The enteropathogenic Escherichia coli effector Cif induces delayed apoptosis in epithelial cells

Abstract

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins, the cyclomodulins, which modulate the host cell cycle. Upon injection into the host cell by the type III secretion system of enteropathogenic Escherichia coli (EPEC), Cif induces both G(2) and G(1) cell cycle arrests. The cell cycle arrests correlate with the accumulation of p21(waf1) and p27(kip1) proteins that inhibit CDK-cyclin complexes, whose activation is required for G(1)/S and G(2)/M transitions. Increases of p21 and p27 levels are independent of p53 transcriptional induction and result from protein stabilization through inhibition of the ubiquitin/proteasome degradation pathway. In this study, we show that Cif not only induces cell cycle arrest but also eventually provokes a delayed cell death. Indeed, 48 h after infection with EPEC expressing Cif, cultured IEC-6 intestinal cells were positive for extracellular binding of annexin V and exhibited high levels of cleaved caspase-3 and lactate dehydrogenase release, indicating evidence of apoptosis. Cif was necessary and sufficient for inducing this late apoptosis, and the cysteine residue of the catalytic site was required for Cif activity. These results highlight a more complex role of Cif than previously thought, as a cyclomodulin but also as an apoptosis inducer.

FIG. 1.

EPEC E22 strains expressing Cif induce the death of IEC-6 cells. Cells were infected for 2 h with EPECwt, EPECΔCif, or EPECΔCif complemented with plasmids coding for wild-type Cif (pCifwt) or a Cif C109A mutant (pCifC109A). Twenty-four, 48, and 72 hours after infection, the culture supernatants were collected to measure the LDH activity. Graphed data represent the means plus standard errors of the means for three independent experiments. Data are significantly different (*, P < 0.05; **, P < 0.001) compared with the control for each time point.

FIG. 2.

EPEC strains induce accumulation of cleaved caspase-3 in IEC-6 cells. (A) IEC-6 cells were infected for 2 h with an EPEC E22 wild-type strain (E22wt), a Cif-deleted mutant (E22ΔCif), or E22ΔCif complemented with a plasmid coding for wild-type Cif (pCif wt) or a Cif C109A mutant. After the indicated times, cell extracts were probed with anti-p21, anti-cleaved caspase-3, and anti-actin antibodies. (B) The same experiment was done with an EPEC B171-8 wild-type strain (B171wt), a Cif-deleted mutant (B171ΔCif), or B171ΔCif complemented with a plasmid coding for wild-type Cif (pCif wt) or a Cif C109A mutant.

FIG. 3.

Cif induces exposure of phosphatidylserine on infected IEC-6 cells. (A) IEC-6 cells were infected with the EPEC B171ΔCif strain. Twenty-four hours after infection, phosphatidylserine exposure was analyzed by flow cytometry as described in Materials and Methods. Viable, dead, and apoptotic cell populations are indicated. (B) Cells were infected or not (control) with the indicated EPEC E22 strains and incubated for 24 h, 48 h, and 72 h. Percentages of apoptotic cells (lower right quadrant in panel A) were measured. The experiment was performed twice with similar results, and data for only one experiment are shown. (C) Cells were infected with the indicated EPEC B171 strains. The experiment was performed three times, and the results are shown as means ± standard errors of the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 4.

Purified Cif induces apoptosis in IEC-6 cells. IEC-6 cells were left untreated (cont) or were incubated with BioPORTER plus PBS, H6-Cifwt, or H6-Cif C109A for 24 h and 48 h, and cellular extracts were probed with the indicated antibodies. p21 and cleaved caspase-3 levels were quantified by densitometry with Quantity One software (Bio-Rad) and normalized to the actin level. Relative levels of p21 and cleaved caspase-3 are shown as increases compared to those in control cells at 24 h.