Glucose metabolism in Giardia intestinalis

doi:10.1016/0166-6851(91)90025-2

Molecular and Biochemical Parasitology

Volume 45, Issue 1, March 1991, Pages 39-47

https://doi.org/10.1016/0166-6851(91)90025-2 Get rights and content

Abstract

The effect of glucose and other monosaccharides on Giardia intestinalis was investigated by growing G. intestinalis trophozoites in Diamond's TYI-S-33 medium modified by changes in the monosaccharide component, and observing changes in the trophozoite growth and product formation (alanine, ethanol and acetate). Reducing the glucose concentration from 50 mM to 10 mM had little effect on trophozoite growth and product formation. Below 10 mM glucose, ethanol production was markedly reduced, there was a lesser effect on alanine, but acetate production was unaffected. In medium in which no glucose had been added, trophozoites grew at about half the rate of controls (50 mM glucose) and continued to form the same products. Growth in medium containing 10 mM ribose or 10 mM fructose substituted for glucose produced a metabolic profile similar to that of the no glucose added condition. The activity of a number of glycolytic and related enzymes was also determined, but the enzymic profile was not affected by the monosaccharide status of the medium. Ethanol production by trophozoites was specifically depressed by the aldehyde reductase inhibitor, valproate; 3 mM valproate reduced ethanol production by 90%. The alcohol dehydrogenase inhibitor pyrazole had no effect on ethanol production or any other parameter. This differential inhibition suggests that ethanol is produced by an aldehyde reductase or related enzyme. The observations that G. intestinalis trophozoites can continue to grow, replicate and produce the same metabolites in medium containing little or no glucose suggest that G. intestinalis is not solely dependent on glucose as a metabolic fuel. It seems likely that G. intestinalis utilises other carbon sources, particularly nitrogenous compounds, and that the energy metabolism is more complex than previously believed.

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Lipid and carbohydrate metabolism of Giardia lamblia
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The pyruvate-phosphate dikinase reaction

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Cited by (45)

<sup>1</sup>H HR-MAS NMR spectroscopy to study the metabolome of the protozoan parasite Giardia lamblia
2018, Talanta
Citation Excerpt :
1H nuclear magnetic resonance (NMR) spectroscopy of solutions is a powerful and well established tool for the direct qualitative and quantitative analysis of small metabolites in mixtures including biological samples without the need of laborious sample preparation steps such as preceding chromatographic separation [19–21]. NMR spectroscopy is less sensitive than other methods like mass spectrometry, but largely benefits from the ease of sample preparation and high reproducibility [21,22] and has successfully been applied for metabolic studies with G. lamblia [11–13]. High resolution magic angle spinning (HR-MAS) NMR spectroscopy allows obtaining well resolved liquid-like 1H NMR spectra from semi-solid materials, which under static conditions would give rise to broad NMR resonances.
Knowledge of the metabolic profile and exchange processes in the protozoan parasite Giardia lamblia is of importance for a better understanding of the biochemical processes and for the development of drugs to control diseases caused by G. lamblia. In the current paper, ¹H High Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy was directly applied to G. lamblia trophozoite suspensions to analyze the detectable small metabolites with a minimum of intervention. Thirty-one components were identified with main contributions from amino acids such as alanine and ornithine. The reproducibility, variability, and stability of the metabolites were investigated. Citrulline was found to be formed as an intermediate and citrulline levels depended on the stage of cell growth. Glucose-1-phosphate was found to be formed in relatively high amounts after cell harvesting if enzymes were not inactivated. In addition, the metabolic footprint of Giardia trophozoites, i.e. changes in the culture medium induced by G. lamblia, was investigated by liquid state NMR spectroscopy of culture media before and after inoculation. A quantitative comparison of the NMR spectra revealed component changes in the culture media during growth. The results suggested that not glucose but rather arginine serves as main energy supply. Biochemical functions of intracellular components and their metabolic exchange with the culture medium are discussed. The results provide an important basis for the design of HR-MAS NMR based metabolomic studies of G. lamblia in particular and any protozoan parasite samples in general.
Proteomic and transcriptional analyses of genes differentially expressed in Giardia duodenalis clones resistant to albendazole
2013, Infection, Genetics and Evolution
Citation Excerpt :
Noteworthy the levels of TPI and tpi mRNA in Abz-resistant G. duodenalis clones were lower than in Abz-sensitive clones. In G. duodenalis, the main source of ATP is the glycolytic pathway (Schofield et al., 1991). TPI is an ubiquitous glycolytic enzyme that catalyzes the reversible isomerization between (R)-glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).
In this study we performed proteomic and transcriptional analyses to identify and characterize genes differentially expressed in Giardia duodenalis clones resistant to albendazole. The expression of proteins and their corresponding mRNAs was analyzed in clones resistant in vitro to different concentrations of albendazole (1.35, 8.0 and 250 μM) and these were compared with albendazole-sensitive clones using two approaches: (1) two-dimensional protein electrophoresis to analyze the proteome by the LC-MS/MS technique, and (2) semi-quantitative RT-PCR to assess the mRNA levels of proteins with the highest levels of differential expression .This strategy allowed the identification of eight proteins differentially expressed in albendazole resistant clones with roles in: (a) the cytoskeletal system (alpha 2-giardin and RanBP1), (b) the antioxidant metabolism (NADH oxidase) and (c) energy metabolism (triosephosphate isomerase, phosphoglycerate kinase and ornithine carbamoyltransferase). Gene expression analyses of these genes correlated well with the proteomics results. These observations suggest that resistance to albendazole in Giardia encompasses a complex response involving an altered expression of genes regulated at the transcriptional level that might have an important role in maintaining cell structural stability, coping with oxidative stress and adapting energy supply to a new metabolic status. These molecules are indeed promising targets for drug development.
Species-specific inhibition of Giardia lamblia triosephosphate isomerase by localized perturbation of the homodimer
2008, Molecular and Biochemical Parasitology
Giardia lamblia depends on glycolysis to obtain ATP, highlighting the suitability of glycolytic enzymes as targets for drug design. We studied triosephosphate isomerase from G. lamblia (GlTIM) as a potential species-specific drug target. Cysteine-reactive agents were used as probes, in order to test those regions near to cysteine residues as targets to perturb enzyme structure and activity. Methyl methanethiosulfonate (MMTS) derivatized three of the five Cys per subunit of dimeric GlTIM and induced 50% of inactivation. The 2-carboxyethyl methanethiosulfonate (MTSCE) modified four Cys and induced 97% of inactivation. Inactivation by MMTS or MTSCE did not affect secondary structure, nor induce dimer dissociation; however, Cys modification decreased thermal stability of enzyme. Inactivation and dissociation of the dimer to stable monomers were reached when four Cys were derivatized by 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB). The effects of DTNB were completely abolished when GlTIM was first treated with MMTS. The effect of thiol reagents on human TIM was also assayed; it is 180-fold less sensitive than GlTIM. Collectively, the data illustrate GlTIM as a good target for drug design.
Disulfide Bridges in the Mesophilic Triosephosphate Isomerase from Giardia lamblia Are Related to Oligomerization and Activity
2007, Journal of Molecular Biology
Triosephosphate isomerase from the mesophile Giardia lamblia (GlTIM) is the only known TIM with natural disulfide bridges. We previously found that oxidized and reduced thiol states of GlTIM are involved in the interconversion between native dimers and higher oligomeric species, and in the regulation of enzymatic activity. Here, we found that trophozoites and cysts have different oligomeric species of GlTIM and complexes of GlTIM with other proteins. Our data indicate that the internal milieu of G. lamblia is favorable for the formation of disulfide bonds. Enzyme mutants of the three most solvent exposed Cys of GlTIM (C202A, C222A, and C228A) were prepared to ascertain their contribution to oligomerization and activity. The data show that the establishment of a disulfide bridge between two C202 of two dimeric GlTIMs accounts for multimerization. In addition, we found that the establishment of an intramonomeric disulfide bond between C222 and C228 abolishes catalysis. Multimerization and inactivation are both reversed by reducing conditions. The 3D structure of the C202A GlTIM was solved at 2.1 Å resolution, showing that the environment of the C202 is prone to hydrophobic interactions. Molecular dynamics of an in silico model of GlTIM when the intramonomeric disulfide bond is formed, showed that S216 is displaced 4.6 Å from its original position, causing loss of hydrogen bonds with residues of the active-site loop. This suggests that this change perturb the conformational state that aligns the catalytic center with the substrate, inducing enzyme inactivation.
Na<sup>+</sup>-dependent pH Regulation by the Amitochondriate Protozoan Parasite Giardia intestinalis
2001, Journal of Biological Chemistry
Citation Excerpt :
In the absence of exogenous substrate, it is likely that the trophozoites were reliant on endogenous glucose stores (e.g. glycogen, Ref. 25) and that the metabolism was therefore essentially the same as that occurring in cells supplemented with glucose. Glucose metabolism in G. intestinalis results in the production of the acidic end-product acetic acid (26, 27), whereas arginine catabolism generates ammonia and ornithine, alkaline and neutral products, respectively (6). The pronounced extracellular acidification observed in suspensions in which the cells were utilizing glucose is likely to be due primarily to the production of acetic acid whereas the lesser acidification observed in cells supplemented with arginine probably represents the combined effects of arginine and glucose (glycogen) metabolism.
Giardia intestinalis is a pathogenic fermentative parasite, which inhabits the gastrointestinal tract of animals and humans. G. intestinalis trophozoites are exposed to acidic fluctuations in vivo and must also cope with acidic metabolic endproducts. In this study, a combination of independent techniques (³¹P NMR spectroscopy, distribution of the weak acid pH marker 5,5-dimethyl-2,4-oxazolidinedione (DMO) and the fluorescent pH indicator 2′,7′-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF)) were used to show thatG. intestinalis trophozoites exposed to an extracellular pH range of 6.0–7.5 maintain their cytosolic pH (pH_i) within the range 6.7–7.1. Maintenance of the resting pH_i was Na⁺-dependent but unaffected by amiloride (or analogs thereof). Recovery of pH_i from an intracellular acidosis was also Na⁺-dependent, with the rate of recovery varying with the extracellular Na⁺concentration in a saturable manner (K_m = 18 mm; V_max = 10 mmH⁺ min⁻¹). The recovery of pH_ifrom an acid load was inhibited by amiloride but unaffected by a number of its analogs. The postulated involvement of one or more Na⁺/H⁺ exchanger(s) in the regulation of pH_i in G. intestinalis is discussed.
Purification and characterization of recombinant pyruvate phosphate dikinase from Giardia
1999, Molecular and Biochemical Parasitology
The gene encoding pyruvate phosphate dikinase (PPDK) from Giardia duodenalis was expressed using a baculovirus system. The recombinant enzyme was purified to homogeneity and its enzymological and solution structure properties characterized. The catalytic constant for the pyruvate-producing reaction was about twice as high (1560 min⁻¹ at 30°C) as that for the reverse reaction (700 min⁻¹) and the k_cat/K_m for PP_i was about two orders of magnitude higher than k_cat/K_m for P_i, indicating that the pyruvate-forming reaction is much more efficient than the reverse, phosphoenolpyruvate (PEP)-forming process. The endogenous substrate levels found for PEP (0.5 mM) and pyruvate (<80 μM) support the assumption that, under physiological conditions, the enzyme primarily performs a catabolic function. The molecular mass of the purified recombinant PPDK was analyzed by analytical ultracentrifugation and size exclusion chromatography using different assay conditions that have been reported to affect the quaternary structure of PPDKs in other organisms. Both methods clearly indicated a dimeric structure for giardial PPDK with a molecular mass of about 197 kDa (monomer mass 97.6 kDa). Several compounds, primarily structural analogs of PP_i, were tested for their ability to inhibit PPDK activity. Most of the bisphosphonates examined showed either no, or only a moderate, inhibitory effect on the enzyme. Imidodiphosphate was the only competitive inhibitor with respect to PP_i (K_ic=0.55 mM), whereas the bisphosphonates produced a mixed type of inhibition. The most active compound in inhibiting PPDK activity was oxalate, with a K_ic value of less than 1 μM with respect to PEP.

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Molecular and Biochemical Parasitology

Abstract

References (17)

Energy metabolism of the anaerobic protozoan Giardia lamblia

Mol. Biochem. Parasitol.

Alanine is a major end product of metabolism by Giardia lamblia: a proton nuclear magnetic resonance study

Mol. Biochem. Parasitol.

Does Giardia intestinalis need glucose as an energy source?

Int. J. Parasitol.

Lipid and carbohydrate metabolism of Giardia lamblia

Mol. Biochem. Parasitol.

Nicotinamide-adenine dinucleotide phosphate dependent alcohol dehydrogenase: the enzyme from Entamoeba histolytica and some enzyme inhibitors

Int. J. Biochem.

Mannose metabolism and demonstration of mannokinase and phosphomannoisomerase activity in the lactating rat mammary gland

Arch. Biochem. Biophys.

The metabolism of phosphoenolpyruvate and pyruvate in adult liver fluke Fasciola hepatica

Biochim. Biophys. Acta

The pyruvate-phosphate dikinase reaction

Cited by (45)

<sup>1</sup>H HR-MAS NMR spectroscopy to study the metabolome of the protozoan parasite Giardia lamblia

Proteomic and transcriptional analyses of genes differentially expressed in Giardia duodenalis clones resistant to albendazole

Species-specific inhibition of Giardia lamblia triosephosphate isomerase by localized perturbation of the homodimer

Disulfide Bridges in the Mesophilic Triosephosphate Isomerase from Giardia lamblia Are Related to Oligomerization and Activity

Na<sup>+</sup>-dependent pH Regulation by the Amitochondriate Protozoan Parasite Giardia intestinalis

Purification and characterization of recombinant pyruvate phosphate dikinase from Giardia