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Characterisation of sterol biosynthesis and validation of 14?-demethylase as a drug target in Acanthamoeba.

ABSTRACT: The soil amoebae Acanthamoeba causes Acanthamoeba keratitis, a severe sight-threatening infection of the eye and the almost universally fatal granulomatous amoebic encephalitis. More effective treatments are required. Sterol biosynthesis has been effectively targeted in numerous fungi using azole compounds that inhibit the cytochrome P450 enzyme sterol 14?-demethylase. Herein, using Gas Chromatography Mass Spectrometry (GCMS), we demonstrate that the major sterol of Acanthamoeba castellanii is ergosterol and identify novel putative precursors and intermediate sterols in its production. Unlike previously reported, we find no evidence of 7-dehydrostigmasterol or any other phytosterol in Acanthamoeba. Of five azoles tested, we demonstrate that tioconazole and voriconazole have the greatest overall inhibition for all isolates of Acanthamoeba castellanii and Acanthamoeba polyphaga tested. While miconazole and sulconazole have intermediate activity econazole is least effective. Through GCMS, we demonstrate that voriconazole inhibits 14?-demethylase as treatment inhibits the production of ergosterol, but results in the accumulation of the lanosterol substrate. These data provide the most complete description of sterol metabolism in Acanthamoeba, provide a putative framework for their further study and validate 14?-demethylase as the target for azoles in Acanthamoeba.

SUBMITTER: Thomson S

PROVIDER: S-EPMC5557935 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Characterisation of sterol biosynthesis and validation of 14α-demethylase as a drug target in Acanthamoeba.

Thomson Scott S Rice Christopher A CA Zhang Tong T Edrada-Ebel RuAngelie R Henriquez Fiona L FL Roberts Craig W CW

Scientific reports 20170815 1

The soil amoebae Acanthamoeba causes Acanthamoeba keratitis, a severe sight-threatening infection of the eye and the almost universally fatal granulomatous amoebic encephalitis. More effective treatments are required. Sterol biosynthesis has been effectively targeted in numerous fungi using azole compounds that inhibit the cytochrome P450 enzyme sterol 14α-demethylase. Herein, using Gas Chromatography Mass Spectrometry (GCMS), we demonstrate that the major sterol of Acanthamoeba castellanii is e ...[more]

PMID: 28811501

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Project description:The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ?8,14-sterol (<95%). The combination of [2H3-methyl]methionine incubation to intact cultures showing C28-ergosterol incorporates 2-2H atoms and C29-7-dehydroporiferasterol incorporates 5 2H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ?24(28)- and ?25(27)-olefin pathways to C28- and C29-sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C28/C29-?5,7-sterols, viable encysted cells (mature cyst) possess mostly C29-?5-sterol and non-viable encysted cells possess C28/C29-?5,7-sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ?5,7-sterol biosynthesis (to ?5,7) - metabolism (to ?5 or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections.

Dataset Information

Characterisation of sterol biosynthesis and validation of 14?-demethylase as a drug target in Acanthamoeba.

Publications

Characterisation of sterol biosynthesis and validation of 14α-demethylase as a drug target in Acanthamoeba.

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