NleL Modulates Pedestal Formation Figure 1. Self-ubiquitination of GST-NleL. (A) NleL-mediated self-ubiquitination requires ATP, ubiquitin, E1 and E2. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL59–782 were incubated at 35uC for 90 min, and the Western blot was performed using polyclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom). (B) NleL C753 residue is required for its E3 ubiquitin ligase activity. Reactions containing ubiquitin, E1 and UbcH5a were incubated with the wild type GST-NleL, GST-NleLC688S or GST-NleLC753S or GST-NleLC753A at 35uC for 90 min. The Western blot was performed using monoclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom). doi:10.1371/journal.pone.0019331.g001 type EPEC or EPEC expressing the catalytically-inactive NleLC753A. For example, the percentage of cells with no pedestals was ,10-fold higher when wild type NleL was delivered. This result suggests that rather than diminish a process specific to Nck or EspFU the E3 ligase activity of NleL functions to down modulate a common step of pedestal formation by EPEC and EHEC. C. rodentium NleL is an E3 ligase and is essential for C. rodentium virulence in mice The efficiency of pedestal formation may influence the bacterial colonization and/or degree of inflammation during natural infection. C. rodentium has been used as a model microorganism to assess the virulence of the A/E pathogens in the mouse infection model. To evaluate whether NleL plays a role in an animal model, we first determined if C. rodentium NleL has E3 ligase activity in an in vitro ubiquitination assay using the in vitro auto-ubiquitination assay. GST-NleL59–782 was added as the potential E3 ligase. Western blotting with either GST or ubiquitin antiserum shows the presence of mono and poly-ubiquitinated C. rodentium NleL (Fig. 5A). No ubiquitination of C. rodentium NleL was observed when GST- NleLC753S was used or in the absence of E1 or UbcH5a indicating that each of these components of the ubiquitination reaction is required for the ubiquitination of NleL. This indicates that C. rodentium NleL is an E3 ubiquitin ligase and C753 is essential for the ligase activity. We next determined if C. rodentium NleL plays a role in C. rodentium virulence in a murine infection model. C. rodentium infection in C57BL/6 mice is characterized by loose stool progressing to diarrhea in severe cases, poor overall body condition, and weight loss, which can be fatal particularly in young animals [50,51]. Colonic lesions consist of epithelial hyperplasia, submucosal edema, mucosal erosion and ulceration, and inflam- matory infiltration varying from submucosal to transmural [50,52]. As shown in Fig. 5B, fecal bacteria shedding of the wild-type strain (DBS130) of C. rodentium reached a maximum at 6 days post-inoculation (DPI) of 66108 CFU/g of feces in agreement with previous reports of C. rodentium infection of C57BL/6J mice [52,53,54]. Fecal shedding of the C. rodentium nleL mutant strain (DBS792), the nleL mutant strain expressing the wild-type C. rodentium NleL (DBS793), or the nleL mutant strain complemented with the catalytically-dead NleLC753S (DBS794) were comparable to the wild-type C. rodentium by 6 DPI and remained comparable until sacrifice at 13 DPI (Fig. 5B). In spite of the observation that NleL had no significant effect on colonization, this effector promoted disease. By 12 DPI uninoc- ulated mice had gained 8% of their initial body weight, whereas mice inoculated with either the wild-type or nleL complemented strain had significant weight loss (8% and 3% of initial weight by 12 DPI, P,0.001, Fig. 5C). In contrast, mice inoculated with the nleL mutant or the non-complementing point mutant NleLC753S gained weight (5% and 4%, respectively) and were not significantly different from uninoculated mice throughout the study. In addition, infection with wild-type C. rodentium resulted in robust colitis with multifocal erosions, infiltration of neutrophils, lym- phocytes and macrophages, hyperplasia, and herniation of glands into local lymphoid tissue. At 13 DPI the histologic colitis index was comparable between mice inoculated with either wild-type strain (median index of 13.25 [13.0 to 14.0]) or nleL-complement- ed strain (10.0 [7.0 to 16.5]) of C. rodentium (Fig. 5D). Deletion of nleL from C. rodentium resulted in reduced lesion severity and colitis in mice (8.5 [8.0–10.0]) compared to wild-type inoculated mice (P,0.05). Similarly, inoculation with the non-complementing NleLC753S point mutant resulted in less severe colitis than wild- type C. rodentium (P,0.01) with a histologic colitis index of 7.0 (5.0– 10.5) that was statistically indistinguishable from the nleL mutant. Discussion EHEC and EPEC are members of the Attaching and Effacing pathogen group of organisms that cause serious food- and water- borne illnesses in humans. A pathogenic infection by EHEC or EPEC involves attachment of the pathogen to the surface of the host cell followed by translocation of key effector proteins into the cytoplasm via the TTSS. Although the molecular events that follow differ somewhat between EHEC and EPEC, they result in the rearrange- ments of host cytoskeletal machinery, which in turn leads to the formation of morphologically similar pedestals, a hallmark of an A/E pathogenic infection. Although a considerable amount of research on PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e19331