NleL Modulates Pedestal Formation Figure 5. Contribution of nleL to C. rodentium virulence. (A) C. rodentium NleL is an E3 ubiquitin ligase. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL or GST-NleLC753S 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) Fecal bacteria shedding of the wild-type strain (DBS130), the nleL mutant strain, the nleL mutant strain expressing the wild-type NleL, or the nleL mutant strain complemented with the catalytically-dead NleLC753S in C. rodentium. (C) Virulence of C. rodentium 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 nleL 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 NleLC753S mutant (DBS794). (D) Contribution of nleL to mouse colitis caused by C. rodentium. Pathology of mouse colon by different C. rodentium 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 nleL 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 NleLC753S mutant (P,0.01). doi:10.1371/journal.pone.0019331.g005 Protein secretion assay Bacterial strains were grown overnight in LB at 37uC. The cultures were then diluted 1:20 in M-9 minimal media supplemented with 0.4% glucose, 8 mM MgSO4, 44 mM NaHCO2, and 0.1% Casamino Acids and grown standing at 37uC in 5% CO2 until the OD600 reached approximately 0.8. The bacterial cultures were centrifuged and the supernatants were filtered through a 0.22 um filter to remove any residual bacteria. Proteins in the supernatants were precipitated with 10% trichloroacetic acid on ice for at least 1 hour. Precipitated proteins were collected by centrifugation and washed twice with ice-cold acetone before being resuspended in SDS-PAGE loading buffer. Adenylate cyclase translocation assay HeLa cells were infected as described. Four hours post- infection, cells were washed with ice-cold PBS and lysed using 0.1 M HCl with gentle agitation for 20 min. Protein concentration in cell lysates was determined using the Bio-Rad Protein Assay Kit (Hercules, CA) according to the manufacturer’s instructions. Equal amount of protein was used to determine levels of Adenosine 39, 59-cyclic monophosphate (cAMP) using the Direct Immunoassay Kit (Assay Designs, MI) according to the manufacturer’s instructions. Adenylate cyclase activity is expressed as pmol per milligram of total protein. PLoS ONE | www.plosone.org 7 April 2011 | Volume 6 | Issue 4 | e19331