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In vitro characterization of Acanthamoeba castellanii cytopathic effect
W M Taylor1, M S Pidherney, H Alizadeh
1Division of Comparative Medicine, UT Southwestern Medical Center at Dallas 75235-9037, USA.
Abstract:
This study examined the mechanism of the cytopathic effect (CPE) of Acanthamoeba castellanii on human target cells. Pathogenic Acanthamoeba trophozoites were incubated with human ocular melanoma (OCM1) cells for 30 min, 1 hr, and 3 hr. The amoebae were treated with a calcium ionophore (A23187), phorbol myristate ester (PMA), calcium channel blocker (Bepridil), cytochalasin D, and L-leucyl-L-leucine methyl ester (leu-leu-OMe). Cytolysis was quantified using a spectrophotometric assay. Cocultures of amoeba and cells were also observed by transmission electron microscopy at 1, 2, and 3 hr. Results show that trophozoites formed pseudopodia that made intimate contact with the target cell membrane. Neither amebostomes nor phagocytosis was seen. The calcium ionophore A23187 increased the cytopathic effect of the trophozoites on the cultured OCM1. In contrast, cytochalasin D, Bepridil, and PMA reduced the cytopathic effect. Leu-leu-OMe did not result in killing of Acanthamoeba trophozoites. The results suggest that the cytopathic effect of Acanthamoeba trophozoites involves calcium channels and cytoskeletal elements. There was no evidence of trogocytosis or phagocytosis as sometimes occurs in cytolysis by other free-living amoeba. Although Acanthamoeba-mediated CPE in some ways resembles CPE produced by cytotoxic lymphocytes, the mechanisms are not identical.
Insights
Acanthamoeba castellanii causes cell damage through direct contact, involving calcium channels and cytoskeletal elements. This cytopathic effect differs from phagocytosis and resembles, but is not identical to, lymphocyte-induced cell death.
Area of Science:
- Cell Biology
- Parasitology
- Immunology
Background:
- Acanthamoeba castellanii is a free-living amoeba known to cause infections in humans.
- The mechanism of Acanthamoeba-induced cytopathic effect (CPE) on human cells is not fully understood.
- Understanding Acanthamoeba CPE is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the mechanism by which Acanthamoeba castellanii trophozoites induce cytopathic effects on human ocular melanoma (OCM1) cells.
- To investigate the role of calcium channels and cytoskeletal elements in Acanthamoeba-mediated cell lysis.
- To differentiate Acanthamoeba CPE from phagocytosis and trogocytosis.
Main Methods:
- Incubation of Acanthamoeba trophozoites with OCM1 cells for varying durations.
- Treatment of amoebae with pharmacological agents affecting calcium signaling (A23187, Bepridil) and cytoskeleton (cytochalasin D, PMA).
- Quantification of cytolysis using spectrophotometry and observation via transmission electron microscopy.
Main Results:
- Acanthamoeba trophozoites formed pseudopodia making direct contact with OCM1 cell membranes, without evidence of phagocytosis or amebastomes.
- The calcium ionophore A23187 enhanced Acanthamoeba's cytopathic effect, while Bepridil, cytochalasin D, and PMA reduced it.
- Leu-leucine methyl ester did not kill Acanthamoeba trophozoites.
Conclusions:
- Acanthamoeba-mediated cytopathic effect on human cells involves calcium channels and cytoskeletal interactions.
- The mechanism of Acanthamoeba CPE is distinct from phagocytosis and trogocytosis observed with other amoebae.
- While sharing some similarities with cytotoxic lymphocyte effects, Acanthamoeba CPE operates via a unique pathway.