Bärbel Bergmann

3-Aminooxy-1-aminopropane and derivatives have an antiproliferative effect on cultured Plasmodium falciparum by decreasing intracellular polyamine concentrations.

ABSTRACT The intraerythrocytic development of Plasmodium falciparum correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the S-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional P. falciparum ODC-AdoMetDC, with Ki values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 μM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against P. falciparum independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in P. falciparum, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC.

The glutathione S-transferase from Plasmodium falciparum.

Liebau, E., Bergmann, B., Campbell, A. M., Teesdale-Spittle, P., Brophy, P. M., Luersen, K., Walter, R. D. (2002). The glutathione S-transferase from Plasmodium falciparum. Molecular and Biochemical Parasitology, 124, (1-2), 85-90

The role of the C-terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum

The thioredoxin system is one of the major thiol reducing systems of the cell. Recent studies have revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, which are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. We identified the cysteines Cys88 and Cys93 as the redox active disulfide and His509 as the active site base [Gilberger, T.‐W., Walter, R.D. and Müller, S., J. Biol. Chem. 272 (1997) 29584–29589]. In addition to the active site thiols Cys88 and Cys93 the P. falciparum enzyme has another pair of cysteines at the C‐terminus: Cys535 and Cys540. To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant PfTrxRC535AC540A and the deletion mutant PfTrxRΔ9 (C‐terminal deletion of the last nine amino acids) were constructed. All mutants are defective in their thioredoxin reduction activity, although they still show reactivity with 5,5′‐dithiobis (2‐nitrobenzoate). These data imply that the C‐terminal cysteines are crucially involved in substrate coordination and/or electron transfer during reduction of the peptide substrate.

Poisoning Pyridoxal 5-Phosphate-Dependent Enzymes: A New Strategy to Target the Malaria Parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum is able to synthesize de novo pyridoxal 5-phosphate (PLP), a crucial cofactor, during erythrocytic schizogony. However, the parasite possesses additionally a pyridoxine/pyridoxal kinase (PdxK) to activate B6 vitamers salvaged from the host. We describe a strategy whereby synthetic pyridoxyl-amino acid adducts are channelled into the parasite. Trapped upon phosphorylation by the plasmodial PdxK, these compounds block PLP-dependent enzymes and thus impair the growth of P. falciparum. The novel compound PT3, a cyclic pyridoxyl-tryptophan methyl ester, inhibited the proliferation of Plasmodium very efficiently (IC50-value of 14 µM) without harming human cells. The non-cyclic pyridoxyl-tryptophan methyl ester PT5 and the pyridoxyl-histidine methyl ester PHME were at least one order of magnitude less effective or completely ineffective in the case of the latter. Modeling in silico indicates that the phosphorylated forms of PT3 and PT5 fit well into the PLP-binding site of plasmodial ornithine decarboxylase (PfODC), the key enzyme of polyamine synthesis, consistent with the ability to abolish ODC activity in vitro. Furthermore, the antiplasmodial effect of PT3 is directly linked to the capability of Plasmodium to trap this pyridoxyl analog, as shown by an increased sensitivity of parasites overexpressing PfPdxK in their cytosol, as visualized by GFP fluorescence.

Plasmodium falciparum glutathione reductase exhibits sequence similarities with the human host enzyme in the core structure but differs at the ligand-binding sites

The homodimeric flavoenzyme glutathione reductase (GR) which catalyzes the reduction of glutathione disulfide is a cornerstone of the malaria parasite antioxidant defense and repair mechanisms. Here we report on the identification of the GR gene from Plasmodium falciparum. A 1.4-kb fragment of the gene was amplified by polymerase chain reaction (PCR). Using this PCR fragment as a probe a full length cDNA clone (2085 bp) was isolated from a P. falciparum gametocyte library. The deduced amino acid sequence of 541 residues shows an overall identity of 35% when compared to the human enzyme. Most amino acids of known function are identical. However, notable differences between human and parasite protein occur in the glutathione-binding pocket (for instance, Glu374 instead of the expected basic residue) and at the intersubunit contact area. These regions are of particular interest since they represent binding sites of known GR inhibitors. Consequently, parasite GR can serve as a target structure for the design of antimalarial drugs.

Pyrimethamin-resistantPlasmodium falciparum lack cross-resistance to methotrexate and 2,4-diamino-5-(substituted benzyl) pyrimidines

Methotrexate resistance induced in culturedPlasmodium falciparum depends on an altered dihydrofolate reductase with decreased affinity for methotrexate as well as for pyrimethamine. In contrast, pyrimethamine-resistant field isolates ofP. falciparum lack cross-resistance to methotrexate and 2,4-diamino-5-(substituted benzyl) pyrimidines. The structure of the latter class was optimized by the use of trimethoprim as a lead and the substitution of methoxy groups at the benzyl ring by 3-(4′-aminophenyl-4-sulfonylphenylamino)propoxy or by (4′-aminophenyl-4-sulfonylphenyl)methoxy, which resulted in antimalarials of high potency. The efficiency of these newly designed 2,4-diamino-5-(substituted benzyl) pyrimidines was confirmed by their strong inhibitory effect on plasmodial dihydrofolate reductase as well as by in vitro screening against drug-sensitive and-resistant strains ofP. falciparum.

Stable Translocation Intermediates Jam Global Protein Export in Plasmodium falciparum Parasites and Link the PTEX Component EXP2 with Translocation Activity.

Protein export is central for the survival and virulence of intracellular P. falciparum blood stage parasites. To reach the host cell, exported proteins cross the parasite plasma membrane (PPM) and the parasite-enclosing parasitophorous vacuole membrane (PVM), a process that requires unfolding, suggestive of protein translocation. Components of a proposed translocon at the PVM termed PTEX are essential in this phase of export but translocation activity has not been shown for the complex and questions have been raised about its proposed membrane pore component EXP2 for which no functional data is available in P. falciparum. It is also unclear how PTEX mediates trafficking of both, soluble as well as transmembrane proteins. Taking advantage of conditionally foldable domains, we here dissected the translocation events in the parasite periphery, showing that two successive translocation steps are needed for the export of transmembrane proteins, one at the PPM and one at the PVM. Our data provide evidence that, depending on the length of the C-terminus of the exported substrate, these steps occur by transient interaction of the PPM and PVM translocon, similar to the situation for protein transport across the mitochondrial membranes. Remarkably, we obtained constructs of exported proteins that remained arrested in the process of being translocated across the PVM. This clogged the translocation pore, prevented the export of all types of exported proteins and, as a result, inhibited parasite growth. The substrates stuck in translocation were found in a complex with the proposed PTEX membrane pore component EXP2, suggesting a role of this protein in translocation. These data for the first time provide evidence for EXP2 to be part of a translocating entity, suggesting that PTEX has translocation activity and provide a mechanistic framework for the transport of soluble as well as transmembrane proteins from the parasite boundary into the host cell.

Secretion of an acid phosphatase provides a possible mechanism to acquire host nutrients by Plasmodium falciparum

As an intracellular proliferating parasite, Plasmodium falciparum exploits the human host to acquire nutrients. However, nutrients such as nucleotides and cofactors are mostly phosphorylated in the host cell cytosol and thus have to be dephosphorylated in order to be taken up by the parasite. Here we report the functional characterization of a unique secreted phosphatase in P. falciparum, which is expressed throughout the developmental stages in the red blood cell. We show that this enzyme, formerly described as anchoring glideosome‐associated protein 50 (GAP50), reveals a broad substrate profile with preference for di‐ and triphosphates at pH 5–7. Bioinformatic studies of the protein sequence identified an N‐terminal signal anchor (SA) as well as a C‐terminal transmembrane domain. By means of live microscopy of parasites transfected with GFP‐fusions of this secreted acid phosphatase (PfSAP), we demonstrate that PfSAP enters the secretory pathway en route to the parasite periphery – mediated by SA – and is subsequently engulfed into the food vacuole. We corroborate this with independent data where acid phosphatase activity is visualized in close proximity to hemozoin. The biochemical as well as the trafficking results support the proposed role of PfSAP in the acquisition of host nutrients by dephosphorylation.

The Vitamin B1 Metabolism of Staphylococcus aureus Is Controlled at Enzymatic and Transcriptional Levels

Vitamin B1 is in its active form thiamine pyrophosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism. The first operon encodes for the thiaminase type II (TenA), 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (ThiD), 5-(2-hydroxyethyl)-4-methylthiazole kinase (ThiM) and thiamine phosphate synthase (ThiE). The second operon encodes a phosphatase, an epimerase and the thiamine pyrophosphokinase (TPK). The open reading frames of the individual operons were cloned, their corresponding proteins were recombinantly expressed and biochemically analysed. The kinetic properties of the enzymes as well as the binding of TPP to the in vitro transcribed RNA of the proposed operons suggest that the vitamin B1 homeostasis in S. aureus is strongly regulated at transcriptional as well as enzymatic levels.

Chemical and genetic validation of thiamine utilization as an antimalarial drug target

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.