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 diversifi- cation 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