Enterotoxigenic Escherichia coli modulates host intestinal cell membrane asymmetry and metabolic activity

Abstract

Enterotoxigenic Escherichia coli (ETEC) is a common cause of travelers' and postweaning diarrhea in humans and swine, respectively. The extent to which ETEC damages host cells is unclear. Experiments are presented that probe the ability of porcine ETEC isolates to induce apoptosis and cell death in porcine intestinal epithelial cells. Quantification of host phosphatidylserine exposure following ETEC infection suggested that ETEC induced changes in plasma membrane asymmetry, independent of the expression of the heat-labile enterotoxin. Significant host cell death was not observed. ETEC infection also caused a drastic inhibition of host esterase activity, as measured by calcein fluorescence. While ETEC infection resulted in activation of host caspase 3, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling of DNA double-strand breakage, indicative of late stages of apoptosis, was not observed. Camptothecin-induced apoptosis markedly increased subsequent ETEC adherence. Transfer of cell-free supernatants from apoptotic cells to bacterial inocula prior to infection of naïve cells increased the transcriptional activity of the regulatory region upstream of the K88ac operon and promoted subsequent adherence to host cells.

FIG. 1.

ETEC induces PS exposure in IPEC-J2 cells. (A) Flow cytometry analysis of PI (y axis) versus annexin V (x axis) staining of IPEC-J2 cells following infection (4 h) with the bacterial strains G58-1, wt 2534-86, 2534-86 ΔeltAB, and 2534-86 ΔeltAB/pLT or intoxication with 100 ng/ml LT. (B) Quantification (% positive cells) (means ± SD; n = 4) of annexin V (open bars) and PI (shaded bars) staining from flow cytometry experiments. (C) Quantification (means ± SD; n = 4) of annexin V (open bars) and PI (shaded bars) staining as a function of time (1 to 4 h) following inoculation of IPEC-J2 cells with wt 2534-86. (D) Quantification (means ± SD; n = 4) of annexin V (open bars) and PI (shaded bars) staining following intoxication of IPEC-J2 cells (4 h) with OMVs (100 ng/ml) purified from the indicated bacterial strains.

FIG. 2.

ETEC infection inhibits calcein-AM conversion in IPEC-J2 cells. (A) Flow cytometry analysis of PI staining (y axis) versus calcein fluorescence (x axis) in IPEC-J2 cells following infection with the bacterial strains G58-1, wt 2534-86, 2534-86 ΔeltAB, and 2534-86 ΔeltAB/pLT or intoxication with 100 ng/ml LT. (B) Quantification (means ± SD; n = 4) of the percentages of calcein-negative cells from flow cytometry experiments. (C) Quantification (means ± SD; n = 4) of the percentages of PI-positive (open bars) and calcein-negative (shaded bars) cells as a function of time (1 to 4 h) following inoculation of IPEC-J2 cells with wt 2534-86. (D) Quantification of the percentages of PI-positive (open bars) and calcein-negative (shaded bars) cells following intoxication of IPEC-J2 cells (4 h) with 100 ng/ml OMVs purified from the indicated bacterial strains. (E) Quantification of calcein fluorescence as a function of time after gentamicin treatment (0 to 60 min) of IPEC-J2 cells infected with wt 2534-86.

FIG. 3.

Diversity of porcine ETEC strains in inducing host cell damage. (A) Quantification of annexin V-positive cells (y axis) (means ± SD; n = 4) following a 4-h infection with the indicated bacterial strains (x axis). (B) Quantification of calcein-negative cells (means ± SD; n = 4).

FIG. 4.

Host cell damage promotes ETEC adherence. (A) Immunoblot of IPEC-J2 cells after 1 h of treatment with 100 ng/ml LT or after 4 h of infection with E. coli G58-1 or wt or ΔeltAB ETEC at an MOI of 10. Blots were probed with rabbit polyclonal antisera against active caspase 3. (B) ETEC adherence (mean CFU/ml ± SD) following treatment of IPEC-J2 cells with 100 μM camptothecin (campt), with or without 100 μM Ac-DEVD-CHO, for 1 h prior to ETEC infection. (C) ETEC adherence (mean CFU/ml ± SD) to naïve host cells following prestimulation of bacterial inocula with sterile cell-free supernatants derived from donor cells treated with camptothecin, with or without Ac-DEVD-CHO. (D) Relative expression of K88ac-CAT in the presence of supernatants derived from host cells treated with camptothecin, with or without Ac-DEVD-CHO. Data are plotted as relative CAT activities versus that of the bacterial culture additive.