Dr Markus Winterberg

Research Area: Global Health
Technology Exchange: Drug discovery, Mass spectrometry and Protein interaction
Scientific Themes: Tropical Medicine & Global Health and Physiology, Cellular & Molecular Biology
Keywords: Clinical Pharmacology, Bioanalysis, Proteomics, Metabolomics, Biomarker, Drug Metabolism, Mass Spectrometry and Biochemistry
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Markus Winterberg heads the biochemistry laboratory and the ISO 15189 accredited routine laboratory for drug quantification in the Department of Clinical Pharmacology at MORU. The key aspect of his research is ‘trop-med-omics’, the application of mass spectrometry-based bioanalysis in tropical medicine.

Having a background in malaria physiology and biochemistry, he pursues his research on the metabolism of antimalarial drugs and the discovery of biomarkers for tropical diseases. The aim of his research is to gain a deeper understanding of the interaction between host, pathogen and drug and uncover markers for the identification of pathogens and their drug resistants mechanisms. The definitive goal is the translation of laboratory research into non-invasive, field-based assays for the identification of pathogens, improving diagnostics and treatment of patients. 

Name Department Institution Country
Professor Joel Tarning Tropical Medicine Oxford University, Bangkok Thailand
Dr Warunee Hanpithakphong Tropical Medicine Oxford University, Bangkok Thailand
Dr Daniel Blessborn Tropical Medicine Oxford University, Bangkok Thailand
Dr Thomas Pouplin Tropical Medicine Oxford University, Bangkok Thailand
Dr Piyanan Assawasuwannakit Tropical Medicine Oxford University, Bangkok Thailand
Dr Richard Hoglund Tropical Medicine Oxford University, Bangkok Thailand
Professor Sir Nicholas J White FRS Tropical Medicine Oxford University, Bangkok Thailand
Professor Nicholas PJ Day FMedSci FRCP Tropical Medicine Oxford University, Bangkok Thailand
Professor Daniel H Paris Tropical Medicine Oxford University, Bangkok Thailand
Dr Kesinee Chotivanich Tropical Medicine Oxford University, Bangkok Thailand
Professor Kiaran Kirk Research School of Biology The Australian National University Australia
Dr Onrapak Reamtong Department of Molecular Tropical Medicine and Genetics Mahidol University Thailand
Dr Poom Adisakwattana Department of Helminthology Mahidol University Thailand
Winterberg M, Kirk K. 2016. A high-sensitivity HPLC assay for measuring intracellular Na(+) and K(+) and its application to Plasmodium falciparum infected erythrocytes. Sci Rep, 6 (1), pp. 29241. | Show Abstract | Read more

The measurement of intracellular ion concentrations, and the screening of chemical agents to identify molecules targeting ion transport, has traditionally involved low-throughput techniques. Here we present a novel HPLC method that allows the rapid, high-sensitivity measurement of cell Na(+) and K(+) content, demonstrating its utility by monitoring the ionic changes induced in the intracellular malaria parasite by the new spiroindolone antimalarial KAE609.

Marchetti RV, Lehane AM, Shafik SH, Winterberg M, Martin RE, Kirk K. 2015. A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum. Nat Commun, 6 pp. 6721. | Show Abstract | Read more

The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formate-nitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate. The transport characteristics of PfFNT in oocytes (pH-dependence, inhibitor-sensitivity and kinetics) are similar to those of the transport of lactate and formate across the plasma membrane of mature asexual-stage P. falciparum trophozoites, consistent with PfFNT playing a major role in the efflux of lactate and hence in the energy metabolism of the intraerythrocytic parasite.

Yabas M, Coupland LA, Cromer D, Winterberg M, Teoh NC, D'Rozario J, Kirk K, Bröer S, Parish CR, Enders A. 2014. Mice deficient in the putative phospholipid flippase ATP11C exhibit altered erythrocyte shape, anemia, and reduced erythrocyte life span. J Biol Chem, 289 (28), pp. 19531-19537. | Show Abstract | Read more

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.

Teng R, Lehane AM, Winterberg M, Shafik SH, Summers RL, Martin RE, van Schalkwyk DA, Junankar PR, Kirk K. 2014. 1H-NMR metabolite profiles of different strains of Plasmodium falciparum. Biosci Rep, 34 (6), pp. e00150. | Show Abstract | Read more

Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite.

Chan XWA, Wrenger C, Stahl K, Bergmann B, Winterberg M, Müller IB, Saliba KJ. 2013. Chemical and genetic validation of thiamine utilization as an antimalarial drug target. Nat Commun, 4 pp. 2060. | Show Abstract | Read more

Thiamine is metabolized into an essential cofactor for several enzymes. Here we show that oxythiamine, a thiamine analog, inhibits proliferation of the malaria parasite Plasmodium falciparum in vitro via a thiamine-related pathway and significantly reduces parasite growth in a mouse malaria model. Overexpression of thiamine pyrophosphokinase (the enzyme that converts thiamine into its active form, thiamine pyrophosphate) hypersensitizes parasites to oxythiamine by up to 1,700-fold, consistent with oxythiamine being a substrate for thiamine pyrophosphokinase and its conversion into an antimetabolite. We show that parasites overexpressing the thiamine pyrophosphate-dependent enzymes oxoglutarate dehydrogenase and pyruvate dehydrogenase are up to 15-fold more resistant to oxythiamine, consistent with the antimetabolite inactivating thiamine pyrophosphate-dependent enzymes. Our studies therefore validate thiamine utilization as an antimalarial drug target and demonstrate that a single antimalarial can simultaneously target several enzymes located within distinct organelles.

Barrand MA, Winterberg M, Ng F, Nguyen M, Kirk K, Hladky SB. 2012. Glutathione export from human erythrocytes and Plasmodium falciparum malaria parasites. Biochem J, 448 (3), pp. 389-400. | Show Abstract | Read more

Glutathione export from uninfected human erythrocytes was compared with that from cells infected with the malaria parasite Plasmodium falciparum using two separate methods that distinguish between oxidized (GSSG) and reduced (GSH) glutathione. One involved enzymatic recycling with or without thiol-masking; the other involved rapid derivatization followed by HPLC. Glutathione efflux from uninfected erythrocytes under physiological conditions occurred predominantly as GSH. On exposure of the cells to oxidative challenge, efflux of GSSG exceeded that of GSH. Efflux of both species was blocked by MK571, an inhibitor of mammalian multidrug-resistance proteins. Glutathione efflux from parasitized erythrocytes was substantially greater than that from uninfected erythrocytes. Under physiological conditions, the exported species was GSH, whereas under energy-depleted conditions, GSSG efflux occurred. Glutathione export from parasitized cells was inhibited partially by MK571 and more so by furosemide, an inhibitor of the 'new permeability pathways' induced by the parasite in the host erythrocyte membrane. Efflux from isolated parasites occurred as GSH. On exposure to oxidative challenge, this GSH efflux decreased, but no GSSG export was detected. These results are consistent with the view that the parasite supplies its host erythrocyte with GSH, much of which is exported from the infected cell via parasite-induced pathways.

Winterberg M, Rajendran E, Baumeister S, Bietz S, Kirk K, Lingelbach K. 2012. Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite-induced glutamate uptake. Blood, 119 (15), pp. 3604-3612. | Show Abstract | Read more

Human erythrocytes have a low basal permeability to L-glutamate and are not known to have a functional glutamate transporter. Here, treatment of human erythrocytes with arsenite was shown to induce the uptake of L-glutamate and D-aspartate, but not that of D-glutamate or L-alanine. The majority of the arsenite-induced L-glutamate influx was via a high-affinity, Na(+)-dependent system showing characteristics of members of the "excitatory amino acid transporter" (EAAT) family. Western blots and immunofluorescence assays revealed the presence of a member of this family, EAAT3, on the erythrocyte membrane. Erythrocytes infected with the malaria parasite Plasmodium falciparum take up glutamate from the extracellular environment. Although the majority of uptake is via a low-affinity Na(+)-independent pathway there is, in addition, a high-affinity uptake component, raising the possibility that the parasite activates the host cell glutamate transporter.

Baumeister S, Winterberg M, Przyborski JM, Lingelbach K. 2010. The malaria parasite Plasmodium falciparum: cell biological peculiarities and nutritional consequences. Protoplasma, 240 (1-4), pp. 3-12. | Show Abstract | Read more

Apicomplexan parasites obligatorily invade and multiply within eukaryotic cells. Phylogenetically, they are related to a group of algae which, during their evolution, have acquired a secondary endosymbiont. This organelle, which in the parasite is called the apicoplast, is highly reduced compared to the endosymbionts of algae, but still contains many plant-specific biosynthetic pathways. The malaria parasite Plasmodium falciparum infects mammalian erythrocytes which are devoid of intracellular compartments and which largely lack biosynthetic pathways. Despite the limited resources of nutrition, the parasite grows and generates up to 32 merozoites which are the infectious stages of the complex life cycle. A large part of the intra-erythrocytic development takes place in the so-called parasitophorous vacuole, a compartment which forms an interface between the parasite and the cytoplasm of the host cell. In the course of parasite growth, the host cell undergoes dramatic alterations which on one hand contribute directly to the symptoms of severe malaria and which, on the other hand, are also required for parasite survival. Some of these alterations facilitate the acquisition of nutrients from the extracellular environment which are not provided by the host cell. Here, we describe the cell biologically unique interactions between an intracellular eukaryotic pathogen and its metabolically highly reduced host cell. We further discuss current models to explain the appearance of pathogen-induced novel physiological properties in a host cell which has lost its genetic programme.

Baumeister S, Winterberg M, Duranton C, Huber SM, Lang F, Kirk K, Lingelbach K. 2006. Evidence for the involvement of Plasmodium falciparum proteins in the formation of new permeability pathways in the erythrocyte membrane. Mol Microbiol, 60 (2), pp. 493-504. | Show Abstract | Read more

The intraerythrocytic developmental stages of the malaria parasite Plasmodium falciparum are responsible for the clinical symptoms associated with malaria tropica. The non-infected human erythrocyte is a terminally differentiated cell that is unable to synthesize proteins and lipids de novo, and it is incapable of importing a number of solutes that are essential for parasite proliferation. Approximately 12-15 h after invasion the parasitized cell undergoes a marked increase in its permeability to a variety of different solutes present in the extracellular milieu. The increase is due to the induction in the erythrocyte membrane of 'new permeability pathways' which have been characterized in some detail in terms of their transport and electrophysiological properties, but which are yet to be defined at a molecular level. Here we show that these pathways are resistant to trypsin but are abolished by treatment of intact infected erythrocytes with chymotrypsin. On resuspension of chymotrypsinized cells in chymotrypsin-free medium the pathways progressively reappear, a process that can be inhibited by cytotoxic agents, and by brefeldin A which inhibits protein secretion. Our results provide evidence for the involvement of parasite encoded proteins in the generation of the pathways, either as components of the pathways themselves or as auxiliary factors.

Winterberg M, Kirk K. 2016. A high-sensitivity HPLC assay for measuring intracellular Na(+) and K(+) and its application to Plasmodium falciparum infected erythrocytes. Sci Rep, 6 (1), pp. 29241. | Show Abstract | Read more

The measurement of intracellular ion concentrations, and the screening of chemical agents to identify molecules targeting ion transport, has traditionally involved low-throughput techniques. Here we present a novel HPLC method that allows the rapid, high-sensitivity measurement of cell Na(+) and K(+) content, demonstrating its utility by monitoring the ionic changes induced in the intracellular malaria parasite by the new spiroindolone antimalarial KAE609.

Marchetti RV, Lehane AM, Shafik SH, Winterberg M, Martin RE, Kirk K. 2015. A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum. Nat Commun, 6 pp. 6721. | Show Abstract | Read more

The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formate-nitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate. The transport characteristics of PfFNT in oocytes (pH-dependence, inhibitor-sensitivity and kinetics) are similar to those of the transport of lactate and formate across the plasma membrane of mature asexual-stage P. falciparum trophozoites, consistent with PfFNT playing a major role in the efflux of lactate and hence in the energy metabolism of the intraerythrocytic parasite.

Yabas M, Coupland LA, Cromer D, Winterberg M, Teoh NC, D'Rozario J, Kirk K, Bröer S, Parish CR, Enders A. 2014. Mice deficient in the putative phospholipid flippase ATP11C exhibit altered erythrocyte shape, anemia, and reduced erythrocyte life span. J Biol Chem, 289 (28), pp. 19531-19537. | Show Abstract | Read more

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.

Teng R, Lehane AM, Winterberg M, Shafik SH, Summers RL, Martin RE, van Schalkwyk DA, Junankar PR, Kirk K. 2014. 1H-NMR metabolite profiles of different strains of Plasmodium falciparum. Biosci Rep, 34 (6), pp. e00150. | Show Abstract | Read more

Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite.

Chan XWA, Wrenger C, Stahl K, Bergmann B, Winterberg M, Müller IB, Saliba KJ. 2013. Chemical and genetic validation of thiamine utilization as an antimalarial drug target. Nat Commun, 4 pp. 2060. | Show Abstract | Read more

Thiamine is metabolized into an essential cofactor for several enzymes. Here we show that oxythiamine, a thiamine analog, inhibits proliferation of the malaria parasite Plasmodium falciparum in vitro via a thiamine-related pathway and significantly reduces parasite growth in a mouse malaria model. Overexpression of thiamine pyrophosphokinase (the enzyme that converts thiamine into its active form, thiamine pyrophosphate) hypersensitizes parasites to oxythiamine by up to 1,700-fold, consistent with oxythiamine being a substrate for thiamine pyrophosphokinase and its conversion into an antimetabolite. We show that parasites overexpressing the thiamine pyrophosphate-dependent enzymes oxoglutarate dehydrogenase and pyruvate dehydrogenase are up to 15-fold more resistant to oxythiamine, consistent with the antimetabolite inactivating thiamine pyrophosphate-dependent enzymes. Our studies therefore validate thiamine utilization as an antimalarial drug target and demonstrate that a single antimalarial can simultaneously target several enzymes located within distinct organelles.

Barrand MA, Winterberg M, Ng F, Nguyen M, Kirk K, Hladky SB. 2012. Glutathione export from human erythrocytes and Plasmodium falciparum malaria parasites. Biochem J, 448 (3), pp. 389-400. | Show Abstract | Read more

Glutathione export from uninfected human erythrocytes was compared with that from cells infected with the malaria parasite Plasmodium falciparum using two separate methods that distinguish between oxidized (GSSG) and reduced (GSH) glutathione. One involved enzymatic recycling with or without thiol-masking; the other involved rapid derivatization followed by HPLC. Glutathione efflux from uninfected erythrocytes under physiological conditions occurred predominantly as GSH. On exposure of the cells to oxidative challenge, efflux of GSSG exceeded that of GSH. Efflux of both species was blocked by MK571, an inhibitor of mammalian multidrug-resistance proteins. Glutathione efflux from parasitized erythrocytes was substantially greater than that from uninfected erythrocytes. Under physiological conditions, the exported species was GSH, whereas under energy-depleted conditions, GSSG efflux occurred. Glutathione export from parasitized cells was inhibited partially by MK571 and more so by furosemide, an inhibitor of the 'new permeability pathways' induced by the parasite in the host erythrocyte membrane. Efflux from isolated parasites occurred as GSH. On exposure to oxidative challenge, this GSH efflux decreased, but no GSSG export was detected. These results are consistent with the view that the parasite supplies its host erythrocyte with GSH, much of which is exported from the infected cell via parasite-induced pathways.

Winterberg M, Rajendran E, Baumeister S, Bietz S, Kirk K, Lingelbach K. 2012. Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite-induced glutamate uptake. Blood, 119 (15), pp. 3604-3612. | Show Abstract | Read more

Human erythrocytes have a low basal permeability to L-glutamate and are not known to have a functional glutamate transporter. Here, treatment of human erythrocytes with arsenite was shown to induce the uptake of L-glutamate and D-aspartate, but not that of D-glutamate or L-alanine. The majority of the arsenite-induced L-glutamate influx was via a high-affinity, Na(+)-dependent system showing characteristics of members of the "excitatory amino acid transporter" (EAAT) family. Western blots and immunofluorescence assays revealed the presence of a member of this family, EAAT3, on the erythrocyte membrane. Erythrocytes infected with the malaria parasite Plasmodium falciparum take up glutamate from the extracellular environment. Although the majority of uptake is via a low-affinity Na(+)-independent pathway there is, in addition, a high-affinity uptake component, raising the possibility that the parasite activates the host cell glutamate transporter.

Baumeister S, Winterberg M, Przyborski JM, Lingelbach K. 2010. The malaria parasite Plasmodium falciparum: cell biological peculiarities and nutritional consequences. Protoplasma, 240 (1-4), pp. 3-12. | Show Abstract | Read more

Apicomplexan parasites obligatorily invade and multiply within eukaryotic cells. Phylogenetically, they are related to a group of algae which, during their evolution, have acquired a secondary endosymbiont. This organelle, which in the parasite is called the apicoplast, is highly reduced compared to the endosymbionts of algae, but still contains many plant-specific biosynthetic pathways. The malaria parasite Plasmodium falciparum infects mammalian erythrocytes which are devoid of intracellular compartments and which largely lack biosynthetic pathways. Despite the limited resources of nutrition, the parasite grows and generates up to 32 merozoites which are the infectious stages of the complex life cycle. A large part of the intra-erythrocytic development takes place in the so-called parasitophorous vacuole, a compartment which forms an interface between the parasite and the cytoplasm of the host cell. In the course of parasite growth, the host cell undergoes dramatic alterations which on one hand contribute directly to the symptoms of severe malaria and which, on the other hand, are also required for parasite survival. Some of these alterations facilitate the acquisition of nutrients from the extracellular environment which are not provided by the host cell. Here, we describe the cell biologically unique interactions between an intracellular eukaryotic pathogen and its metabolically highly reduced host cell. We further discuss current models to explain the appearance of pathogen-induced novel physiological properties in a host cell which has lost its genetic programme.

Baumeister S, Winterberg M, Duranton C, Huber SM, Lang F, Kirk K, Lingelbach K. 2006. Evidence for the involvement of Plasmodium falciparum proteins in the formation of new permeability pathways in the erythrocyte membrane. Mol Microbiol, 60 (2), pp. 493-504. | Show Abstract | Read more

The intraerythrocytic developmental stages of the malaria parasite Plasmodium falciparum are responsible for the clinical symptoms associated with malaria tropica. The non-infected human erythrocyte is a terminally differentiated cell that is unable to synthesize proteins and lipids de novo, and it is incapable of importing a number of solutes that are essential for parasite proliferation. Approximately 12-15 h after invasion the parasitized cell undergoes a marked increase in its permeability to a variety of different solutes present in the extracellular milieu. The increase is due to the induction in the erythrocyte membrane of 'new permeability pathways' which have been characterized in some detail in terms of their transport and electrophysiological properties, but which are yet to be defined at a molecular level. Here we show that these pathways are resistant to trypsin but are abolished by treatment of intact infected erythrocytes with chymotrypsin. On resuspension of chymotrypsinized cells in chymotrypsin-free medium the pathways progressively reappear, a process that can be inhibited by cytotoxic agents, and by brefeldin A which inhibits protein secretion. Our results provide evidence for the involvement of parasite encoded proteins in the generation of the pathways, either as components of the pathways themselves or as auxiliary factors.

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