Multiple Triclosan Targets in Trypanosoma brucei

Abstract

Trypanosoma brucei genes encoding putative fatty acid synthesis enzymes are homologous to those encoding type II enzymes found in bacteria and organelles such as chloroplasts and mitochondria. It was therefore not surprising that triclosan, an inhibitor of type II enoyl-acyl carrier protein (enoyl-ACP) reductase, killed both procyclic forms and bloodstream forms of T. brucei in culture with 50% effective concentrations (EC 50 s) of 10 and 13 M, respectively. Triclosan also inhibited cell-free fatty acid synthesis, though much higher concentrations were required (EC 50 s of 100 to 200 M). Unexpectedly, 100 M triclosan did not affect the elongation of [ 3 H]laurate (C 12:0 ) to myristate (C 14:0 ) in cultured bloodstream form parasites, suggesting that triclosan killing of trypanosomes may not be through specific inhibition of enoyl-ACP reductase but through some other mechanism. Interestingly, 100 M triclosan did reduce the level of incorporation of [ 3 H]myristate into glycosyl phosphatidylinositol species (GPIs). Furthermore, we found that triclosan inhibited fatty acid remodeling in a cell-free assay in the same concentration range required for killing T. brucei in culture. In addition, we found that a similar concentration of triclosan also inhibited the myristate exchange pathway, which resides in a distinct subcellular compartment. However, GPI myristoylation and myristate exchange are specific to the bloodstream form parasite, yet triclosan kills both the bloodstream and procyclic forms. Therefore, triclosan killing may be due to a nonspecific perturbation of subcellular membrane structure leading to dysfunction in sensitive membrane-resident biochemical pathways.

A Pathogenic Fungi Diphenyl Ether Phytotoxin Targets Plant Enoyl (Acyl Carrier Protein) Reductase

“…Triclosan is a relatively small antimicrobial diphenyl ether that is absorbed via diffusion into the bacterial cell wall and causes light-independent disruption of the cellular membranes (Guillén et al, 2004;Paul et al, 2004). The first evidence that this diphenyl ether is a strong inhibitor of fatty acid biosynthesis came when genetic analysis of an E. coli strain resistant to triclosan linked the resistance to the type II fatty acid synthase gene, which encodes for ENR (McMurry et al, 1998).…”

“…Inhibition of ENR interrupts the critical process of fatty acid elongation. It has also been reported that this compound can intercalate itself within the lipid bilayer and compromise the functional integrity of the cell membranes of microorganisms (Villalain et al, 2001;Paul et al, 2004). The objective of this study was to determine whether cyperin had the same molecular target site as triclosan.…”

Mitochondrial Fatty Acid Synthesis in Trypanosoma brucei

“…Some reactions contained 50 M butyryl-CoA as a potential primer. Lipids were then extracted in chloroform/methanol/water (10:10:3) and converted to methyl esters for chain-length analysis by fractionation on reversed-phase TLC (10,14,30). TLCs were exposed to a phosphorimaging screen and quantitated on a Fujifilm BAS-2500 phosphorimaging device.…”

“…Fatty Acid Synthesis Assays Using Total Lysates-BSF knockouts of either ELO1 or -3 (14) were hypotonically lysed (5 ϫ 10 8 cell equivalents/ml) as previously described (10,14,30), except cells were grown in culture (instead of in rats) and lysates were incubated for 20 min on ice before being restored to isotonic conditions. In addition, the membrane-washing steps used previously were omitted; instead, total lysates were used.…”

“…Lysate preparation for these experiments resembled methods previously used to assay ELO activity (10,14,30), except washed membranes were replaced with total lysates (which were 40% more active than washed membranes (data not shown)). For these assays, we often left out exogenous primers, normally required for ELO activity, and we included NAD(P)H, ATP, and CoA, which individually enhanced mitochondrial FAS by 2-, 4-, and 2-fold, respectively (data not shown).…”