Plasmodial sugar transporters as anti-malarial drug targets and comparisons with other protozoa

Glucose is the primary source of energy and a key substrate for most cells. Inhibition of cellular glucose uptake (the first step in its utilization) has, therefore, received attention as a potential therapeutic strategy to treat various unrelated diseases including malaria and cancers. For malaria, blood forms of parasites rely almost entirely on glycolysis for energy production and, without energy stores, they are dependent on the constant uptake of glucose. Plasmodium falciparum is the most dangerous human malarial parasite and its hexose transporter has been identified as being the major glucose transporter. In this review, recent progress regarding the validation and development of the P. falciparum hexose transporter as a drug target is described, highlighting the importance of robust target validation through both chemical and genetic methods. Therapeutic targeting potential of hexose transporters of other protozoan pathogens is also reviewed and discussed

Host Cell Egress and Invasion Induce Marked Relocations of Glycolytic Enzymes in Toxoplasma gondii Tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K+] experienced during egress and invasion, a signal that requires changes of [Ca2+]c in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility.

BACKGROUND: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. METHODS: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. RESULTS: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. CONCLUSIONS: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization

Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae)

The green alga Haematococcus pluvialis produces large amounts of the pink carotenoid astaxanthin under high photon flux density (PFD) and other oxidative stress conditions. However, the regulation and physiological role of carotenogenesis leading to astaxanthin formation is not well understood. Comparative transcriptional expression of five carotenoid genes along with growth and pigment composition as a function of PFD was studied using a wild-type and an astaxanthin-overproduction mutant of H. pluvialis NIES144. The results indicate that astaxanthin biosynthesis was mainly under transcriptional control of the gene encoding carotenoid hydroxylase, and to a lesser extent, the genes encoding isopentenyl isomerase and phytoene desaturase, and to the least extent, the genes encoding phytoene synthase and carotenoid oxygenase. The expression of a plastid terminal oxidase (PTOX) gene ptox2 underwent transient up-regulation under elevated PFDs, suggesting that PTOX may be functionally coupled with phytoene desaturase through the plastoquinone pool and may play a role in reducing redox-potential-dependent and oxygen-concentration-dependent formation of reactive oxygen species in the chloroplast. Over-expression of both the carotenogenic and PTOX genes confers to the astaxanthin-overproduction mutant more effective photoprotective capability than that of the wild type under photooxidative stress

Targeting pathogen metabolism without collateral damage to the host

The development of drugs that can inactivate disease-causing cells (e.g. cancer cells or parasites) without causing collateral damage to healthy or to host cells is complicated by the fact that many proteins are very similar between organisms. Nevertheless, due to subtle, quantitative differences between the biochemical reaction networks of target cell and host, a drug can limit the flux of the same essential process in one organism more than in another. We identified precise criteria for this â €network-based' drug selectivity, which can serve as an alternative or additive to structural differences. We combined computational and experimental approaches to compare energy metabolism in the causative agent of sleeping sickness, Trypanosoma brucei, with that of human erythrocytes, and identified glucose transport and glyceraldehyde-3-phosphate dehydrogenase as the most selective antiparasitic targets. Computational predictions were validated experimentally in a novel parasite-erythrocytes co-culture system. Glucose-transport inhibitors killed trypanosomes without killing erythrocytes, neurons or liver cells

Identification, expression and characterisation of a Babesia bovis hexose transporter

Babesia are tick-transmitted haemoprotozoan parasites that infect cattle, with an estimated 500 million at risk worldwide. Here, two predicted hexose transporters (BboHT1 and 2) have been identified within the Babesia bovis genome. BboHT1, having 40% and 47% amino acid sequence similarity compared with the human (GLUT1) and Plasmodium falciparum (PfHT) hexose transporters, respectively, is the only one that could be characterised functionally after expression in Xenopus laevis oocytes. Radiotracer studies on BboHT1 showed that it is a saturable, Na+-independent, stereo-specific hexose transporter, with a Km value for glucose of 0.84 ± 0.54 mM (mean ± SEM). Using d-glucose analogues, hydroxyl positions at O-4 and O-6 have been identified as important for ligand binding to BboHT1. d-Glucose transport was inhibited maximally by cytochalasin B (50 μM). A long-chain O-3 hexose derivative (compound 3361) that selectively inhibits PfHT also inhibited relatively potently BboHT1, with an apparent Ki value of 4.1 ± 0.9 μM (mean ± SEM). Compound 3361 did not inhibit B. bovis proliferation in in vitro growth assays but inhibited invasion of glucose-depleted bovine erythrocytes. Taken together with results of inhibition studies with cytochalasin B and β-glucogallin, these data provide new insights into glucose metabolism of erythrocytic-stage Babesia infections

Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues

Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (−)-Epicatechin-gallate (ECG) and (−)-epigallocatechin-gallate (EGCG) inhibited d-glucose transport by GLUT1 and PfHT, and d-fructose transport by GLUT5, with apparent Ki values between 45 and 117 μM. BboHT1 was more potently inhibited by the ungallated catechins (−)-epicatechin (EC) and (−)-epigallocatechin (EGC), with apparent Ki values of 108 and 168 μM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external d-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents