Hemolysin-positive enteroaggregative and cell-detaching Escherichia coli strains cause oncosis of human monocyte-derived macrophages and apoptosis of murine J774 cells

Abstract

Infection of human monocyte-derived macrophages (HMDM) and J774 cells (murine macrophage cell line) with several enteroaggregative and cytodetaching Escherichia coli (EAggEC and CDEC, respectively) strains demonstrated that some strains could induce macrophage cell death accompanied by release of lactate dehydrogenase activity and interleukin 1beta (IL-1beta) into culture supernatants. The mode of cell death differed in the two types of macrophages. Damage to macrophage plasma membrane integrity without changes in nuclear morphology resulted in cytolysis of HMDM. This mechanism of cell death has been previously described for virulent Shigella infection of HMDM and is termed oncosis. In contrast, infection of J774 cells by EAggEC and CDEC strains resulted in apoptosis. The presence of alpha-hemolysin (Hly) in EAggEC and CDEC strains appears to be critical for both oncosis in HMDM and apoptosis in J774 cells. Bacteria lacking Hly, including Hly- EAggEC strains as well as enterotoxigenic, enteropathogenic, and enterohemorrhagic E. coli strains, behaved like avirulent Shigella flexneri in that the macrophage monolayers were intact, with no release of lactate dehydrogenase activity or IL-1beta into the culture supernatants.

FIG. 1

Survival of bacterial strains in HMDM; HMDM infected with various E. coli and Shigella strains (A) and various EAggEC strains (B). CFU represents the total number of bacteria in macrophage cell lysates. M9OT-W, virulent S. flexneri 5; M9OT-55, isogeneic plasmid-cured strain; NF705, normal E. coli strain. The rest of the strains are described in Table 1 and in the text.

FIG. 2

Light microscopic analysis of HMDM and J774 murine macrophages infected with EAggEC and CDEC strains. Bacteria were left in contact with HMDM (A, B, and C) and J774 macrophages (D, E, and F) for 1 h. Macrophages were stained with a modified Wright’s stain after infection with Hly⁺ CDEC 55-3 (A and E), Hly⁻ CDEC 55-3 (B and F), S. flexneri 5 M9OT-W (C), and uninfected J774 (D). Magnification, ×910.

FIG. 3

DNA fragmentation assays on agarose gels. DNA was isolated from macrophages infected with different EAggEC and CDEC strains. M9OT-W and M9OT-55 were included for comparative purposes. The DNA was electrophoresed on 1.2% agarose gels for 3 h at 100 V. DNA was isolated from HMDM (A) or J774 (B) murine macrophages infected with M9OT-55 (lanes 3), EAggEC 697 (Hly⁺) (lanes 4), CDEC 55-3 (Hly⁺) (lanes 5), CDEC 55-3 (Hly⁻) (lanes 6), and M9OT-W (lanes 7). Lanes 1, 123-bp DNA ladder molecular-weight-marker (BRL); lanes 2, DNA isolated from noninfected macrophages.

FIG. 4

TEM of HMDM and J774 cells infected with hemolysin-positive and -negative EAggEC and CDEC strains. (A and B) HMDM infected with Hly⁺ EAggEC shown in a state of necrosis; (C) HMDM infected with Hly⁻ EAggEC or CDEC strains; (D and E) J774 cells infected with Hly⁺ CDEC strain 55-3; (F) J774 cells infected with Hly⁻ CDEC strain 55-3. Bar markers: 2 μm (A and C) and 1 μm (B, D, E, and F). b, bacteria; n, nucleus; and sn, swollen nucleus; an, apoptotic nucleus.