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Comparative Study
, 67 (5), 2575-84

Host Cell Death Due to Enteropathogenic Escherichia Coli Has Features of Apoptosis

Affiliations
Comparative Study

Host Cell Death Due to Enteropathogenic Escherichia Coli Has Features of Apoptosis

J K Crane et al. Infect Immun.

Abstract

Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen.

Figures

FIG. 1
FIG. 1
EPEC-induced cell killing by trypan blue uptake. T84 cells were grown in Lab-Tek chamber slides, changed to antibiotic-free adherence medium, treated with inhibitors and/or infected with an E. coli strain for 2 h, and then stained with trypan blue as described in Materials and Methods. Cells were viewed by differential interference contrast microscopy at a magnification of ×200. (A) Uninfected control; (B) cells treated with 100 nM wortmannin alone; (C) cells infected with EPEC strain E851/71; (D) cells infected with E851/71 in the presence of 100 nM wortmannin; (E) cells infected with laboratory strain HB101; (F) cells infected with HB101 in the presence of wortmannin.
FIG. 2
FIG. 2
Colocalization of cell death with adherent EPEC. HeLa cells were left uninfected (A), infected with nonadherent strain E. coli HB101 (B), or infected with EPEC strain B171-8 (C and D) for 2 h. Bacteria had been fluorescently labeled by subculturing in the presence of acridine orange; after infection, HeLa cells were stained with propidium iodide for an additional 30 min as described in Materials and Methods. Original magnifications: A through C, ×600 (under oil); D, ×1,000. Propidium iodide-stained HeLa cell nuclei are 16 to 20 μm in size and fluoresce reddish orange (black arrows on white background), and clumps of adherent EPEC appear greenish yellow (yellow arrowheads). The apparent size of an individual bacterial cell (E. coli B171-8) is 1.8 μm (propidium iodide stains the central nucleoid, not the cell membrane or wall).
FIG. 3
FIG. 3
Early expression of phosphatidylserine on the host cell surface after EPEC infection. HeLa cells were grown to confluency on Lab-Tek slides, infected with EPEC strain E2348 in the presence or absence of wortmannin, and then incubated with FITC-labeled annexin V to reveal phosphatidylserine on the cell surface as described in Materials and Methods. (A) Normal, uninfected control HeLa cells; (B) cells treated with 100 nM wortmannin for 3 h; (C) cells infected with E2348 for 1 h; (D) cells infected with E2348 for 3 h; (E) cells infected with E2348 in the presence of 100 nM wortmannin for 3 h; (F) cells treated for 20 h with doxorubicin (1 μg/ml) as a positive control. Magnifications: A through E, ×200; F, ×400.
FIG. 4
FIG. 4
Internucleosomal breakdown of host cell DNA after EPEC infection. T84 cells were treated with inhibitors or infected with E. coli for 6 h; medium was replenished once at 2 h, at which time wortmannin was readded. Cells were recovered in 70% ethanol and subjected to extraction with phosphate-citric acid as described in Materials and Methods and then analyzed by agarose gel electrophoresis. A negative image of the ethidium bromide-stained gel is shown. (A) Extracts from human cells; (B) extracts from E. coli alone; MW, 100-bp DNA molecular size markers, with the 600-bp marker indicated. (A) Lane 1, uninfected, control T84 cells; lane 2, cells treated with 100 nM wortmannin alone; lane 3, cells treated with 200 μM genistein alone; lane 4, cells infected with EPEC strain E2348; lane 5, cells treated with EPEC plus wortmannin; lane 6, cells treated with E2348 plus genistein; lane 7, cells infected with strain HS alone; lane 8, H9 leukemia cells treated with doxorubicin (2 μg/ml) for 16 h as a positive control. (B) DNA extracts from E. coli HS (lane 1) and EPEC strain E2348 (lane 2). In panel B, the number of bacteria subjected to the extraction was about five times greater than that calculated to be present in panel A.
FIG. 5
FIG. 5
Quantitation of EPEC-induced cell death by LDH release. (A) LDH activity was measured as described in Materials and Methods in E. coli bacteria alone and in T84 cells treated as indicated for 3 h. uninf, uninfected control. (B) The data in panel A expressed as percent cell death. *, statistically increased in Salmonella-infected compared to both EPEC-infected conditions (P < 0.05).
FIG. 6
FIG. 6
Quantitation of EPEC-induced cell death by propidium iodide uptake. T84 cells grown to confluency in 48-well plates were treated with or without inhibitors or infected with E. coli in adherence medium for 2 h, and then the medium was replaced with phenol red-free medium containing propidium iodide (2 μg/ml) as described in Materials and Methods; measurements were taken at various times afterward, as indicated on the graph. Abbreviations: uninf, uninfected control; E2348, infected with E2348 alone; E23 filtr, cells treated with a sterile filtrate of EPEC subculture; killed E23, E2348 bacteria killed by incubation with ciprofloxacin (15 μg/ml) for 5 min prior to addition to the T84 cells; wort, treated with 100 nM wortmannin; JPN15 and CVD206, the plasmid-cured and eae-deleted derivatives of E2348, respectively; H2O2, cells treated with 0.1% H2O2 as a positive control. In panel B, the single asterisk indicates statistical significance at P < 0.05 compared to HB101 plus wortmannin and the double asterisk indicates statistical significance (P < 0.05) compared to E2348 alone. In panel C, the three infected conditions gave results which were significantly different from each other (P < 0.05). In panel D, ethidium homodimer was used instead of propidium iodide; the concentration of MG-132 used was 40 μM, and the data shown are at 6 h after infection; the asterisk indicates statistical significance (P < 0.05) compared to E2348 alone.
FIG. 7
FIG. 7
Scheme for understanding the slow host cell killing by EPEC and enhanced killing in the presence of inhibitors. When antiapoptotic signals are blocked by genistein, wortmannin, staurosporine, or the NF-κB inhibitor MG-132, death-promoting pathways act unopposed and killing is enhanced.

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