Experimental and in Silico Analyses of Glycolytic Flux Control in Bloodstream Form Trypanosoma brucei

Abstract

A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity (but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes (the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.Trypanosomatid parasites (Trypanosoma and Leishmania) are responsible for serious diseases of mankind in tropical and subtropical countries worldwide. These diseases affect millions of people, and hundreds of millions are at risk of becoming infected. Unfortunately, available treatments are largely inadequate. Currently used drugs are inefficacious and toxic. There is a desperate need for new effective and safe drugs, particularly in view of the development and spreading of drug resistance (1).Glycolysis plays an important role in the energy metabolism of these protozoan organisms, notably of Trypanosoma brucei when it lives in the blood of its mammalian host, causing a disease called sleeping sickness or human African trypanosomiasis in man and nagana in cattle (2, 3). This bloodstream form of T. brucei is entirely dependent on the conversion of the blood sugar glucose into pyruvate for its ATP supply. Oxidative metabolism involving mitochondrial Krebs cycle enzymes and oxidative phosphorylation are largely repressed. Therefore, glycolysis has been perceived as a potentially good target for anti-trypanosome drugs. Moreover, the glycolytic pathway in trypanosomatids is organized in a unique manner: the majority of the glycolytic enzymes are sequester...

Glycerol 3-Phosphate Alters Trypanosoma brucei Hexokinase Activity in Response to Environmental Change

“…Reflecting metabolic differences in the life stages, glycolytic proteins make up ϳ90% of the protein content in BSF glycosomes, whereas glycolytic proteins make up only ϳ40 -50% of the protein content in PF glycosomes (24 -26). Nevertheless, TbHKs are important to both life stages, as TbHK1 and TbHK2 have been shown to be essential to BSF parasites (27,28), whereas TbHK2 Ϫ/Ϫ PF parasites are viable, but the knockout has a pleiotropic impact on the cells. Viable TbHK1 Ϫ/Ϫ PF cells have not yet been isolated despite repeated attempts under different growth conditions, 4 suggesting that TbHK1 is also essential to PF cells.…”

The silicon trypanosome

“…Protein phosphorylation may contribute, since a glycosomal phosphatase has been identified in developmental signalling (Szoor and Matthews, unpublished data). The inhibition of anaerobic glycolysis by glycerol was also reproduced by the model, first qualitatively and then quantitatively (Bakker et al 1997 ;Albert et al 2005). …”

“…Since the publication of the first version of the glycolysis model (Bakker et al 1997), there have been two major updates (Helfert et al 2001 ;Albert et al 2005). Both of these involved updates and extensions of the enzyme kinetic dataset, e.g.…”

“…the form that replicates in the mammalian bloodstream) has been developed (Bakker et al 1997) and iteratively updated after experimental testing Albert et al 2005 ;Haanstra et al 2008 a). This model yields quantitative predictions of the flux through glycolysis, the concomitant ATP production flux, and the concentrations of glycolytic metabolites, at steady state as well as following a perturbation.…”

“…the explicit inclusion of individual enzymes that were previously grouped into a net multi-step conversion. In the second update (Albert et al 2005) the enzyme expression levels (V max ) were adapted to reflect the concentrations observed in trypanosomes obtained from controlled state-of-the-art in vitro cultivation. Key missing pieces of information remain the mechanism and kinetics of the transport of glycolytic metabolites across the glycosomal membrane.…”