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

Heather Piscatelli,Megan E Mcbee,Sureshkumar Muthupalani,Robert E Mandrell,Daoguo Zhou,David B Schauer,Shalaka A Kotkar,John M Leong,F Gisou Van Der Goot

doi:10.1371/journal.pone.0019331

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.

Highlights

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of food- and water-borne illnesses in developed countries and in the world
Autoubiquitination is often used in the absence of a physiological substrate to measure the ubiquitin E3 ligase activity, we first tested if glutathione S-transferase (GST)-Non-LEE-encoded Ligase (NleL) has the E3 ligase activity in an in vitro autoubiquitination assay using E1, E2 (UbcH5a), ATP, and ubiquitin in the presence of purified recombinant GST-NleL59–782
A pathogenic infection by EHEC or Enteropathogenic E. coli (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 type III secretion system (TTSS)

Summary

Introduction

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of food- and water-borne illnesses in developed countries and in the world. A/E lesions are characterized by effacement of the brush border microvilli, intimate attachment of the bacterium to the plasma membrane of the enterocytes, and the formation of actinrich pedestals within the host cell beneath the adherent bacteria [4,5,6,7]. Pedestal formation requires virulence-related EHEC proteins to be injected directly into the host cell through a type III secretion system (TTSS) [8]. Two EHEC O157:H7 type III secreted effectors, Tir and EspFu/TccP, are known to be required for pedestal formation [9,10,11]. EspM was shown to inhibit pedestal formation via an unknown mechanism [12,13]

Methods

Results

Conclusion