Interaction of enteropathogenic or enterohemorrhagic Escherichia coli with HeLa cells results in translocation of cortactin to the bacterial adherence site

Abstract

Infection of cultured HeLa epithelial cells with enteropathogenic Escherichia coli (EPEC) or enterohemorrhagic E. coli (EHEC) O157:H7 results in accumulation of cortactin under the adherent bacteria. Tyrosine phosphorylation of cortactin is not induced following HeLa cell infection with EHEC or EPEC, contrary to what has been reported to occur with Shigella flexneri.

FIG. 1

EPEC and EHEC induce cortactin accumulation in cultured HeLa cells. Noninfected HeLa cells (A, D, and G) and HeLa cells infected for 4 h with EHEC O157:H7 (B, E, and H) or EPEC (C, F, and I) were visualized with anticortactin antibodies labeled with rhodamine (D, E and F) by confocal laser scanning microscopy. Panels A, B, and C are phase-contrast images. Panels G, H, and I are superimposed images A and D, B and E, and C and F, respectively. The arrows indicate a bacterial cluster (phase-contrast images) and the corresponding accumulation of cortactin at the adherence site (confocal images).

FIG. 2

(A) EPEC or EHEC does not induce tyrosine phosphorylation of cortactin during HeLa cell infection. Cortactin was immunoprecipitated from noninfected HeLa cell extracts (A, D) or from extracts of HeLa cells infected with EPEC (B, E) or EHEC O157:H7 (C, F), blotted, and probed with antiphosphotyrosine serum (A, B, and C). The same membrane was reprobed with anticortactin serum (D, E, and F). The arrowhead indicates the cortactin bands. (B) Induction of tyrosine phosphorylation of cortactin during HeLa cell infection with S. flexneri. Cortactin was immunoprecipitated from HeLa cells infected with S. flexneri (C, D), vanadate-treated HeLa cells (B), or control cells (A). A Western blot membrane was probed with antiphosphotyrosine antibodies (PY) and reprobed with anticortactin antibodies (Cort). The arrowheads indicate the cortactin bands.

FIG. 3

(A) Effects of inhibitors on EHEC- or EPEC-induced cortactin accumulation observed by confocal laser scanning microscopy. HeLa cells were treated with Cyt-D, staurosporine (STAU), or the drug PP1 (see the text for details) and were either not infected (CONTROL) or infected with EHEC O157:H7 or EPEC organisms. Cortactin (pink) was stained with anticortactin serum labeled with appropriate rhodamine-conjugated goat anti-mouse antibodies. The arrows indicate a bacterial cluster and the corresponding accumulation of cortactin at the bacterial adherence site. (B) Confocal images of cortactin accumulation by S. flexneri. HeLa cells were infected with S. flexneri for 30 min (A and B) or 1 h (C). Cortactin accumulation was observed in the absence (A and C) or presence (B) of PP1. The arrows show cortactin accumulation at the bacterial adherence site. See the text for details. Bar, 10 μm.