similarity between the N-terminal NleG5-1 domain and several layered α/β domains (SI Appendix, Tables S1 and S2). However, this analysis did not suggest possible functions that could be inferred from structural similarity.

Fig. 1.
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Fig. 1.

The NleG5-1 NMR and crystal structures reveal a conserved N-terminal fold. (A) Bundle of the 20 lowest-energy NMR models representing 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 core of the fold. Depiction of the most conserved residues between NleGs 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 (PDB ID code 5VGC) with the N-terminal domain colored in blue as in A and the U-box domain in purple. Due to slight secondary structure annotation differences the first β-strand in the NMR structure is annotated as β0.

Although the structure of NleG5-1[1–100] along with the NleG2-3 U-box domain, reported previously by Wu et al. (4), provide structural details for understanding their function individually, how these domains are linked and their function in context of full-length NleG remained elusive. To understand the architecture of NleGs, we determined the crystal structure of full-length NleG5-1 to 2.6 Å by molecular replacement (PDB ID code 5VGC). Each NleG5-1 molecule featured distinct N- and C-terminal domains connected by two consecutive α-helices, designated α3 and α4, which are tethered by an unstructured linker (Fig. 1C). The NMR-derived NleG5-1[1–100] N-terminal domain and the C-terminal U-box domain of NleG2-3 (4) superimposed with the NleG5-1 full-length crystal structure with an rmsd of 2.14 (over 89 Cα atoms) and 1.35 Å (over 91 Cα atoms), respectively (SI Appendix, Fig. S3A). To interpret this full-length NleG structure in the context of host target ubiquitination, we modeled NleG5-1 against the structure of an activated eukaryotic E3–E2∼Ub complex, RNF4–UbcH5a∼Ub (13). Superimposition of the NleG5-1 U-box domain to the RING domain of RNF4 revealed no major clashes between NleG5-1 and the E2-conjugating enzyme UbcH5a, or the conjugated ubiquitin (SI Appendix, Fig. S3B). Thus, our structural analysis of NleG5-1 has established the two-domain architecture shared by NleG effectors and is consistent with eukaryotic ubiquitination processes.

Different NleG Effectors Target Unique Host Proteins.

To test the functional diversification among NleG effectors, we searched for human protein targets of the EHEC O157:H7 effectors NleG5-1 and NleG2-3, which share only ∼20% sequence identity in their N-terminal domain. For this, affinity-purified NleGs immobilized on magnetic beads were used to capture potential interaction partners from human U937 cell lysate, which were then identified using mass spectrometry (Fig. 2 A and B). For NleG5-1, we identified 23 subunits of human Mediator complex and several additional Mediator complex-affiliated proteins as possible interaction targets (Fig. 2A) (14). In contrast, NleG2-3 coprecipitated with only three human proteins, Hexokinase-2 (HK2), Synaptosomal-associated protein 29 (SNAP29), and Hexokinase-1 (HK1), with HK2 accounting for the majority of the identified peptides (Fig. 2B).

Fig. 2.
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Fig. 2.

Human protein targets of NleG5-1 and NleG2-3 are MED15 and HK2, respectively. AP-MS experiments with U937 cell lysate reveals the human protein target candidates for NleG5-1 (A) and NleG2-3 (B). AP-MS results are 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 experiment). To identify the direct interactors from the AP-MS candidate pools, Y2H experiments were performed with human protein candidates and NleG2-3 (C) or NleG5-1 (D), confirming interactions between MED15 and NleG5-1 and between HK2 and NleG2-3.

The human Mediator complex is composed of ∼26 subunits and plays a critical role in transcriptional regulation for eukaryotes (15). Hexokinase-2 (HK2) is an enzyme involved in glycolysis and regulation of intrinsic apoptosis (16, 17). SNAP29 is a SNARE protein involved in vesicle fusion and transport, as well as phagocytosis (18). To determine which of these proteins were involved in direct interactions with NleG5-1 and NleG2-3 we used a yeast two-hybrid (Y2H) approach (19). To control for the E3 ligase activity of the NleGs, which could lead to ubiquitination and degradation of their interacting partner, the NleG variants NleG5-1V144K and NleG2-3I121K were tested alongside the wild-type NleGs as they have previously shown to be deficient in ubiquitination due to disruption of the hydrophobic E2-binding surface (4). Coexpression of wild-type NleG2-3 with HK2 or SNAP29 did not result in yeast growth on selective media (Fig. 2C). However, the coexpression of NleG2-3I121K with HK2, but not with SNAP29, consistently showed yeast growth, implying that NleG2-3 directly interacts with HK2, consistent with HK2 representing the most highly enriched NleG2-3 target identified in affinity purification (AP)-MS experiments. This observation suggested that the absence of growth for strains coexpressing wild-type NleG2-3 with HK2 is a false negative result, due to degradation of HK2 via NleG-triggered ubiquitination.

The Y2H screens for interaction between NleG5-1 and individual Mediator complex subunits were complicated by autoactivation of yeast growth for DNA-binding (DB) domain fusions of NleG5-1V144K (SI Appendix, Fig. S4A) and of the Mediator subunits MED1, MED4, MED15, MED26, and MED31 (Fig. 2D). Coexpression of the activating domain (AD) fusion of NleG5-1 with the rest of the tested Mediator subunits did not reveal any specific interactions. However, when plated on selective media containing 3-amino-1,2,4-triazole (3AT), which increases the stringency of the Y2H readout, coexpression of 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 subsequent ubiquitination-mediated degradation of MED15, analogous to the results observed with coexpression of wild-type NleG2-3 and HK2 (Fig. 2C). This hypothesis was examined further by monitoring the growth of yeast coexpressing MED15 with wild-type NleG5-1 or NleG5-1V144K in the presence of increasing concentrations of 3AT. Under these conditions, the MED15-triggered autoactivation of yeast growth was decreased only by the presence of wild-type NleG5-1, while growth with the NleG5-1V144K mutant was amplified (SI Appendix, Fig. S4B). Considering that MED15 was the most represented human protein coprecipitated with NleG5-1 from human cell lysate (Fig. 2A), these results strongly suggest that the MED15 subunit of the Mediator complex is a direct target of NleG5-1.

To determine whether the N-terminal domain of NleGs mediate host target interactions, we tested the ability of the individual N- and C-terminal domains of NleG2-3 and NleG5-1 to bind their respective targets using Y2H. In this assay, yeast growth indicating direct binding was observed between the N-terminal domains of NleG2-3 with HK2 and NleG5-1 with MED15 (for details see SI Appendix, Supplementary Results and Fig. S5 A and B). Considering the NleG2-3 and NleG5-1 N-terminal domains mediate interactions with specific human protein targets, we then hypothesized that sequence variability in their N-terminal surface-exposed residues mediates the specificity of these interactions. A model of the N-terminal domain of NleG2-3 was constructed based on the crystal structure of NleG5-1 using MODELER (20) and used to select a panel of NleG2-3 N-terminal surface-exposed residues that are not conserved with NleG5-1, which were then probed for their role in HK2 interaction. These NleG2-3 residues were individually substituted with alanine and tested for their interaction with HK2 by Y2H in the NleG2-3I121K ubiquitination-deficient variant. The substitutions D30A, T32A, G35A, T37A, V41A, Y42A, S44A, L63A, and L64A resulted in a detectable decrease of Y2H growth, particularly when tested on the medium containing 3AT (SI Appendix, Fig. S5 C and D). Of these, Y42A, L63A, and L64A mutations had the most severe effect on Y2H signal. Since these residues are conserved in a subset of NleG effectors, but are not found in NleG5-1, we postulated that these residues may be part of a common target interaction interface in several NleGs, while the rest of the identified NleG2-3 residues are responsible for specificity of NleG2-3 toward HK2. The large number of NleG2-3 N-terminal residues that appeared to be involved in interaction with HK2 is indicative of a broad NleG2-3–HK2 interface. Intriguingly, the mutation H10A in NleG2-3 resulted in increased Y2H growth on selective medium, suggesting that this substitution may strengthen the NleG2-3 interactions with HK2.

Our results identify distinct host targets for two EHEC NleG effectors, revealing the functional diversification in this family and identify a number of N-terminal residues specific to NleG2-3 that are critical for HK2 interaction, supporting the hypothesis that sequence plasticity of this domain is responsible for host target specificity of NleG effectors.

NleG Effectors Bind and Induce Degradation of Their Targets in Human Cells.

To determine whether NleG2-3 and NleG5-1 affected 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 MED15 and HK2 were probed by immunoblotting. Cells expressing 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 NleG2-3I121K 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 NleG5-1V144A 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 target the human proteins MED15 and HK2 and induce their degradation in human cells.

Fig. 3.
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Fig. 3.

NleG5-1 and NleG2-3 bind and degrade host targets in human cells. (A) NleG2-3 and NleG5-1 bind and degrade their host targets following ectopic expression in HEK293T cells using the vector pcDNA3.1/nFLAG-DEST and their subsequent immunoprecipitation. (B) The indicated FLAG NleG2-3 and NleG5-1 constructs were expressed in EPEC O126:H7 ΔnleG and used to infect HeLa 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, and their indicated mutants were expressed in EPEC O126:H7 ΔnleG and used to infect HeLa cells in culture along with wild-type and secretion-deficient (ΔescN) EPEC and probed for target degradation. Immunoblotting of the targets reveals specific degradation by both NleG2-3 and NleG5-1 during infection. GAPDH is a loading control in A and C.

To further address individual roles of the NleG N- and C-terminal domains in interaction with host proteins, we constructed a chimeric NleG effector composed of the N-terminal binding domain of NleG5-1 (residues 1–112) and the U-box domain of NleG2-3 (residues 90–191), which we called NleG5-1chi. This chimeric effector was transiently expressed in HEK293T cells and the level of MED15 was measured by immunoblotting (SI Appendix, Fig. S5E). While NleG5-1chi did not degrade MED15 as efficiently as wild-type 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. Furthermore, the N-terminal fragment of NleG5-1 spanning residues 1–112 transiently expressed in HEK293T cells also coimmunoprecipitated with MED15 and not with HK2. In the reciprocal experiment, we constructed a chimeric effector composed of the N-terminal domain of NleG2-3 (residues 1–88) and the C-terminal U-box of NleG5-1 (residues 105–213), called NleG2-3chi, although we were not able to obtain substantial expression of NleG2-3chi or NleG2-3chi/V144K. However, the N-terminal fragment of NleG2-3 individually expressed in HEK293T cells coimmunoprecipitated with HK2 and not with MED15 (SI Appendix, Fig. S5E). Combined, these results show that the N-terminal domain in NleG2-3 and NleG5-1 are sufficient for specific interactions with human HK2 and MED15, respectively.

To test whether ubiquitination of the identified targets by NleG effectors triggers their degradation by the proteasome, we transiently expressed NleG2-3 or NleG5-1 in HEK293T cells and tested the effect of the proteasome inhibitor MG132 on the levels of HK2 and MED15. Treatment of cells with MG132 for 2 h resulted in restoration of HK2 levels in cells carrying NleG2-3WT, although the levels of HK2 slowly lowered after this point, potentially showing the rate of HK2 turnover (SI Appendix, Fig. S6A). Similarly, a 2-h treatment with MG132 restored the levels of MED15 in cells expressing NleG5-1WT (SI Appendix, Fig. S6B), together supporting our hypothesis that NleG-mediated ubiquitination directs their specific host protein targets for degradation by the proteasome. To confirm that NleGs are directly ubiquitinating their targets, we transiently expressed NleG5-1WT or NleG5-1V144K in combination with HA-tagged ubiquitin in HEK293T cells and treated these cells with MG132 for 6 h. Next, MED15 was immunoprecipitated under denaturing conditions (21) and probed for ubiquitinated MED15 species by immunoblotting using anti-HA antibodies. The amount of ubiquitinated MED15 was noticeably higher in cells expressing the NleG5-1WT relative to the cells expressing NleG5-1V144K variant and the vector control (SI Appendix, Fig. S6C), thus confirming that NleG5-1 directly ubiquitinates MED15 to trigger its degradation by the proteasome.

NleG5-1 and NleG2-3 Induce Specific Host Target Degradation During EPEC Infection.

The repertoire of NleG effectors varies between pathogenic E. coli, ranging from 14 members in EHEC O157:H7 to a single NleG (also known as NleI) in the EPEC strain E2348/69 (22, 23). We decided to take advantage of the low representation of NleGs in EPEC to test the individual function of NleG5-1 and NleG2-3 during infection. An EPEC ΔnleG strain was constructed and transformed with plasmids expressing FLAG-tagged NleG2-3, NleG5-1, or their ubiquitination-inactive variants. The secretion of NleG5-1 and NleG2-3 by the EPEC ΔnleG strain was confirmed by stimulating T3SS-dependent secretion in defined media (SI Appendix, Fig. S7 A and B) (24). Next, to test whether the identified interactions of NleG2-3 and NleG5-1 with host proteins were in line with their cellular localization during infection, we infected HeLa cells with EPEC expressing FLAG-tagged NleG2-3, NleG5-1, or empty vector. Consistent with its interaction with the nuclear Mediator complex, NleG5-1 predominantly localized to the nucleus, while NleG2-3 displayed nonspecific localization and was generally present in the cytosol, in line with the fact that HK2 is cytosolic and commonly localized to the surface of mitochondria (Fig. 3B) (14, 16, 25).

To determine whether NleG2-3 and NleG5-1 triggered degradation of their identified targets in the context of infection, we infected HeLa cells with EPEC strains expressing NleG2-3, NleG5-1, or empty vector, and monitored the levels of HK2 and MED15. The infection of HeLa cells by EPEC carrying wild-type NleG2-3, but not the inactive NleG2-3I121K variant, resulted in a decrease of HK2 levels (Fig. 3C). Unexpectedly, the level of SNAP29 was also reduced during infection with EPEC expressing NleG2-3. Similarly, infection by EPEC expressing NleG5-1WT led to complete degradation of MED15 (Fig. 3C). Additional probing for the levels of another Mediator subunit, MED6, showed no change between infections with EPEC expressing NleG5-1WT or NleG5-1V144K, indicating that NleG5-1–mediated degradation of MED15 does not lead to degradation of the entire Mediator complex. We did not observe any detectable changes in the level of MED15 for EPEC carrying NleG2-3, nor for HK2 or SNAP29 following infection by EPEC expressing NleG5-1. Next, we followed the infection of HeLa cells with EPEC expressing FLAG-tagged NleG2-3, NleG5-1, or a vector control with an anti-FLAG immunoprecipitation and immunoblotting for HK2, MED15, and SNAP29. We detected HK2 and MED15 coimmunoprecipitating with NleG2-3 and NleG5-1, respectively, consistent with our other results, suggesting that these are the direct targets of NleG2-3 and NleG5-1 during infection. This is in line with our hypothesis that NleG2-3 and NleG5-1 bind and ubiquitinate HK2 and MED15 during infection, respectively (SI Appendix, Fig. S7C). Taken together, our results demonstrate the EHEC effectors NleG2-3 and NleG5-1 are functionally diverse E3 ubiquitin ligases that are able to trigger the ubiquitination-mediated degradation of specific human proteins during infection.

Discussion

NleG effectors represent an expanded family that is part of the core arsenal of type III-secreted effectors in EHEC and EPEC. Our previous work established these effectors as E3 ubiquitin ligases with conserved C-terminal U-box domains (4). Phylogenetic analysis of the NleG family of effectors revealed conserved residues in the N-terminal domain, which, as revealed by our structural analysis, form part of the hydrophobic core in the NleG5-1 N-terminal domain. We further demonstrated that the N-terminal domain of NleG2-3 and NleG5-1 is required and sufficient for binding of specific host targets. This suggests that, while diverse in sequence, NleGs share a core N-terminal structural fold that has evolved to interact with distinct host targets via variation in the N-terminal domain. This hypothesis is supported by site-directed mutagenesis studies of the NleG2-3 N-terminal domain surface based on a model established using the NleG5-1 structures, with a number of surface-exposed residues revealed to be crucial for interaction between NleG2-3 and HK2, all of which are residues that are not conserved with NleG5-1. A number of these residues are conserved between members of the NleG2 family, which share 60% or greater overall identity in their primary sequence, suggesting that these effectors may share a common target recognition interface.

The two-domain architecture established for NleG effectors by our structural analysis is reminiscent of other characterized effector E3 protein ubiquitin ligases (26⇓–28). However, the NleG5-1 N-terminal domain fold bears no resemblance to domains found in other E3 ubiquitin ligases or other characterized proteins in general. The expansion of the NleG family in pathogenic E. coli strains such as EHEC O157:H7 through apparent diversification of the N-terminal region suggests that this domain is a versatile interface for host protein recognition. Comparing against an activated E3–E2∼ubiquitin complex, RNF4–UbcH5a∼Ub (13), shows the positioning of the NleG5-1 N-terminal domain is consistent with the N-terminal domain’s role of bringing the substrate into contact with the eukaryotic ubiquitination machinery. However, the true conformation adopted by NleG5-1 during target ubiquitination remains to be determined through further structural studies.

We identified distinct human proteins targeted for degradation by two EHEC NleGs with divergent N-terminal domains, NleG2-3 and NleG5-1. HK2, a target of NleG2-3, is a major metabolic enzyme that converts glucose to glucose-6-phosphate, the initial step of glucose metabolism. In addition, HK2 carries apoptosis-preventing cellular functions, whereby consistent binding of HK2 to mitochondria is crucial to inhibit cytochrome C release and subsequent apoptosis (16, 17, 29). Both the metabolic and apoptotic roles of HK2 are implicated in E. coli infection. HK2 is up-regulated in host cells in the presence of uropathogenic E. coli, possibly to support the energy demands of fighting infection (30), while other effectors have been shown to manipulate mitochondrial apoptosis, in particular EspF, which induces cytochrome C release from mitochondria and causes apoptosis (31). Interestingly, we found that expression of NleG2-3 in host cells also leads to degradation of SNAP29, a SNARE protein involved in vesicle fusion and transport (18). Although SNAP29 coprecipitated with purified NleG2-3 in AP experiments we were not able to demonstrate a direct interaction between these two proteins using Y2H or immunoprecipitation. It is therefore tempting to suggest that NleG2-3 triggering the degradation of SNAP29 is indicative of an interaction between SNAP29 and HK2, the primary identified target of NleG2-3, or the presence of additional targets for this effector, although the functional implications of this interaction remain to be determined.

NleG5-1 targets the Mediator complex member MED15, which functions as an end point for many signaling pathways, with a number of transcription factors (TFs) mediating transcription of their target genes through interaction with specific Mediator subunits (15, 32). Although NleG5-1 does not possess an identifiable nuclear localization signal, it is able to localize to the nucleus and target MED15. This may be due to the passive diffusion of NleG5-1 into the nucleus as its size of ∼24 kDa is well under the presumed nuclear pore diffusion limit of at least 60 kDa (33). MED15 is specifically involved in integration of various transcriptional signals, including TGF-β and SREBP1 signaling (15, 32, 34). Intriguingly, TGF-β is implicated in E. coli infection, as TGF-β treatment of epithelial cells has been shown to inhibit the ability of EHEC to disrupt tight junctions, and elevated TGF-β levels are associated with reduced incidence of hemolytic uremic syndrome induced by EHEC O157:H7 (35, 36). The potential consequence of impaired SREBP1 signaling, which is involved in responding to a sensor of intracellular cholesterol and regulating of lipid homeostasis (37, 38) in the context of E. coli infection has not been addressed so far.

The infection strategy of attaching and effacing pathogens is dependent on a specific arsenal of virulence factors that manipulate and control host cells. Thus, it is plausible that the dramatic expansion of the NleG effectors in EHEC species is a direct contributor to the high virulence of these bacteria in humans or efficient asymptomatic colonization of cattle, their natural reservoir. Similarly, the human-specific bacterial pathogen Shigella also has a large arsenal of sequence-related yet functionally diverse E3 ubiquitin ligase effectors, the IpaHs (26, 27, 39). Functionally similar to NleGs but structurally distinct, the IpaH effectors contain a conserved C-terminal catalytic E3 ligase domain and a variable N-terminal leucine rich repeat (LRR) domain that enables them to target a variety of discrete human proteins for degradation (39). This is indicative of the expanded arsenal of E3 ubiquitin ligase effectors, representing a versatile evolutionary mechanism that allows bacterial pathogens to efficiently adapt to a specific host niche. Our current work provides insight into the broad range of host proteins targeted by EHEC NleG effectors and contributes to the diversity of host cell processes targeted during bacterial infection.

Materials and Methods

Please refer to SI Appendix, Supplementary Materials and Methods for full extensive details on cloning, protein expression, AP-MS, Y2H, structural determination, cell culture, transfection experiments, EPEC infection experiments, immunofluorescence, generation of EPEC mutants, and T3SS translocation assays.

Acknowledgments

We thank Zdzislaw Wawrzak for assistance in X-ray data collection for the crystal structure of NleG5-1 and Joan and Ron Conaway for their gift of HeLa cell lines expressing FLAG-tagged MED26 and CDK8. The structural information was obtained as part of the Center for Structural Genomics of Infectious Diseases (https://csgid.org/). This project has been funded in whole or in part by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract nos. HHSN272201200026C and HHSN272201700060C. The C.H.A. NMR facility is generously supported with operating funds provided by the Princess Margaret Cancer Centre. The functional data were obtained using a Canadian Institutes of Health Research (CIHR) operating grant to A.S. (principal investigator) and B.K.C. (coprincipal investigator). B.K.C. is supported by a Canada Research Chair in Infectious Disease Pathogenesis. D.J.L. has been supported in part by Michael G. DeGroote and CIHR fellowships.

Footnotes

  • ↵1To whom correspondence should be addressed. Email: alexei.savchenko{at}ucalgary.ca.
  • Author contributions: D.V., D.J.L., C.H.A., B.K.C., and A.S. designed research; D.V., D.J.L., T.S., A.T.Q., R.D.L., and S.H. performed research; R.D.L. contributed new reagents/analytic 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 A.S. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The solution NMR and crystal structures for NleG5-1 have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 6B3N and 5VGC).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1718350115/-/DCSupplemental.

Published under the PNAS license.

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Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli
Dylan Valleau, Dustin J. Little, Dominika Borek, Tatiana Skarina, Andrew T. Quaile, Rosa Di Leo, Scott Houliston, Alexander Lemak, Cheryl H. Arrowsmith, Brian K. Coombes, Alexei Savchenko
Proceedings of the National Academy of Sciences Oct 2018, 115 (40) 10004-10009; DOI: 10.1073/pnas.1718350115

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Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli
Dylan Valleau, Dustin J. Little, Dominika Borek, Tatiana Skarina, Andrew T. Quaile, Rosa Di Leo, Scott Houliston, Alexander Lemak, Cheryl H. Arrowsmith, Brian K. Coombes, Alexei Savchenko
Proceedings of the National Academy of Sciences Oct 2018, 115 (40) 10004-10009; DOI: 10.1073/pnas.1718350115
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