Immunofluorescence microscopy HeLa cells infected for 3 hours were maintained in fresh media for an additional 3 hours. Cells were washed with cold PBS, fixed in 3% formaldehyde and permeabilized with 0.1% Triton X-100 [4]. Cells were incubated with either rabbit anti-O antibody (EHEC O157, Difco Laboratories, Detroit, MI; EPEC O111, Denka Seiken Co., Tokyo, Japan) as primary and Alexa Flour 488 conjugated anti-rabbit (1:300 dilution) secondary antibody or Texas-red Phalloidin (1:300; Molecular Probes, Carlsbad, CA). All images represent black and white projections of z-section slices obtained on a Zeiss LSM 700 confocal microscope. Approximately 300 cells were counted from each infection. Based on the number of pedestals per cluster, the clusters were divided into groups; extra large cluster (.20 pedestals/cluster for EPEC), large clusters (.10 pedestals/cluster), medium clusters (6–10 pedestals/cluster) and small clusters (1–5 pedestals/cluster). In vitro ubiquitination assay In vitro ubiquitination experiments were carried out as described [64]. Briefly, a reaction mixture containing 40 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM ATP, 2 mM dithiothreitol, 300 ng/ ml ubiquitin [65], 0.1 mM E1 activating enzyme, 0.5 mM UbcH5a, E2 ubiquitin-conjugating enzyme (Boston Biochem, Boston, MA), 2 mg GST-NleL, GST-NleLC688S, GST-NleLC753S, or GST- NleLC753A were incubated at 35uC for 90 min and subjected to SDS-PAGE (8%) and Western blot analysis using anti-GST or anti-MBP antibodies (New England Biolabs, Ipswich, MA). A similar in vitro ubiquitination assay was performed for each of the E2 ubiquitin-conjugating enzymes of interest: UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7 and UbcH10 (Boston Biochem, Boston, MA). Mice Infection Female 5-week old C57BL/6J (The Jackson Laboratory, Bar Harbor, Maine) mice were fed a rodent diet and water ad libitum and housed in micro-isolator cages that were maintained specific- pathogen-free of known murine bacterial, viral and parasitic infections including all known Helicobacter spp. in facilities at MIT approved by the Association for Assessment and Accreditation of References 1. Garmendia J, Frankel G, Crepin VF (2005) Enteropathogenic and enterohe- morrhagic Escherichia coli infections: translocation, translocation, translocation. Infect Immun 73: 2573–2585. 2. Kaper JB, Nataro JP, Mobley HL (2004) Pathogenic Escherichia coli. Nat Rev Microbiol 2: 123–140. 3. Griffin PM, Ostroff SM, Tauxe RV, Greene KD, Wells JG, et al. (1988) Illnesses associated with Escherichia coli O157:H7 infections. A broad clinical spectrum. Ann Intern Med 109: 705–712. 4. 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Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee at the Massachusetts Institute of Technology (Protocol Number: 0207-020-10). All efforts were made to minimize suffering. Author Contributions Conceived and designed the experiments: HP SAK MEM SM DBS REM JML DZ. Performed the experiments: HP SAK MEM SM DBS REM JML DZ. Analyzed the data: HP SAK MEM SM DBS REM JML DZ. Contributed reagents/materials/analysis tools: HP SAK MEM SM DBS REM JML DZ. Wrote the paper: HP SAK MEM SM DBS REM JML DZ. 11. Abe A, de Grado M, Pfuetzner RA, Sanchez-Sanmartin C, Devinney R, et al. (1999) Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion. Mol Microbiol 33: 1162–1175. 12. Simovitch M, Sason H, Cohen S, Zahavi EE, Melamed-Book N, et al. 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