title: The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation.
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.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana, United States of America.
authors.forename: Heather
authors.initials: H
authors.lastname: Piscatelli
authors.forename: Shalaka A
authors.initials: SA
authors.lastname: Kotkar
authors.forename: Megan E
authors.initials: ME
authors.lastname: McBee
authors.forename: Sureshkumar
authors.initials: S
authors.lastname: Muthupalani
authors.forename: David B
authors.initials: DB
authors.lastname: Schauer
authors.forename: Robert E
authors.initials: RE
authors.lastname: Mandrell
authors.forename: John M
authors.initials: JM
authors.lastname: Leong
authors.forename: Daoguo
authors.initials: D
authors.lastname: Zhou
mesh_terms: D000818:Animals; D022081:Cell Surface Extensions / Q000378:metabolism*; D044082:Citrobacter rodentium / Q000472:pathogenicity; D015536:Down-Regulation; D019453:Escherichia coli O157 / Q000201:enzymology* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination; D014774:Virulence
chemical_list: D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; C555414:NleL protein, E coli; D044767:Ubiquitin-Protein Ligases