Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites

Abstract

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

Keywords: Naegleria fowleri; cell death; cytotoxicity; microglial cells.

Fig. 1

Light microscopic images. These images show the cytopathic effects of N. fowleri trophozoites on U87MG cells in a contact or a non-contact culture system. N. fowleri lysate treated directly at concentration of 1 mg/ml for 4 hr. The control was cultured with U87MG cells only at the same condition. (A, B) control (×100, ×200), (C, E, and G) U87MG cells co-cultured with N. fowleri trophozoite in a non-contact system for 30 min, 2 hr, and 4 hr, respectively (×200), (D, F, H) The U87MG cells treated with N. fowleri lysate for 30 min, 2 hr, and 4 hr, respectively (×200).

Fig. 2

FACS analysis for apoptosis of U87MG cells. (A) The U87MG cells were co-cultured with N. fowleri trophozoites for 30 min, 2 hr, and 4 hr. The U87MG cells (1 mg/ml) were directly treated with N. fowleri lysate or TRAIL (100 ng/ml) for 4 hr. Untreated U87MG cells and TRAIL was used as a control and positive control for apoptosis, respectively. (B) Quantitative analysis calculated from FACS data. The values were significantly different from the value for the negative control for all samples.

Fig. 3

In vitro cytotoxicity of N. fowleri against U87MG cells in a non-contact system by LDH release assay. The N. fowleri trophozoites showed higher cytotoxicity than those of N. gruberi trophozoites, N. fowleri lysate and N. gruberi lysate used as the control group.