Effects of the green tea catechin (-)-epigallocatechin gallate on Trypanosoma brucei

Abstract

The current pharmacopeia to treat the lethal human and animal diseases caused by the protozoan parasite Trypanosoma brucei remains limited. The parasite's ability to undergo antigenic variation represents a considerable barrier to vaccine development, making the identification of new drug targets extremely important. Recent studies have demonstrated that fatty acid synthesis is important for growth and virulence of Trypanosoma brucei brucei, suggesting this pathway may have therapeutic potential. The first committed step of fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC), which is a known target of (-)-epigallocatechin-3-gallate (EGCG), an active polyphenol compound found in green tea. EGCG exerts its effects on ACC through activation of AMP-dependent protein kinase, which phosphorylates and inhibits ACC. We found that EGCG inhibited TbACC activity with an EC50 of 37 μM and 55 μM for bloodstream form and procyclic form lysates, respectively. Treatment with 100 μM EGCG induced a 4.7- and 1.7- fold increase in TbACC phosphorylation in bloodstream form and procyclic lysates. EGCG also inhibited the growth of bloodstream and procyclic parasites in culture, with a 48 h EC50 of 33 μM and 27 μM, respectively, which is greater than the EGCG plasma levels typically achievable in humans through oral dosing. Daily intraperitoneal administration of EGCG did not reduce the virulence of an acute mouse model of T. b. brucei infection. These data suggest a reduced potential for EGCG to treat T. brucei infections, but suggest that EGCG may prove to be useful as a tool to probe ACC regulation.

Keywords: ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; Acetyl-CoA carboxylase; BF, bloodstream form; DMSO, dimethyl sulfoxide; EGCG, (−)-epigallocatechin gallate; Epigallocatechin gallate; PF, procyclic form; Phosphorylation; RNAi, RNA interference; SA-HRP, streptavidin conjugated to horseradish peroxidase; Trypanosoma brucei.

Graphical abstract

Fig. 1

Inhibition of TbACC activity by EGCG. TbACC activity in lysates of (A) BF and (B) PF trypanosomes was measured in the presence of 5–100 μM EGCG in the absence (black bars) or presence (grey bars) of HALT phosphatase inhibitor cocktail. As a negative control, ATP was omitted from the reaction (No ATP). Values are expressed as a percentage of the DMSO solvent control. DMSO concentrations were maintained at 1% v/v for all conditions. The mean of 3 independent experiments is shown. Error bars indicate ±SEM. The * indicates P < 0.01 for the difference between DMSO control and EGCG-treated conditions (student’s t-test).

Fig. 2

EGCG induces phosphorylation of TbACC. BF and PF ACC-myc lysates were treated with 100 μM EGCG or DMSO as solvent control in the presence of HALT phosphatase inhbitor cocktail. (A) BF and (B) PF TbACC-myc immunoprecipitates (∼5 × 10⁸ cell equivalents/lane) were resolved by SDS–PAGE and stained with Pro Q Diamond phosphoprotein gel stain to detect phosphorylated ACC (ACC-p; upper panels). Identically loaded gels were blotted to nitrocellulose and probed for total ACC with SA-HRP (ACC-t; bottom panels). Representative gels and blots are shown. Unlabeled upper band in panel A is an unknown phosphorylated contaminant. (C) Densitometric quantitation of phosphorylated ACC from phospho-stained gels normalized to total ACC detected by SA-HRP blotting. The mean of 3 independent experiments is shown. Error bars indicate the ±SEM. The * indicates P < 0.05 and ** indicates P < 0.00001 for the difference between DMSO control and EGCG-treated conditions (student’s t-test).

Fig. 3

Effect of EGCG on in vitro growth of T. brucei. (A and B) WT BF cells were diluted back to 1 × 10⁶ cells/ml into fresh media containing 0.1–1 μM EGCG or DMSO as the solvent control and culture cell densities were monitored for 6 days. (A) Representative growth curve showing cumulative culture densities. (B) Mean culture doubling times of 3 independent experiments. Error bars show ±SEM. (C) WT BF and (D) WT PF cells were back-diluted into fresh media containing 5–50 μM EGCG or DMSO control. Culture cell densities were determined after 48 h. Values are expressed as a percentage of DMSO control. The mean of 4 independent experiments is shown. Error bars show ±SEM. The * indicates P < 0.01 for the difference between DMSO and EGCG-treated conditions (student’s t-test).