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Papers by Verena Brand

In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation ... more In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation (NSC) permeability and of sphingomyelinase by Plasmodium falciparum infection. Both are involved in programmed cell death in erythrocytes, also referred to as eryptosis [Lang et al., 2006; Lang et al., 2005], and meet four requirements for parasite growth at the same time: i) Development of an inwardly directed Na+ and an outwardly directed K+ ion gradient across the parasite plasma membrane, this in turn making the parasite dependent on high Na+ and low K+ ion concentration extracellularly [Brand et al., 2003]. ii) The presence of Ca2+ ions in the culture medium and a Ca2+ permeability conferred by a NSC conductance [Brand et al., 2003]. iii) Increased ceramide formation (detected in the blood of P. berghei ANKA infected mice). iv) Activity of parasitic neutral sphingomyelinase and host acid sphingomyelinase, probably to provide ceramide for membrane synthesis [Hanada et al., 2000]. In ...

bioRxiv, 2021

The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen with sig... more The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen with significant impact in children and during pregnancy. The majority of the T. gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we utilize a biochemical fractionation strategy coupled with mass spectrometry to predict interactions by correlation profiling. Key to this approach is the integration of additional datasets based on gene co-expression as well as phylogenetic profiles that eliminate poorly supported interactions and reduce the number of false positive interactions. In addition to a supervised machine learning strategy, we employed an unsupervised approach in data integration, based on similarity network fusion, to overcome the deficit of high-quality training data in non-model organisms. The resulting high confidence network, we term ToxoNet, comprises 2,063 interactions connecting 652 proteins. Cluste...

In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation ... more In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation (NSC) permeability and of sphingomyelinase by Plasmodium falciparum infection. Both are involved in programmed cell death in erythrocytes, also referred to as eryptosis [Lang et al., 2006; Lang et al., 2005], and meet four requirements for parasite growth at the same time: i) Development of an inwardly directed Na+ and an outwardly directed K+ ion gradient across the parasite plasma membrane, this in turn making the parasite dependent on high Na+ and low K+ ion concentration extracellularly [Brand et al., 2003]. ii) The presence of Ca2+ ions in the culture medium and a Ca2+ permeability conferred by a NSC conductance [Brand et al., 2003]. iii) Increased ceramide formation (detected in the blood of P. berghei ANKA infected mice). iv) Activity of parasitic neutral sphingomyelinase and host acid sphingomyelinase, probably to provide ceramide for membrane synthesis [Hanada et al., 2000]. In the second part of my doctoral thesis I investigated differences in the induction of eryptosis in normal (HbA/A) and sickle trait (HbA/S) P. falciparum infected red blood cells (RBCs). The results were as follows: i) P. falciparum did not develop differently in HbA/A and HbA/S RBCs, but infected HbA/S RBCs showed enhanced PS exposure. ii) The host ceramide pathway played only a minor role in causing PS exposure in infected RBCs under normal culturing conditions. iii) Although parasite growth was dependent on ceramide, extragenously applied ceramide inhibited P. falciparum in vitro growth, and more so in HbA/S RBCs than in HbA/A RBCs. iv) Infected HbA/S RBCs secreted more prostaglandin E2 (PGE2) than did infected HbA/A RBCs, probably significantly contributing to the selective advantage of P. falciparum infected sickle cell trait carriers. Increased PGE2 levels are associated with a mild course of malaria infection [Perkins et al., 2001]. v) The increased PGE2 levels enhanced the infection-induced Ca2+ influx into ring-stage infected HbA/S RBCs. This promoted the observed increase in PS exposure on the outer membrane leaflet of infected HbA/S RBCs as compared to infected HbA/A RBCs. These observations show that the accelerated eryptosis of P. falciparum infected HbA/S RBCs is caused by enhanced activation of a NSC permeability during ring stage. Enhanced PS exposure acts as an 'eat-me' signal for macrophages. Together with other signals [Ayi et al., 2004], it triggers the early recognition and removal of ring-stage infected HbA/S RBCs [Kasinathan, 2007].; Im ersten Teil meiner Doktorarbeit untersuchte ich die Aktivierung einer nichtselektiven Kationenkanalaktivitat und von Sphingomyelinase in Plasmodium falciparum infizierten Erythrozyten. Beide Prozesse sind am programmierten Zelltod in Erythrozyten beteiligt, der auch Eryptose genannt wird [Lang et al., 2006; Lang et al., 2005], und erfullen gleichzeitig vier Bedingungen fur das Parasitenwachstum: i) Entwicklung eines einwarts gerichteteten Natrium- und eines auswarts gerichteten…

Molecular and biochemical parasitology, Jan 22, 2015

FIKKs are protein kinases with distinctive sequence motifs found exclusively in Apicomplexa. FIKK... more FIKKs are protein kinases with distinctive sequence motifs found exclusively in Apicomplexa. FIKK8 is the sole predicted cytosolic member of the subfamily and also the only one conserved amongst some non-Plasmodium parasites. Here, we report on the biochemical characterization of Plasmodium falciparum FIKK8 (PfFIKK8) and its Cryptosporidium parvum orthologue (CpFIKK). Recombinant protein samples of both were catalytically active. We characterized their phosphorylation ability using an enzymatic assay and substrate specificities using an arrayed positional scanning peptide library. Our results show that FIKK8 targets serine, preferably with arginine in the +3 and -3 positions. Furthermore, the soluble and active FIKK constructs in our experiments contained an N-terminal extension (NTE) conserved in FIKK8 orthologues from other apicomplexan species. Based on our results, we propose that this NTE is an integral feature of the FIKK subfamily.

Respiration Physiology, 2001

NADPH oxidase isoforms with different gp91phox subunits as well as an unusual cytochrome aa3 with... more NADPH oxidase isoforms with different gp91phox subunits as well as an unusual cytochrome aa3 with a heme a/a3 relationship of 9:91 are discussed as putative oxygen sensor proteins influencing gene expression and ion channel conductivity. Reactive oxygen species (ROS) are important second messengers of the oxygen sensing signal cascade determining the stability of transcription factors or the gating of ion channels. The formation of ROS by a perinuclear Fenton reaction is imaged by 1 and 2 photon confocal microscopy revealing mitochondrial and non-mitochondrial generation.

Molecular and Biochemical Parasitology, 2007

Synthesis of the modified thymine base, β-D-glucosyl-hydroxymethyluracil or J, within telomeric D... more Synthesis of the modified thymine base, β-D-glucosyl-hydroxymethyluracil or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. In order to analyze the function of base J in the regulation of antigenic variation, we are characterizing the regulatory mechanism of J biosynthesis. We have recently proposed a model in which chromatin remodeling by a SWI2/SNF2-like protein (JBP2) regulates the developmental and de-novo site-specific localization of J synthesis within bloodstream-form trypanosome DNA. Consistent with this model, we now show that JBP2 (−/−) bloodstream-form trypanosomes contain 5-fold less base J and are unable to stimulate de-novo J synthesis in newly generated telomeric arrays.

The FASEB Journal, 2005

In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stres... more In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stress induces a new organic osmolyte and anion permeability. To examine a role for autocrine purinoceptor signaling during this induction process, erythrocytic purinoceptor expression, and ATP release were determined. Furthermore, using pharmacological and genetic approaches the dependence on purinoceptor signaling of osmolyte permeability and Plasmodium development, both in vitro and in vivo, were assessed. Extracellular ATP did not induce an osmolyte permeability in non-infected or non-oxidized erythrocytes. ATP and other purinoceptor agonists increased the induction of osmolyte permeability during infection or oxidation as measured by isosmotic hemolysis and patch-clamp recording. Purinoceptor antagonists and apyrase decreased the induced permeability. The observed pharmacology suggested the involvement of P2Y purinoceptors. Accordingly, human erythrocytes expressed P2Y 1 protein. Moreover, P2Y 1deficient mouse erythrocytes exhibited a delayed appearance of the osmolyte permeability during P. berghei infection-or oxidation compared with wild-type erythrocytes. Furthermore, the nonspecific purinoceptor antagonist suramin decreased in vitro growth and DNA/RNA amplification of P. falciparum in human erythrocytes and decreased in vivo growth of P. berghei. P. berghei developed slower in P2Y 1-deficient mice in vivo compared with wild-type animals. In conclusion, induction of the osmolyte permeability in Plasmodium-infected erythrocytes involves autocrine purinoceptor signaling. Key words: purinergic receptors • ATP release • oxidative stress • new permeability pathways • red blood cells n human red blood cells (RBCs) infected by Plasmodium falciparum, tracer flux measurements disclosed a dramatic increase of erythrocyte cell membrane permeability reflecting the generation of so-called New Permeability Pathways (NPP) (1). Functionally, the NPP are organic osmolyte and anion channels. They transport a wide variety of solutes, I

Pflügers Archiv - European Journal of Physiology, 2008

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in ... more Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 microM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl(-) or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na-ATP (NMDG-Cl pipette solution) shifted the current reversal potential (V (rev)) from -2 +/- 1 to +51 +/- 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V (rev) to significantly larger (p < or = 0.05, two-tailed t test) values (from -3 +/- 1 to +66 +/- 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 +/- 0.1 nS to 0.2 +/- 0.05 nS and further shifted V (rev) to +87 +/- 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 +/- 4 mV suggesting a relative permeability of P (ATP)/P (Cl) approximately 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway.

Pflügers Archiv - European Journal of Physiology, 2005

Intraerythrocytic survival of the malaria pathogen Plasmodium falciparum requires delivery of nut... more Intraerythrocytic survival of the malaria pathogen Plasmodium falciparum requires delivery of nutrients and disposal of waste products across the host erythrocyte membrane. Recent patch-clamp experiments have demonstrated inwardly and outwardly rectifying anion conductances in infected but not in control erythrocytes. A ClC-2-generated fraction of the inwardly rectifying current is activated by cell swelling and presumably subserves host cell volume regulation. In contrast, the outwardly rectifying current is insensitive to cell volume but allows the passage of lactate and is involved in the transport of nutrients. The present study was performed to characterize the permselectivity and pH sensitivity of the anion conductances using wholecell recording. The outwardly rectifying and the inwardly rectifying currents exhibited permselectivities of Cl À ‡Br À %I À >SCN À and SCN À >I À >Br À >Cl À , respectively, as evident from the reversal potentials recorded under biionic conditions. While the inwardly rectifying current was not affected significantly by alterations of pH between 6.0 and 8.4, the outward rectifier was inhibited strongly by alkalinization to pH ‡7.8. Fluxes of 14 C-lactate and parasite growth were decreased markedly by the increase of bath pH, an effect that may at least in part be due to inhibition of the outward rectifier and subsequently impaired transport across the erythrocyte membrane.

Pfl�gers Archiv European Journal of Physiology, 2004

Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of se... more Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of several distinct anion channels and a non-selective, Ca 2+-permeable cation channel. All channel types are presumably activated by the oxidative stress generated by the pathogen. Similar or identical channels are activated by oxidation of non-infected erythrocytes. Activation of the non-selective cation channel allows entry of Ca 2+ and Na + , both of which are required for intracellular growth of the pathogen. The entry of Ca 2+ stimulates an intraerythrocytic scramblase that facilitates bi-directional phospholipid migration across the bilayer, resulting in breakdown of the phosphatidylserine asymmetry of the cell membrane. The exposure of phosphatidylserine at the outer surface of the cell membrane is presumably followed by binding to phosphatidylserine receptors on macrophages and subsequent phagocytosis of the affected erythrocyte. The lysosomal degradation may eventually eliminate the pathogen. The channel may thus play a dual role in pathogen survival. Absence of the channels is not compatible with pathogen growth, enhanced channel activity accelerates erythrocyte "apoptosis" that may represent a host defence mechanism serving to eliminate infected erythrocytes.

Nanomedicine: Nanotechnology, Biology and Medicine, 2005

Previous patch-clamp studies have demonstrated inwardly and outwardly rectifying anion currents, ... more Previous patch-clamp studies have demonstrated inwardly and outwardly rectifying anion currents, ClC-2 Cl- currents, and nonselective Ca(++)-permeable cation currents in Plasmodium falciparum-infected human erythrocytes. The current work studied the effect of the potent antimalarial drug artemisinin on the P falciparum infection-induced whole cell currents in human erythrocyte. Artemisinin had no significant effect on the outwardly rectifying anion currents but inhibited the cation-selective currents with an apparent half-maximal inhibitory concentration of < or =10 micromol/L. Because artemisinin reportedly inhibits the asexual parasite amplification with much higher potency, the antimalarial action of the drug cannot be attributed to the artemisinin effect on the cation currents. However, artemisinin may be used as a pharmacologic tool to dissect different current fractions in P falciparum-infected erythrocytes.

Journal of General Physiology, 2004

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new perme... more Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new permeability pathways (NPPs) in the host cell membrane. Isotopic flux measurements demonstrated that the NPP are permeable to a wide variety of molecules, thus allowing uptake of nutrients and release of waste products. Recent patch-clamp recordings demonstrated the infection-induced up-regulation of an inwardly and an outwardly rectifying Cl− conductance. The present experiments have been performed to explore the sensitivity to cell volume and the organic osmolyte permeability of the two conductances. It is shown that the outward rectifier has a high relative lactate permeability (Plactate/PCl = 0.4). Sucrose inhibited the outward-rectifier and abolished the infection-induced hemolysis in isosmotic sorbitol solution but had no or little effect on the inward-rectifier. Furosemide and NPPB blocked the outward-rectifying lactate current and the sorbitol hemolysis with IC50s in the range of 0.1 ...

Journal of Biological Chemistry, 2004

1 The abbreviations used are: RBC, red blood cell; PBS, phosphatebuffered saline; FACS, fluoresce... more 1 The abbreviations used are: RBC, red blood cell; PBS, phosphatebuffered saline; FACS, fluorescent-activated cell sorter.

Cellular Physiology and Biochemistry, 2008

Plasmodia express a sphingomyelinase, which is apparently required for their development. On the ... more Plasmodia express a sphingomyelinase, which is apparently required for their development. On the other hand, the sphingomyelinase product ceramide has previously been shown to delay parasite development. Moreover, ceramide triggers suicidal erythrocyte death or eryptosis, characterized by exposure of phosphatidylserine at the erythrocyte surface and cell shrinkage. Accelerated eryptosis of infected erythrocytes is considered to clear infected erythrocytes from circulating blood and, thus, to favourably influence the clinical course of malaria. The present experiments explored whether the sphingomyelinase inhibitor amitriptyline or genetic knockout of host acid sphingomyelinase influence in vitro parasite growth, eryptosis of Plasmodium falciparum-infected human erythrocytes, in vivo parasitemia and survival of P. berghei-infected mice. Phosphatidylserine exposure was determined by annexin V-binding and cell volume by forward scatter in FACS analysis. In vitro infection of human erythrocytes increased annexin-binding, an effect blunted in the presence of amitriptyline (≥ 50 µM). Amitriptyline did not significantly alter intraerythrocytic parasite development but significantly (≥ 1 µM) delayed the increase in parasitemia in vitro. Most importantly, amitriptyline treatment (1 mM in drinking water) resulted in a significant delay of parasitemia and death of infected mice. However, upon infection, ceramide formation was stimulated in both, acid sphingomyelinase knockout mice (Smpd1-/-) and their wild type littermates (Smpd1 +/+). Parasitemia following P. berghei infection was significantly lower in Smpd1-/-than in Smpd1 +/+ mice but did not significantly extend the life span of infected animals. In conclusion, mammalian and parasite sphingomyelinase contribute to ceramide formation during malaria, whereby the parasite sphingomyelinase ultimately determines the course of the infection. Amitriptyline presumably blocks both sphingomyelinases and, thus, its use might be a novel strategy to treat malaria.

Cellular Physiology and Biochemistry, 2008

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the... more The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cland lactate outward currents with an EC 50 of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Cellular Physiology and Biochemistry, 2003

Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca2+-permeable un... more Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca2+-permeable unselective cation conductance in the host cell membrane which is inhibited by ethylisopropylamiloride (EIPA) and is paralleled by an exchange of K+ by Na+ in the host cytosol. The present study has been performed to elucidate the functional significance of the electrolyte exchange. Whole-cell patch-clamp experiments confirmed the Ca2+ permeability and EIPA sensitivity of the Plasmodium falciparum induced cation channel. In further experiments, ring stage-synchronized parasites were grown in vitro for 48 h in different test media. Percentage of Plasmodium-infected and phosphatidylserine-exposing erythrocytes was measured with FACS analysis by staining with the DNA-dye Syto16 and annexin V, respectively. The increase of infected cells was not significantly affected by an 8 h replacement of NaCl in the culture medium with Na-gluconate but was significantly blunted by replacement of NaCl with KCl, NMDG-Cl or raffinose. Half maximal growth was observed at about 25 mM Na+. The increase of infected cells was further inhibited by EIPA (IC50< 10 microM) and at low extracellular free Ca2+. Infected cells displayed significantly stronger annexin binding, an effect mimicked by exposure of noninfected erythrocytes to oxidative stress (1 mM T-butylhydroperoxide for 15 min) or to Ca2+ ionophore ionomycin (1 microM for 60 min). The observations indicate that parasite growth requires the entry of both, Na+ and Ca2+ cations into the host erythrocyte probably through the EIPA sensitive cation channel. Ca2+ entry further induces break-down of the phospholipid asymmetry in the host membrane.

Cellular Physiology and Biochemistry, 2009

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also o... more The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE 2 formation, Ca 2+ permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7-/-) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7-/mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7-/mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE 2-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stageinfected HbA/S or annexin-A7-/erythrocytes and thus confers partial protection against malaria in vivo.

In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation ... more In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation (NSC) permeability and of sphingomyelinase by Plasmodium falciparum infection. Both are involved in programmed cell death in erythrocytes, also referred to as eryptosis [Lang et al., 2006; Lang et al., 2005], and meet four requirements for parasite growth at the same time: i) Development of an inwardly directed Na+ and an outwardly directed K+ ion gradient across the parasite plasma membrane, this in turn making the parasite dependent on high Na+ and low K+ ion concentration extracellularly [Brand et al., 2003]. ii) The presence of Ca2+ ions in the culture medium and a Ca2+ permeability conferred by a NSC conductance [Brand et al., 2003]. iii) Increased ceramide formation (detected in the blood of P. berghei ANKA infected mice). iv) Activity of parasitic neutral sphingomyelinase and host acid sphingomyelinase, probably to provide ceramide for membrane synthesis [Hanada et al., 2000]. In ...

bioRxiv, 2021

The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen with sig... more The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen with significant impact in children and during pregnancy. The majority of the T. gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we utilize a biochemical fractionation strategy coupled with mass spectrometry to predict interactions by correlation profiling. Key to this approach is the integration of additional datasets based on gene co-expression as well as phylogenetic profiles that eliminate poorly supported interactions and reduce the number of false positive interactions. In addition to a supervised machine learning strategy, we employed an unsupervised approach in data integration, based on similarity network fusion, to overcome the deficit of high-quality training data in non-model organisms. The resulting high confidence network, we term ToxoNet, comprises 2,063 interactions connecting 652 proteins. Cluste...

In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation ... more In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation (NSC) permeability and of sphingomyelinase by Plasmodium falciparum infection. Both are involved in programmed cell death in erythrocytes, also referred to as eryptosis [Lang et al., 2006; Lang et al., 2005], and meet four requirements for parasite growth at the same time: i) Development of an inwardly directed Na+ and an outwardly directed K+ ion gradient across the parasite plasma membrane, this in turn making the parasite dependent on high Na+ and low K+ ion concentration extracellularly [Brand et al., 2003]. ii) The presence of Ca2+ ions in the culture medium and a Ca2+ permeability conferred by a NSC conductance [Brand et al., 2003]. iii) Increased ceramide formation (detected in the blood of P. berghei ANKA infected mice). iv) Activity of parasitic neutral sphingomyelinase and host acid sphingomyelinase, probably to provide ceramide for membrane synthesis [Hanada et al., 2000]. In the second part of my doctoral thesis I investigated differences in the induction of eryptosis in normal (HbA/A) and sickle trait (HbA/S) P. falciparum infected red blood cells (RBCs). The results were as follows: i) P. falciparum did not develop differently in HbA/A and HbA/S RBCs, but infected HbA/S RBCs showed enhanced PS exposure. ii) The host ceramide pathway played only a minor role in causing PS exposure in infected RBCs under normal culturing conditions. iii) Although parasite growth was dependent on ceramide, extragenously applied ceramide inhibited P. falciparum in vitro growth, and more so in HbA/S RBCs than in HbA/A RBCs. iv) Infected HbA/S RBCs secreted more prostaglandin E2 (PGE2) than did infected HbA/A RBCs, probably significantly contributing to the selective advantage of P. falciparum infected sickle cell trait carriers. Increased PGE2 levels are associated with a mild course of malaria infection [Perkins et al., 2001]. v) The increased PGE2 levels enhanced the infection-induced Ca2+ influx into ring-stage infected HbA/S RBCs. This promoted the observed increase in PS exposure on the outer membrane leaflet of infected HbA/S RBCs as compared to infected HbA/A RBCs. These observations show that the accelerated eryptosis of P. falciparum infected HbA/S RBCs is caused by enhanced activation of a NSC permeability during ring stage. Enhanced PS exposure acts as an 'eat-me' signal for macrophages. Together with other signals [Ayi et al., 2004], it triggers the early recognition and removal of ring-stage infected HbA/S RBCs [Kasinathan, 2007].; Im ersten Teil meiner Doktorarbeit untersuchte ich die Aktivierung einer nichtselektiven Kationenkanalaktivitat und von Sphingomyelinase in Plasmodium falciparum infizierten Erythrozyten. Beide Prozesse sind am programmierten Zelltod in Erythrozyten beteiligt, der auch Eryptose genannt wird [Lang et al., 2006; Lang et al., 2005], und erfullen gleichzeitig vier Bedingungen fur das Parasitenwachstum: i) Entwicklung eines einwarts gerichteteten Natrium- und eines auswarts gerichteten…

Molecular and biochemical parasitology, Jan 22, 2015

FIKKs are protein kinases with distinctive sequence motifs found exclusively in Apicomplexa. FIKK... more FIKKs are protein kinases with distinctive sequence motifs found exclusively in Apicomplexa. FIKK8 is the sole predicted cytosolic member of the subfamily and also the only one conserved amongst some non-Plasmodium parasites. Here, we report on the biochemical characterization of Plasmodium falciparum FIKK8 (PfFIKK8) and its Cryptosporidium parvum orthologue (CpFIKK). Recombinant protein samples of both were catalytically active. We characterized their phosphorylation ability using an enzymatic assay and substrate specificities using an arrayed positional scanning peptide library. Our results show that FIKK8 targets serine, preferably with arginine in the +3 and -3 positions. Furthermore, the soluble and active FIKK constructs in our experiments contained an N-terminal extension (NTE) conserved in FIKK8 orthologues from other apicomplexan species. Based on our results, we propose that this NTE is an integral feature of the FIKK subfamily.

Respiration Physiology, 2001

NADPH oxidase isoforms with different gp91phox subunits as well as an unusual cytochrome aa3 with... more NADPH oxidase isoforms with different gp91phox subunits as well as an unusual cytochrome aa3 with a heme a/a3 relationship of 9:91 are discussed as putative oxygen sensor proteins influencing gene expression and ion channel conductivity. Reactive oxygen species (ROS) are important second messengers of the oxygen sensing signal cascade determining the stability of transcription factors or the gating of ion channels. The formation of ROS by a perinuclear Fenton reaction is imaged by 1 and 2 photon confocal microscopy revealing mitochondrial and non-mitochondrial generation.

Molecular and Biochemical Parasitology, 2007

Synthesis of the modified thymine base, β-D-glucosyl-hydroxymethyluracil or J, within telomeric D... more Synthesis of the modified thymine base, β-D-glucosyl-hydroxymethyluracil or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. In order to analyze the function of base J in the regulation of antigenic variation, we are characterizing the regulatory mechanism of J biosynthesis. We have recently proposed a model in which chromatin remodeling by a SWI2/SNF2-like protein (JBP2) regulates the developmental and de-novo site-specific localization of J synthesis within bloodstream-form trypanosome DNA. Consistent with this model, we now show that JBP2 (−/−) bloodstream-form trypanosomes contain 5-fold less base J and are unable to stimulate de-novo J synthesis in newly generated telomeric arrays.

The FASEB Journal, 2005

In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stres... more In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stress induces a new organic osmolyte and anion permeability. To examine a role for autocrine purinoceptor signaling during this induction process, erythrocytic purinoceptor expression, and ATP release were determined. Furthermore, using pharmacological and genetic approaches the dependence on purinoceptor signaling of osmolyte permeability and Plasmodium development, both in vitro and in vivo, were assessed. Extracellular ATP did not induce an osmolyte permeability in non-infected or non-oxidized erythrocytes. ATP and other purinoceptor agonists increased the induction of osmolyte permeability during infection or oxidation as measured by isosmotic hemolysis and patch-clamp recording. Purinoceptor antagonists and apyrase decreased the induced permeability. The observed pharmacology suggested the involvement of P2Y purinoceptors. Accordingly, human erythrocytes expressed P2Y 1 protein. Moreover, P2Y 1deficient mouse erythrocytes exhibited a delayed appearance of the osmolyte permeability during P. berghei infection-or oxidation compared with wild-type erythrocytes. Furthermore, the nonspecific purinoceptor antagonist suramin decreased in vitro growth and DNA/RNA amplification of P. falciparum in human erythrocytes and decreased in vivo growth of P. berghei. P. berghei developed slower in P2Y 1-deficient mice in vivo compared with wild-type animals. In conclusion, induction of the osmolyte permeability in Plasmodium-infected erythrocytes involves autocrine purinoceptor signaling. Key words: purinergic receptors • ATP release • oxidative stress • new permeability pathways • red blood cells n human red blood cells (RBCs) infected by Plasmodium falciparum, tracer flux measurements disclosed a dramatic increase of erythrocyte cell membrane permeability reflecting the generation of so-called New Permeability Pathways (NPP) (1). Functionally, the NPP are organic osmolyte and anion channels. They transport a wide variety of solutes, I

Pflügers Archiv - European Journal of Physiology, 2008

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in ... more Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 microM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl(-) or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na-ATP (NMDG-Cl pipette solution) shifted the current reversal potential (V (rev)) from -2 +/- 1 to +51 +/- 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V (rev) to significantly larger (p < or = 0.05, two-tailed t test) values (from -3 +/- 1 to +66 +/- 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 +/- 0.1 nS to 0.2 +/- 0.05 nS and further shifted V (rev) to +87 +/- 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 +/- 4 mV suggesting a relative permeability of P (ATP)/P (Cl) approximately 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway.

Pflügers Archiv - European Journal of Physiology, 2005

Intraerythrocytic survival of the malaria pathogen Plasmodium falciparum requires delivery of nut... more Intraerythrocytic survival of the malaria pathogen Plasmodium falciparum requires delivery of nutrients and disposal of waste products across the host erythrocyte membrane. Recent patch-clamp experiments have demonstrated inwardly and outwardly rectifying anion conductances in infected but not in control erythrocytes. A ClC-2-generated fraction of the inwardly rectifying current is activated by cell swelling and presumably subserves host cell volume regulation. In contrast, the outwardly rectifying current is insensitive to cell volume but allows the passage of lactate and is involved in the transport of nutrients. The present study was performed to characterize the permselectivity and pH sensitivity of the anion conductances using wholecell recording. The outwardly rectifying and the inwardly rectifying currents exhibited permselectivities of Cl À ‡Br À %I À >SCN À and SCN À >I À >Br À >Cl À , respectively, as evident from the reversal potentials recorded under biionic conditions. While the inwardly rectifying current was not affected significantly by alterations of pH between 6.0 and 8.4, the outward rectifier was inhibited strongly by alkalinization to pH ‡7.8. Fluxes of 14 C-lactate and parasite growth were decreased markedly by the increase of bath pH, an effect that may at least in part be due to inhibition of the outward rectifier and subsequently impaired transport across the erythrocyte membrane.

Pfl�gers Archiv European Journal of Physiology, 2004

Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of se... more Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of several distinct anion channels and a non-selective, Ca 2+-permeable cation channel. All channel types are presumably activated by the oxidative stress generated by the pathogen. Similar or identical channels are activated by oxidation of non-infected erythrocytes. Activation of the non-selective cation channel allows entry of Ca 2+ and Na + , both of which are required for intracellular growth of the pathogen. The entry of Ca 2+ stimulates an intraerythrocytic scramblase that facilitates bi-directional phospholipid migration across the bilayer, resulting in breakdown of the phosphatidylserine asymmetry of the cell membrane. The exposure of phosphatidylserine at the outer surface of the cell membrane is presumably followed by binding to phosphatidylserine receptors on macrophages and subsequent phagocytosis of the affected erythrocyte. The lysosomal degradation may eventually eliminate the pathogen. The channel may thus play a dual role in pathogen survival. Absence of the channels is not compatible with pathogen growth, enhanced channel activity accelerates erythrocyte "apoptosis" that may represent a host defence mechanism serving to eliminate infected erythrocytes.

Nanomedicine: Nanotechnology, Biology and Medicine, 2005

Previous patch-clamp studies have demonstrated inwardly and outwardly rectifying anion currents, ... more Previous patch-clamp studies have demonstrated inwardly and outwardly rectifying anion currents, ClC-2 Cl- currents, and nonselective Ca(++)-permeable cation currents in Plasmodium falciparum-infected human erythrocytes. The current work studied the effect of the potent antimalarial drug artemisinin on the P falciparum infection-induced whole cell currents in human erythrocyte. Artemisinin had no significant effect on the outwardly rectifying anion currents but inhibited the cation-selective currents with an apparent half-maximal inhibitory concentration of < or =10 micromol/L. Because artemisinin reportedly inhibits the asexual parasite amplification with much higher potency, the antimalarial action of the drug cannot be attributed to the artemisinin effect on the cation currents. However, artemisinin may be used as a pharmacologic tool to dissect different current fractions in P falciparum-infected erythrocytes.

Journal of General Physiology, 2004

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new perme... more Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new permeability pathways (NPPs) in the host cell membrane. Isotopic flux measurements demonstrated that the NPP are permeable to a wide variety of molecules, thus allowing uptake of nutrients and release of waste products. Recent patch-clamp recordings demonstrated the infection-induced up-regulation of an inwardly and an outwardly rectifying Cl− conductance. The present experiments have been performed to explore the sensitivity to cell volume and the organic osmolyte permeability of the two conductances. It is shown that the outward rectifier has a high relative lactate permeability (Plactate/PCl = 0.4). Sucrose inhibited the outward-rectifier and abolished the infection-induced hemolysis in isosmotic sorbitol solution but had no or little effect on the inward-rectifier. Furosemide and NPPB blocked the outward-rectifying lactate current and the sorbitol hemolysis with IC50s in the range of 0.1 ...

Journal of Biological Chemistry, 2004

1 The abbreviations used are: RBC, red blood cell; PBS, phosphatebuffered saline; FACS, fluoresce... more 1 The abbreviations used are: RBC, red blood cell; PBS, phosphatebuffered saline; FACS, fluorescent-activated cell sorter.

Cellular Physiology and Biochemistry, 2008

Plasmodia express a sphingomyelinase, which is apparently required for their development. On the ... more Plasmodia express a sphingomyelinase, which is apparently required for their development. On the other hand, the sphingomyelinase product ceramide has previously been shown to delay parasite development. Moreover, ceramide triggers suicidal erythrocyte death or eryptosis, characterized by exposure of phosphatidylserine at the erythrocyte surface and cell shrinkage. Accelerated eryptosis of infected erythrocytes is considered to clear infected erythrocytes from circulating blood and, thus, to favourably influence the clinical course of malaria. The present experiments explored whether the sphingomyelinase inhibitor amitriptyline or genetic knockout of host acid sphingomyelinase influence in vitro parasite growth, eryptosis of Plasmodium falciparum-infected human erythrocytes, in vivo parasitemia and survival of P. berghei-infected mice. Phosphatidylserine exposure was determined by annexin V-binding and cell volume by forward scatter in FACS analysis. In vitro infection of human erythrocytes increased annexin-binding, an effect blunted in the presence of amitriptyline (≥ 50 µM). Amitriptyline did not significantly alter intraerythrocytic parasite development but significantly (≥ 1 µM) delayed the increase in parasitemia in vitro. Most importantly, amitriptyline treatment (1 mM in drinking water) resulted in a significant delay of parasitemia and death of infected mice. However, upon infection, ceramide formation was stimulated in both, acid sphingomyelinase knockout mice (Smpd1-/-) and their wild type littermates (Smpd1 +/+). Parasitemia following P. berghei infection was significantly lower in Smpd1-/-than in Smpd1 +/+ mice but did not significantly extend the life span of infected animals. In conclusion, mammalian and parasite sphingomyelinase contribute to ceramide formation during malaria, whereby the parasite sphingomyelinase ultimately determines the course of the infection. Amitriptyline presumably blocks both sphingomyelinases and, thus, its use might be a novel strategy to treat malaria.

Cellular Physiology and Biochemistry, 2008

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the... more The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cland lactate outward currents with an EC 50 of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Cellular Physiology and Biochemistry, 2003

Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca2+-permeable un... more Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca2+-permeable unselective cation conductance in the host cell membrane which is inhibited by ethylisopropylamiloride (EIPA) and is paralleled by an exchange of K+ by Na+ in the host cytosol. The present study has been performed to elucidate the functional significance of the electrolyte exchange. Whole-cell patch-clamp experiments confirmed the Ca2+ permeability and EIPA sensitivity of the Plasmodium falciparum induced cation channel. In further experiments, ring stage-synchronized parasites were grown in vitro for 48 h in different test media. Percentage of Plasmodium-infected and phosphatidylserine-exposing erythrocytes was measured with FACS analysis by staining with the DNA-dye Syto16 and annexin V, respectively. The increase of infected cells was not significantly affected by an 8 h replacement of NaCl in the culture medium with Na-gluconate but was significantly blunted by replacement of NaCl with KCl, NMDG-Cl or raffinose. Half maximal growth was observed at about 25 mM Na+. The increase of infected cells was further inhibited by EIPA (IC50< 10 microM) and at low extracellular free Ca2+. Infected cells displayed significantly stronger annexin binding, an effect mimicked by exposure of noninfected erythrocytes to oxidative stress (1 mM T-butylhydroperoxide for 15 min) or to Ca2+ ionophore ionomycin (1 microM for 60 min). The observations indicate that parasite growth requires the entry of both, Na+ and Ca2+ cations into the host erythrocyte probably through the EIPA sensitive cation channel. Ca2+ entry further induces break-down of the phospholipid asymmetry in the host membrane.

Cellular Physiology and Biochemistry, 2009

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also o... more The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE 2 formation, Ca 2+ permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7-/-) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7-/mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7-/mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE 2-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stageinfected HbA/S or annexin-A7-/erythrocytes and thus confers partial protection against malaria in vivo.