Figure 2. Secretion of Tir and translocation of NleL into HeLa cells. (A) The expression and secretion levels of Tir in either the wild- type EHEC or the mutant EHEC expressing the chromosomal catalytically-dead mutant NleLC753A was examined by Western blot. (B) Intracellular cAMP levels are an indication of the translocation of CyaA fusion proteins in EHEC WT (ZP250) and EHEC TTSS deficient mutant (escF; ZP2251) strains. Cells were infected for 4 hrs, and the adenylate cyclase activity was determined. The data were from three independent experiments, with standard deviations shown as error bars. cAMP values are presented as pmol per milligram of total cellular protein. The expression levels of the NleL-CyaA fusions were found to be similar by Western blot as shown on the bottom panel. doi:10.1371/journal.pone.0019331.g002 the mechanism of pedestal formation has been carried out, little is known about how this process is regulated. We demonstrated here that EHEC utilize the host ubiquitination pathway to down-regulate the pedestal formation, perhaps to maintain a balance to co-exist with the host cells. Thus, the bacteria is involved actively not only in the formation, but also the modulation of pedestal formation. Consistent with the hypothesis that unregulated pedestal formation may alter disease, we demonstrate the importance of NleL and its E3 ubiquitin ligase activity in development of clinical and pathological disease caused by C. rodentium. EHEC NleL shares sequence similarity to Salmonella SopA mainly toward the C-terminus where the E3 ligase activity lies [28,30]. A recent study found that both NleL and SopA form the bilobed catalytic domain reminiscent of the N- and C-lobe architecture of HECT E3 ligases [45]. Although both NleL and SopA possess poly- ubiquitination activities in vitro, NleL form free (unanchored) poly- ubiquitin chains when the GST-free NleL is used in the ubiquitination assay [45]. Salmonella is able to invade the host cells and survive inside the host cells. In contrary, EHEC exerts its virulence by adhering to the surface of the host cells via actin-rich NleL Modulates Pedestal Formation pedestals. It is not clear whether the different biochemical E3 ligase activities contribute to the different life-style of the two intestinal pathogens. We speculate that NleL may exploit the free ubiquitin pools in the host cell to exert its function without the canonical substrates. Further studies are needed to examine this possibility. Our data demonstrate that loss of the NleL E3 ligase activity leads to increased pedestal formation. Preliminary work showed that the loss of the NleL E3 ligase activity did not alter the expression of Tir or EspFu (Fig. 2A and data not shown), suggesting that NleL may exert its function after their transloca- tion. EPEC infection has been shown to lead to pedestal formation much more efficiently than that of EHEC in vitro [55]. A significant difference between the two pathogenic types lies in the nature of the involvement of Tir during pedestal formation. While TirEPEC is tyrosine phosphorylated upon entry into the host cell and interacts with host adaptor protein Nck, which stimulates Arp2/3- mediated actin polymerization [49,56], TirEHEC lacks the homologous tyr residue, Y474, and depends on another effector EspFu/TccP to induce actin polymerization [9,10,23]. Such studies highlight the differences in the mechanism of pedestal formation between the two organisms. Our data indicate that NleL probably functions at steps common to EPEC and EHEC- mediated pedestal formation. It is reasonable to speculate that NleL ubiquitinates an unknown factor of bacterial or host origin involved in pedestal formation. One such scenario would be that host cell proteins involved in pedestal formation are ubiquitinated by NleL which, in turn, corresponds to a decrease in the level of host proteins or alters their localization, resulting in a decrease in pedestal formation. Alternatively, NleL may ubiquitinate Tir and promote its endocytosis, thus decreasing the surface availability of Tir. Our preliminary studies have shown that Tir is not a substrate for the E3 ligase activity of NleL in an in vitro ubiquitination assay (data not shown). However, we cannot rule out that a third factor is required, but absent, in our in vitro reaction. A recent structural study showed that another non-LEE-encoded effector, NleG, is a RING finger ubiquitin E3 ligase [57]. It is tempting to speculate that NleL, NleG and Tir may work together to modulate the actin pedestal formation. In addition, Tir is inserted into the plasma membrane of the host cell in a hairpin loop structure. It is also possible that NleL is affecting Tir localization. Further studies are required to dissect the exact molecular mechanism on how the E3 ligase activity of NleL modulates the pedestal formation. Materials and Methods Bacterial strains, plasmids and mammalian cell lines The Enterohemorrhagic strain designated E. coli O157:H7 (ZP250), a spontaneous nalidixic acid mutant of an outbreak strain isolated from apple juice (RM1484) [58,59]. Using ZP250 as a parent, strains harboring in-frame chromosomal deletions of genes escF (ZP251) were generated with the help of an allelic-exchange suicide vector as described previously [60]. The EHEC strain expressing mutant NleLC753A (ZP254) was generated similarly using a plasmid encoding the NleLC753A (pZP2111). C. rodentium nleL deletion strain was similarly generated. Complementing strains were created by introducing the plasmid expressing the wild-type C. rodentium NleL (pZP1661) or the catalytically-dead NleLC753S (pZP1664) into DBS792 generating DBS793 and DBS794 respec- tively. The EHEC nleL-M45 complementation plasmid (pZP1666) was constructed by cloning DNA fragments encoding full length NleL and its promoter sequence (250 bp upstream of the translation start site) into pSB1136, a pBAD derivative [61]. The nleL translocation plasmid (pZP1671) was subcloned by ligating nleL DNA sequences from the complementation plasmid with appropri- PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e19331