Probing sterol biosynthesis chokepoint enzymes in Naegleria gruberi for treatment of amoeba diseases
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Naegleria gruberi (NG) is a non-pathogenic amoeba that is closely related to Naegleria fowleri a.k.a. the brain-eating amoeba, responsible for primary amebic meningoencephalitis (PAM). Currently the treatment involves several combinations of antibiotics, usually including Amphotericin B which is known to complex with ergosterol in the membrane. In studies described herein, NG strain NEG-M, grown axenically in ATCC 1034 media supplemented with heat-inactivated fetal bovine serum (FBS), was shown to synthesize C28 ergosterol in trophozoites and to uptake C27 cholesterol in cysts from the growth medium. The distinct differentiated forms of trophozoite and cyst were examined microscopically by using light microscopy, scanning electron microscopy, and transmission electron microscopy. The physiological significance of two potential chockepoint enzymes in the NG ergosterol biosynthesis pathway C24-sterol methyl transferase (24-SMT) and 14α-demethylase (CYP51) was probed using the ergosterol biosynthesis inhibitors 25-azalanosterol, 25-azacyloartenol, itraconazole, voriconazole. Both sterol inhibitors inhibited the ergosterol biosynthesis pathway causing accumulation of Δ24-sterol corresponding to 24-SMT inhibition and 14-methyl sterol accumulation corresponding to CYP51 inhibition and inhibition of amoeba growth in nanomolar range. The results show that SMT and CYP51 are essential enzymes in the ergosterol biosynthesis pathway of N. gruberi. Interference of these enzymes by high affinity inhibitors can lead to cell death.