The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation Heather Piscatelli1, Shalaka A. Kotkar1, Megan E. McBee2¤, Sureshkumar Muthupalani2, David B. Schauer2, Robert E. Mandrell3, John M. Leong 4 , Daoguo Zhou 1 * 1 Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America, 2 Department of Biological Engineering and Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3 Agricultural Research Service, United States Department of Agriculture, Albany, California, United States of America, 4 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America Abstract Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation. Citation: Piscatelli H, Kotkar SA, McBee ME, Muthupalani S, Schauer DB, et al. (2011) The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation. PLoS ONE 6(4): e19331. doi:10.1371/journal.pone.0019331 Editor: F. Gisou van der Goot, Ecole Polytechnique Federale de Lausanne, Switzerland Received December 9, 2010; Accepted March 27, 2011; Published April 26, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: Research was supported by National Institutes of Health (NIH) grants AI078092 and AI068655 to D.Z. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: zhoud@purdue.edu ¤ Current address: Department of Pathology, The University of Chicago, Chicago, Illinois, United States of America Introduction Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of food- and water-borne illnesses in developed countries and in the world. EHEC O157:H7 infections cause hemorrhagic colitis and can result in potentially fatal hemolytic uremia syndrome [1,2,3]. EHEC along with Enteropathogenic E. coli (EPEC) and Citrobacter rodentium form a group of pathogens called A/E pathogens that are able to colonize the intestinal mucosa and produce characteristic ‘attaching and effacing’ (A/E) lesions. A/E lesions are characterized by effacement of the brush border microvilli, intimate attachment of the bacterium to the plasma membrane of the enterocytes, and the formation of actin- rich pedestals within the host cell beneath the adherent bacteria [4,5,6,7]. Pedestal formation requires virulence-related EHEC proteins to be injected directly into the host cell through a type III secretion system (TTSS) [8]. Two EHEC O157:H7 type III secreted effectors, Tir and EspFu/TccP, are known to be required for pedestal formation [9,10,11]. EspM was shown to inhibit pedestal formation via an unknown mechanism [12,13]. Tir is a bacteria-made receptor that binds bacterial surface protein intimin to facilitate the pedestal formation [14,15,16]. Binding to intimin is followed by coordinated events that lead to rearrangement and/or assembly of actin to form pedestals on the host cell surface. The N-terminus of the Tir domain binds several host focal adhesion proteins, including a-actinin, talin and vinculin, and cortactin to which may promote actin networks that support the pedestal [17,18]. EHEC and EPEC Tir C-termini appear to be modified by serine/threonine phosphorylation upon entry into the host cell [19,20]. In addition, the C-terminus of EPEC Tir is tyrosine phosphorylated, leading to the recruitment of the host adaptor protein Nck, which in turn stimulates actin assembly [21,22]. In contrast, EHEC Tir is not tyrosine phosphorylated and does not recruit Nck. Instead, EHEC translocates a second effector, EspFU/TccP, which co-localizes with Tir at the site of EHEC attachment. EspFU activates N- WASP by binding to its CDC42-binding domain [9,10,23]. Ubiquitination is a reversible posttranslational modification of cellular proteins, in which a 76 amino acid polypeptide, ubiquitin, is attached to the e-amino group of lysines in target proteins. Ubiquitination is a major player in regulating a broad range of cellular processes, including cell division, differentiation, signal transduction, protein trafficking, and quality control [24,25]. Ubiquitination involves a multienzyme cascade consisting of classes of enzymes known as ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin protein ligases (E3). The E3 ubiquitin ligases play pivotal roles in defining the specificity of target proteins to be ubiquitinated. Most E3 ubiquitin PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e19331