The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation

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.National Institutes of Health (U.S.) (grant AI078092)National Institutes of Health (U.S.) (grant AI068655

Molecular mechanisms of type III secretion effector NleL in enterohemorrhagic Escherichia coli O157:H7 pathogenesis

EHEC, a member of the attaching and effacing (A/E) pathogen group of bacteria, causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome. Effective infection of host intestinal epithelial cells by EHEC is a result of coordinated interactions of bacterial effectors and/or host cellular proteins. Several key effectors involved in the process of infection have been identified yet certain aspects of the pathogenicity of EHEC remain unclear. While we understand much of the molecular dynamics of cytoskeletal rearrangements, we still lack comprehensive knowledge of all bacterial effectors involved in pedestal formation. Therefore, identification and functional characterization of novel effectors is important for understanding the mechanism of pedestal formation during EHEC infection and A/E pathogenesis in general. We report discovery of a novel effector protein called Non-LEE encoding Ligase (NleL) encoded by the ECs1560 locus of EHEC. In vitro, we show that NleL translocates into the host cell via the TTSS and is an E3 ubiquitin ligase with the C753 residue being critical for its activity. While E3 ubiquitin ligases have been reported from intracellular animal pathogens like Salmonella and Shigella, NleL is the first E3 ubiquitin ligase identified from an extracellular animal pathogen. Functionally, we show that not only is NleL required but it is its E3 ubiquitin ligase activity that is essential for pedestal formation under conditions of sub optimal expression of TTSS effectors. Furthermore, we report that NleL specifically interacts with another EHEC effector Tir and a host cell derived E3 ubiquitin ligase HsRma1. However, the exact nature and role of these interactions still remains unclear. More significantly, we have discovered a homolog of NleL from Citrobacter rodentium. We show that NleLc is a bona fide E3 ubiquitin ligase and that function of NleLc is required to carry out efficient infection of murine colonic epithelial cells In vivo. Ongoing research focuses on characterization of molecular interactions of NleL with Tir and HsRma1 and identification of other putative binding partners of NleL

Contribution of <i>nleL</i> to <i>C. rodentium</i> virulence.

<p>(<b>A</b>) <i>C. rodentium</i> NleL is an E3 ubiquitin ligase. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL or GST-NleL^C753S were incubated at 35°C for 90 min, and the Western blot was performed using polyclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom). (<b>B)</b> Fecal bacteria shedding of the wild-type strain (DBS130), the <i>nleL</i> null mutant strain, the <i>nleL</i> mutant strain expressing the wild-type NleL, or the <i>nleL</i> mutant strain complemented with the catalytically-dead NleL^C753S in <i>C. rodentium</i>. (<b>C</b>) Virulence of <i>C. rodentium</i> strains as indicated by weight of the mice post-inoculation. The percent weight change of the mice over the 13 days post-inoculation was measured. The error bars indicate standard errors. Mice infected with <i>nleL</i> deletion strain (DBS792) had significantly reduced weight loss when compared to the wild type (DBS130). This could be partially rescued by plasmid expressing the full length NleL (DBS793) but not by plasmid expressing the catalytically inactive NleL^C753S mutant (DBS794). (<b>D</b>) Contribution of <i>nleL</i> to mouse colitis caused by <i>C. rodentium</i>. Pathology of mouse colon by different <i>C. rodentium</i> strains, as indicated by histologic activity index (sum of lesion scores). At day 13 post-inoculation colon tissue was scored for lesions: inflammation, edema, epithelial defects, crypt atrophy, hyperplasia, and dysplasia. Mice infected with <i>nleL</i> deletion strain showed significantly reduced lesions when compared to the wild type (P<0.05). This could be rescued by plasmid expressing the full length NleL but not by plasmid expressing the catalytically inactive NleL^C753S mutant (P<0.01).</p

Self-ubiquitination of GST-NleL.

<p>(<b>A</b>) NleL-mediated self-ubiquitination requires ATP, ubiquitin, E1 and E2. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL^59–782 were incubated at 35°C for 90 min, and the Western blot was performed using polyclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom). (<b>B</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-NleL^C688S or GST-NleL^C753S or GST-NleL^C753A at 35°C for 90 min. The Western blot was performed using monoclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom).</p

Secretion of Tir and translocation of NleL into HeLa cells.

<p>(<b>A</b>) The expression and secretion levels of Tir in either the wild-type EHEC or the mutant EHEC expressing the chromosomal catalytically-dead mutant NleL^C753A was examined by Western blot. (<b>B</b>) Intracellular cAMP levels are an indication of the translocation of CyaA fusion proteins in EHEC WT (ZP250) and EHEC TTSS deficient mutant (<i>escF</i>; 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.</p

The E3 ligase activity of EHEC NleL down modulates EPEC pedestal formation.

<p>HeLa cells were infected at a multiplicity of infection of 100 with wild type EPEC, or EPEC harboring plasmid expressing wild-type EHEC NleL or NleL^C753A. Cells were infected for 4 hours. (<b>A</b>) Bacteria were visualized by staining with an anti-EPEC LPS antibody (green). Actin was detected with a Texas Red Phalloidin (red). (<b>B</b>) The number of micro-clusters of pedestals on HeLa cells were counted and were grouped into clusters having 1–10 pedestals, 11–20 pedestals and >20 pedestals. Quantitative analysis includes three independent experiments. A minimum of 300 cells were counted from each experiment with standard deviation shown as error bars.</p