ligases belong to two families: one contains the HECT domain and the other possesses the RING finger domain [24]. The HECT family E3 ubiquitin ligases contain a 350-residue region that maintains a strictly conserved cysteine residue that is located approximately 35 residues from the C-terminus [26,27]. Many pathogenic microbes have developed means to interfere with different stages of ubiquitin pathways to promote their survival and replication. These include SopA, SlrP, SspH1, and SspH2 from Salmonella [28,29,30,31,32]; AvrPtoB from Pseudomonas syringae [33,34,35]; IpaH 9.8 and IpaH3 from Shigella flexneri [31,36,37]; and LubX from Legionella pneumophila [38]. More than 60 putative EHEC type III effectors have been proposed using the proteomic approach [39] and bioinformatic studies [40]. Previous studies have suggested that the EHEC EspX7 (ECs1560) as an type III effector [40,41,42]. EHEC EspX7 is predicted to encode a 782 amino acid peptide and is found on prophage Sp6 [43]. EspX7 homolog is found in the closely related A/E pathogen, C. rodentium [44] but not in EPEC E2348/69. We report here that EspX7 is an E3 ubiquitin ligase with C753 being critical for its ligase activity. This E3 ligase activity plays an important role in down-modulating the pedestal formation. We have renamed EspX7 as NleL (Non-Lee-Encoded effector Ligase) to reflect its novel biochemical activity. Results Self-ubiquitination of GST-NleL A study using a bioinformatic approach and validation with various translocation assays has identified multiple translocated effectors in EHEC including the NleL (ECs1560) locus [40]. A sequence comparison and structural studies have identified NleL as a bacterial HECT-like E3 ubiquitin ligase [28,30,45]. The substrate and biological function of NleL remains unknown. Auto- ubiquitination is often used in the absence of a physiological substrate to measure the ubiquitin E3 ligase activity, we first tested if GST-NleL has the E3 ligase activity in an in vitro auto- ubiquitination assay using E1, E2 (UbcH5a), ATP, and ubiquitin in the presence of purified recombinant GST-NleL59–782. Consistent with our previous work [45], poly-ubiquitinated GST-NleL was observed by Western blot when E1, E2 or ubiquitin were added to the reaction (Fig. 1A). No ubiquitination of GST-NleL was detected in the absence of E1, E2 or ubiquitin, indicating that each of these components were essential for GST- NleL ubiquitination. Furthermore, the mutant GST-NleLC753S (pZP1658) or the GST-NleLC753A (pZP2129) failed to form the poly-ubiquitination pattern seen with the wild-type GST-NleL in the in vitro ubiquitination assay (Fig. 1B). To assess the specificity of the cysteine mutation, a substitution mutant targeting C688 (C688S, pZP1657) of NleL was used in the same ubiquitination assay. Western blot analysis shows presence of mono- and poly- ubiquitinated GST-NleLC688S species similar to those observed when the wild-type GST-NleL was used (Fig. 1B). Mutant NleLC753A is translocated at similar efficiency as the wild-type NleL into mammalian host cells To test whether NleLC753A mutant affected the secretion of other type III effectors, we examined the secretion of Tir, a key player in EHEC-mediated actin pedestal formation upon infection. The expression and secretion levels of Tir in EHEC expressing the chromosomal catalytically-dead mutant NleLC753A (ZP254) was found to be similar to that of the wild type EHEC (Fig. 2A). Previous studies have shown that NleL (EspX7) might be a type III EHEC effector [40,41,42]. We further tested whether NleL was translocated using an adenylate cyclase (CyaA) reporter NleL Modulates Pedestal Formation system [46]. The CyaA from B. pertussis requires host cell derived calmodulin for its activity, hence fusion proteins of CyaA only with proteins that translocate into the host cell have catalytic activity which can be examined by measuring cAMP levels. We compared cAMP levels from HeLa cells infected with either the wild-type strain (ZP250) or a TTSS-deficient EHEC strain (escF, ZP251), both harboring the reporter plasmid expressing NleL-CyaA in- frame fusion (pZP1671). Cells infected with the wild-type EHEC strain show approximately 400-fold higher cAMP levels as compared to those infected with the TTSS-deficient strain (Fig. 2B). These results confirmed that NleL is indeed an effector protein translocated via the EHEC O157:H7 type III-secretion system. Similarly, we tested the translocation of NleLC753A using the cyaA reporter gene assay. HeLa cells were infected with wild-type EHEC harboring the plasmid encoding either the wild type NleL- CyaA, or the NleLC753A-CyaA fusion protein. We found that the cAMP levels were similar to those detected in cells infected with Salmonella harboring the wild-type NleL. This indicates that the C753A mutation did not significantly alter the translocation of NleL (Fig. 2). Expression of the wild-type and the C753A mutant NleL-CyaA fusion proteins in bacterial whole cell lysates was further confirmed to be at similar levels by Western analysis (Fig. 2, lower panel). E3 ligase activity of NleL down-regulates the EHEC pedestal formation A central function of the EHEC translocated effectors is to promote pedestal formation, which is a hallmark of A/E pathogens [47]. Pedestals are actin-rich structures formed as a direct result of effectors that bring about cytoskeletal rearrange- ments upon translocation by the EHEC TTSS [23]. Two EHEC effectors, Tir and EspFu are sufficient to induce the formation of pedestals [9,10,17,48,49]. To test whether the ligase activity of NleL is involved in pedestal formation, an EHEC strain expressing the chromosomal catalytically-dead mutant NleLC753A was used to infect HeLa cells to examine its ability to form pedestals. The number of pedestals formed in HeLa cells infected with ZP254 was increased significantly as compared to those in cells infected with the wild-type ZP250 (Fig. 3). For example, the percentage of cells with no pedestals decreased two-fold, and the percentage with more than 10 pedestals increased more than two-fold. The phenotype of ZP254 infected HeLa cells could be rescued by introducing wild-type NleL expressing plasmid (pZP1666) into ZP254. Together, these data indicate that the E3 ligase activity of NleL plays a major role in down-regulating pedestal formation during EHEC infection. The E3 ligase activity of EHEC NleL down modulate EPEC pedestal formation A genome-wide search of the A/E pathogens revealed that EPEC strains do not harbor the nleL gene. We showed that the E3 ligase activity of NleL down modulates the pedestal formation during EHEC infection. Unlike EHEC, canonical EPEC strains do not encode EspFU, and generate pedestals by recruiting the host adaptor Nck after phosphorylation of a tyrosine residue in the Tir C-terminus, one that is lacking in EHEC Tir [21,22]. To determine if, in spite of this mechanistic difference, EHEC NleL also acts to diminish pedestal formation during EPEC infection, we introduced EHEC NleL into the wild-type EPEC strain and assessed their ability to form pedestals. As shown in Fig. 4, an EPEC strain expressing the wild type EHEC NleL induced significantly less pedestals compared to that induced by the wild- PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e19331