Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth

Abstract

Abbreviations: AZA, 25-azalanosterol; BSF, bloodstream form; DOX, doxycycline; ED, effective dose; FGM, full-growth medium; ITC, itraconazole; LDM, lipid-depleted medium; PCF, procyclic form; RRTc, relative retention time with cholesterol used as standard; SAM, S-adenosyl-lmethionine; SDM, sterol C14-demethylase; SMT, sterol C24-methyltransferase; Tb SDM, Trypanosoma brucei analyzed by RNAi silencing and inhibition of sterol C14-demethylase; Tb SMT, Trypanosoma brucei analyzed by RNAi silencing and inhibition of sterol C24-methyltransferase; TetR, tetracycline repressor .

Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei

“…The compound added at various concentrations to the medium of T. brucei procyclic (PCF) and bloodstream (BSF) forms and human epithelial kidney (HEK) cell cultures generated IC 50 values of approximately 3 μM for PCF and 16 μM for BSF (Figure 2) while 26FL had no effect on HEK cell growth up to 100 μM (data not shown). The dose of 26FL causing 50% cell growth inhibition correlates to a moderate therapeutic index [ED 50HEK /ED 50bloodstream ] of 6.2 with cells remaining viable even at the IC99 value of the drug (Haubrich et al, 2015). …”

“…This diversity in sterol metabolism between T. brucei and its human host has presented the intriguing possibility that protozoan biochemistry could be selectively inhibited. Indeed, our recent discovery shows there is an essential requirement for hormonal levels of ergosterol to “spark” (signal) cell proliferation of T. brucei bloodstream forms (Figure 1A) which cannot be satisfied solely by cholesterol salvaged from the host (Haubrich et al, 2015). These studies, plus the demonstration that inhibitors shown to block enzymes in the post-squalene segment of ergosterol biosynthesis pathway prevent growth of many protozoan parasites without effect on cultured animal cells (Gigante et al, 2009; Gros et al, 2006; Gunatilleke et al, 2012; Lepesheva et al, 2007; Lepesheva and Waterman, 2011; Lorente et al, 2004; Lovieno et al, 2014; Porta et al, 2014; Rahman and Pascal., 1990; Zhou et al, 2007) confirms our idea.…”

Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY‐2 Cells

“…This scenario might not hold true for other organisms, like the protozoan Trypanosoma brucei. In this organism, whose sterol biosynthesis depends on ergosterol, inducible amiRNA silencing of TbSMT (coding for C24-β-methyl transferase) and of TbSDM (coding for sterol 14α-demethylase) was shown to be more efficient than in our system, albeit not complete [30]. Nonetheless, some effects on sterol accumulation in estradiol-induced cells versus vector BY-2 control cells could be seen.…”

“…A further approach to study the functional importance of a particular enzyme reaction is its inhibition by specific inhibitors [24][25][26][27][28][29][30], or by down-regulation of gene expression [30][31][32]. In this contribution we report on the inhibition by RO 48-8071 [35] and gene down-regulation strategies for the functional study of tobacco CAS in relation to growth and phytosterol accumulation.…”

In Vitro and In Vivo Investigation of the Inhibition of Trypanosoma brucei Cell Growth by Lipophilic Bisphosphonates

“…For example, in most trypanosomatid parasites, such as Trypanosoma cruzi and the Leishmania spp., an important drug target is ergosterol biosynthesis, since ergosterol is the primary membrane sterol and plays a structural role similar to that of cholesterol in mammalian cells. In T. brucei, the main membrane sterol is cholesterol, which is imported from the mammalian host (or culture medium) through receptor-mediated endocytosis of low-density lipoproteins (6,7); however, low levels of endogenously synthesized ergosterol-related sterols are also present, and some sterol biosynthesis inhibitors inhibit T. brucei cell growth (8).…”