Figure 1.
Self-ubiquitination of GST-NleL.
(A) NleL-mediated self-ubiquitination requires ATP, ubiquitin, E1 and E2. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL59–782 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) NleL C753 residue is required for its E3 ubiquitin ligase activity. Reactions containing ubiquitin, E1 and UbcH5a were incubated with the wild type GST-NleL, GST-NleLC688S or GST-NleLC753S or GST-NleLC753A at 35°C for 90 min. The Western blot was performed using monoclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom).
Figure 2.
Secretion of Tir and translocation of NleL into HeLa cells.
(A) The expression and secretion levels of Tir in either the wild-type EHEC or the mutant EHEC expressing the chromosomal catalytically-dead mutant NleLC753A was examined by Western blot. (B) Intracellular cAMP levels are an indication of the translocation of CyaA fusion proteins in EHEC WT (ZP250) and EHEC TTSS deficient mutant (escF; ZP2251) strains. Cells were infected for 4 hrs, and the adenylate cyclase activity was determined. The data were from three independent experiments, with standard deviations shown as error bars. cAMP values are presented as pmol per milligram of total cellular protein. The expression levels of the NleL-CyaA fusions were found to be similar by Western blot as shown on the bottom panel.
Figure 3.
E3 ligase activity of NleL is involved in modulating EHEC pedestal formation.
HeLa cells were infected at a multiplicity of infection of 100 with wild type EHEC, nleLC753A, or nleLC753A harboring plasmid expressing wild-type NleL. Cells were infected for 6 hours. (A) Bacteria were visualized by staining with an anti-EHEC LPS antibody (green). Actin was detected with a Texas Red Phalloidin (red). (B) The number of micro-clusters of pedestals on HeLa cells were counted and were grouped into clusters having 1–4 pedestals, 5–10 pedestals and >10 pedestals. Quantitative analysis includes three independent experiments. A minimum of 300 cells were counted from each experiment with standard deviation shown as error bars.
Figure 4.
The E3 ligase activity of EHEC NleL down modulates EPEC pedestal formation.
HeLa cells were infected at a multiplicity of infection of 100 with wild type EPEC, or EPEC harboring plasmid expressing wild-type EHEC NleL or NleLC753A. Cells were infected for 4 hours. (A) Bacteria were visualized by staining with an anti-EPEC LPS antibody (green). Actin was detected with a Texas Red Phalloidin (red). (B) The number of micro-clusters of pedestals on HeLa cells were counted and were grouped into clusters having 1–10 pedestals, 11–20 pedestals and >20 pedestals. Quantitative analysis includes three independent experiments. A minimum of 300 cells were counted from each experiment with standard deviation shown as error bars.
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).