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Metalloprotease NleC Suppresses Host NF-κB/Inflammatory Responses by Cleaving p65 and Interfering with the p65/RPS3 Interaction

Fig 4

NleC cleaves p65 during C. rodentium and EPEC infections.

A. HEK293T cells expressing N-terminal GFP-tagged p65 were mock infected or infected with indicated C. rodentium strains at 100 MOI for 3 h. Whole cell lysates were derived, separated by SDS/PAGE, and immunoblotted for indicated proteins. The GFP-tagged p651–10 cleavage products are indicated by an arrow, and an asterisk labels nonspecific bands. B. Caco-2 cells were mock infected or infected with indicated EPEC strains at 100 MOI for 1 h. Whole cell lysates were derived, separated by SDS/PAGE, and immunoblotted for indicated proteins. C. Mouse colon epithelial cells (CECs) were mock infected or infected in suspension with indicated C. rodentium strains at 100 MOI for 3 h. Whole cell lysates were derived, separated by SDS/PAGE, and immunoblotted for indicated proteins. The full-length p65 and cleaved p65 C-terminal fragment are indicated by filled and open triangles, respectively. D. Mouse CECs infected as in C were separated from free C. rodentium by Percoll gradient centrifugation. Whole cell lysates from CECs and attached C. rodentium were derived, separated by SDS/PAGE, and immunoblotted for indicated proteins. E. Representative immunofluorescence micrographs of mouse CECs infected as in C that were centrifuged onto cover slips and stained for C. rodentium LPS, with nuclei counterstained by DAPI. Scale bars, 10 μm. F. The numbers of C. rodentium attached to mouse CECs as in E (from 6 random fields) were quantified and normalized to the perimeter of individual CEC.

Fig 4

doi: https://doi.org/10.1371/journal.ppat.1004705.g004