Autophagy Inhibitors as a Potential Antiamoebic Treatment for Acanthamoeba Keratitis

Abstract

ABSTRACTAcanthamoebacysts are resistant to extreme physical and chemical conditions. Autophagy is an essential pathway for encystation ofAcanthamoebacells. To evaluate the possibility of an autophagicAcanthamoebaencystation mechanism, we evaluated autophagy inhibitors, such as 3-methyladenine (3MA), LY294002, wortmannin, bafilomycin A, and chloroquine. Among these autophagy inhibitors, the use of 3MA and chloroquine showed a significant reduction in the encystation ratio inAcanthamoebacells. Wortmannin also inhibited the formation of mature cysts, while LY294002 and bafilomycin A did not affect the encystation ofAcanthamoebacells. Transmission electron microscopy revealed that 3MA and wortmannin inhibited autophagy formation and that chloroquine interfered with the formation of autolysosomes. Inhibition of autophagy or autolysosome formation resulted in a significant block in the encystation inAcanthamoebacells. Clinical treatment with 0.02% polyhexamethylene biguanide (PHMB) showed high cytopathic effects onAcanthamoebatrophozoites and cysts; however, it also revealed high cytopathic effects on human corneal epithelial cells. In this study, we investigated effects of the combination of a low (0.00125%) concentration of PHMB with each of the autophagy inhibitors 3MA, wortmannin, and chloroquine onAcanthamoebaand human corneal epithelial cells. These new combination treatments showed low cytopathic effects on human corneal cells and high cytopathic effects onAcanthamoebacells. Taken together, these results provide fundamental information for optimizing the treatment ofAcanthamoebakeratitis.