The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That 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 35°C 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 null 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).