Effects of mannose on Acanthamoeba castellanii proliferation and cytolytic ability to corneal epithelial cells

Michael Hurt, Jerry Niederkorn, Hassan Alizadeh

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

PURPOSE. Acanthamoeba trophozoites express a mannose binding receptor that facilitates adhesion of trophozoites to mannosylated proteins on corneal epithelial cells. This study was undertaken to determine the role that mannose stimulation has in the amoeba's growth, secreted products, and ability to desquamate the corneal epithelium. METHODS. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose (PYG) and PYG with 100 mM methyl α-D-mannopyranoside or galactose. The proliferation of trophozoites and cysts was examined by optical density and direct counts. The molecular weight of the mannose-stimulated protein was examined by SDS/PAGE. The cytolytic protein was purified by fast protein liquid chromatography (FPLC) size exclusion and ionic exchange and then tested for cytopathic effect (CPE) and collagenolytic activity in vitro. Collagenolytic activity was examined by zymography. Proteases and protease inhibitors were used to characterize the nature of the cytolytic protein. RESULTS. Methyl α-D-mannopyranoside inhibited the growth of A. castellanii by 50% (P < 0.05) and concomitantly induced a threefold increase in the formation of cysts. SDS-PAGE analysis revealed a mannose-induced protein of ∼133 kDa (MIP-133). The MIP-133 protein was found to be highly cytolytic against corneal epithelial cells, but not human intestinal epithelial cells and also degraded collagen in vitro. Serine protease inhibitors abrogated both CPE and collagenolytic activity of the MIP-133 protein (P < 0.001). CONCLUSIONS. The results suggest that binding of trophozoites to mannosylated proteins on the corneal surface induces A. castellanii to secrete a ∼133-kDa serine protease that kills both human and hamster corneal epithelium and degrades collagen.

Original languageEnglish (US)
Pages (from-to)3424-3431
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number8
DOIs
StatePublished - Aug 1 2003

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Acanthamoeba castellanii
Mannose
Epithelial Cells
Trophozoites
Proteins
Peptones
Corneal Epithelium
Cysts
Polyacrylamide Gel Electrophoresis
Collagen
Yeasts
Acanthamoeba
Glucose
Serine Proteinase Inhibitors
Amoeba
Serine Proteases
Growth
Protease Inhibitors
Galactose
Liquid Chromatography

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Effects of mannose on Acanthamoeba castellanii proliferation and cytolytic ability to corneal epithelial cells. / Hurt, Michael; Niederkorn, Jerry; Alizadeh, Hassan.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 8, 01.08.2003, p. 3424-3431.

Research output: Contribution to journalArticle

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abstract = "PURPOSE. Acanthamoeba trophozoites express a mannose binding receptor that facilitates adhesion of trophozoites to mannosylated proteins on corneal epithelial cells. This study was undertaken to determine the role that mannose stimulation has in the amoeba's growth, secreted products, and ability to desquamate the corneal epithelium. METHODS. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose (PYG) and PYG with 100 mM methyl α-D-mannopyranoside or galactose. The proliferation of trophozoites and cysts was examined by optical density and direct counts. The molecular weight of the mannose-stimulated protein was examined by SDS/PAGE. The cytolytic protein was purified by fast protein liquid chromatography (FPLC) size exclusion and ionic exchange and then tested for cytopathic effect (CPE) and collagenolytic activity in vitro. Collagenolytic activity was examined by zymography. Proteases and protease inhibitors were used to characterize the nature of the cytolytic protein. RESULTS. Methyl α-D-mannopyranoside inhibited the growth of A. castellanii by 50{\%} (P < 0.05) and concomitantly induced a threefold increase in the formation of cysts. SDS-PAGE analysis revealed a mannose-induced protein of ∼133 kDa (MIP-133). The MIP-133 protein was found to be highly cytolytic against corneal epithelial cells, but not human intestinal epithelial cells and also degraded collagen in vitro. Serine protease inhibitors abrogated both CPE and collagenolytic activity of the MIP-133 protein (P < 0.001). CONCLUSIONS. The results suggest that binding of trophozoites to mannosylated proteins on the corneal surface induces A. castellanii to secrete a ∼133-kDa serine protease that kills both human and hamster corneal epithelium and degrades collagen.",
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N2 - PURPOSE. Acanthamoeba trophozoites express a mannose binding receptor that facilitates adhesion of trophozoites to mannosylated proteins on corneal epithelial cells. This study was undertaken to determine the role that mannose stimulation has in the amoeba's growth, secreted products, and ability to desquamate the corneal epithelium. METHODS. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose (PYG) and PYG with 100 mM methyl α-D-mannopyranoside or galactose. The proliferation of trophozoites and cysts was examined by optical density and direct counts. The molecular weight of the mannose-stimulated protein was examined by SDS/PAGE. The cytolytic protein was purified by fast protein liquid chromatography (FPLC) size exclusion and ionic exchange and then tested for cytopathic effect (CPE) and collagenolytic activity in vitro. Collagenolytic activity was examined by zymography. Proteases and protease inhibitors were used to characterize the nature of the cytolytic protein. RESULTS. Methyl α-D-mannopyranoside inhibited the growth of A. castellanii by 50% (P < 0.05) and concomitantly induced a threefold increase in the formation of cysts. SDS-PAGE analysis revealed a mannose-induced protein of ∼133 kDa (MIP-133). The MIP-133 protein was found to be highly cytolytic against corneal epithelial cells, but not human intestinal epithelial cells and also degraded collagen in vitro. Serine protease inhibitors abrogated both CPE and collagenolytic activity of the MIP-133 protein (P < 0.001). CONCLUSIONS. The results suggest that binding of trophozoites to mannosylated proteins on the corneal surface induces A. castellanii to secrete a ∼133-kDa serine protease that kills both human and hamster corneal epithelium and degrades collagen.

AB - PURPOSE. Acanthamoeba trophozoites express a mannose binding receptor that facilitates adhesion of trophozoites to mannosylated proteins on corneal epithelial cells. This study was undertaken to determine the role that mannose stimulation has in the amoeba's growth, secreted products, and ability to desquamate the corneal epithelium. METHODS. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose (PYG) and PYG with 100 mM methyl α-D-mannopyranoside or galactose. The proliferation of trophozoites and cysts was examined by optical density and direct counts. The molecular weight of the mannose-stimulated protein was examined by SDS/PAGE. The cytolytic protein was purified by fast protein liquid chromatography (FPLC) size exclusion and ionic exchange and then tested for cytopathic effect (CPE) and collagenolytic activity in vitro. Collagenolytic activity was examined by zymography. Proteases and protease inhibitors were used to characterize the nature of the cytolytic protein. RESULTS. Methyl α-D-mannopyranoside inhibited the growth of A. castellanii by 50% (P < 0.05) and concomitantly induced a threefold increase in the formation of cysts. SDS-PAGE analysis revealed a mannose-induced protein of ∼133 kDa (MIP-133). The MIP-133 protein was found to be highly cytolytic against corneal epithelial cells, but not human intestinal epithelial cells and also degraded collagen in vitro. Serine protease inhibitors abrogated both CPE and collagenolytic activity of the MIP-133 protein (P < 0.001). CONCLUSIONS. The results suggest that binding of trophozoites to mannosylated proteins on the corneal surface induces A. castellanii to secrete a ∼133-kDa serine protease that kills both human and hamster corneal epithelium and degrades collagen.

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