Rolf D. Walter

Thiol-based redox metabolism of protozoan parasites

The review considers redox enzymes of Plasmodium spp., Trypanosomatida, Trichomonas, Entamoeba and Giardia, with special emphasis on their potential use as targets for drug development. Thiol-based redox systems play pivotal roles in the success and survival of these parasitic protozoa. The synthesis of cysteine, the key molecule of any thiol metabolism, has been elucidated in trypanosomatids and anaerobes. In trypanosomatids, trypanothione replaces the more common glutathione system. The enzymes of trypanothione synthesis have recently been identified. The role of trypanothione in the detoxification of reactive oxygen species is reflected in the multiplicity of trypanothione-dependent peroxidases. In Plasmodium falciparum, the crystal structures of glutathione reductase and glutamate dehydrogenase are now available; another drug target, thioredoxin reductase, has been demonstrated to be essential for the malarial parasite.

Regulation of intracellular glutathione levels in erythrocytes infected with chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum

Malaria is one of the most devastating tropical diseases despite the availability of numerous drugs acting against the protozoan parasite Plasmodium in its human host. However, the development of drug resistance renders most of the existing drugs useless. In the malaria parasite the tripeptide glutathione is not only involved in maintaining an adequate intracellular redox environment and protecting the cell against oxidative stress, but it has also been shown that it degrades non-polymerized ferriprotoporphyrin IX (FP IX) and is thus implicated in the development of chloroquine resistance. Glutathione levels in Plasmodium -infected red blood cells are regulated by glutathione synthesis, glutathione reduction and glutathione efflux. Therefore the effects of drugs that interfere with these metabolic processes were studied to establish possible differences in the regulation of the glutathione metabolism of a chloroquine-sensitive and a chloroquine-resistant strain of Plasmodium falciparum. Growth inhibition of P. falciparum 3D7 by D,L-buthionine-( S, R )sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), and by Methylene Blue (MB), an inhibitor of gluta thione reductase (GR), was significantly more pronounced than inhibition of P. falciparum Dd2 growth by these drugs. These results correlate with the higher levels of total glutathione in P. falciparum Dd2. Short-term incubations of Percoll-enriched trophozoite-infected red blood cells in the presence of BSO, MB and N, N (1)-bis(2-chloroethyl)- N -nitrosourea and subsequent determinations of gamma-GCS activities, GR activities and glutathione disulphide efflux revealed that maintenance of intracellular glutathione in P. falciparum Dd2 is mainly dependent on glutathione synthesis whereas in P. falciparum 3D7 it is regulated via GR. Generally, P. falciparum Dd2 appears to be able to sustain its intracellular glutathione more efficiently than P. falciparum 3D7. In agreement with these findings is the differential susceptibility to oxidative stress of both parasite strains elicited by the glucose/glucose oxidase system.

Pathogenic and nonpathogenicEntamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase

Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytica isolate HM-1:IMSS. Under anaerobic culture conditions, 18.7 (+/- 4.9) units SOD activity (mg protein)-1 were found. By inhibition studies the activity was attributed to an iron-containing type of SOD (FeSOD). Using degenerate oligonucleotide primers derived from regions highly conserved in prokaryotic FeSOD sequences, a genomic DNA fragment was amplified by the polymerase chain reaction. The fragment was used to isolate FeSOD specific cDNA clones from a pathogenic and a nonpathogenic E. histolytica isolate. A comparison of the 2 sequences revealed 5% nucleotide differences resulting in a single amino acid exchange. The primary structure showed the characteristics of an iron-containing type of SOD with a homology of approximately 55% with other FeSOD sequences. The enzyme was found to be encoded by single copy genes in both the pathogenic and the nonpathogenic E. histolytica, but restriction fragment lengths differed between the 2 groups. In 5 isolates studied, no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA.

Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO-1)

To elucidate the function of Omega class glutathione transferases (GSTs) (EC 2.5.1.18) in multicellular organisms, the GSTO‐1 from Caenorhabditis elegans (GSTO‐1; C29E4.7) was investigated. Disc diffusion assays using Escherichia coli overexpressing GSTO‐1 provided a test of resistance to long‐term exposure under oxidative stress. After affinity purification, the recombinant GSTO‐1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity. Microinjection of the GSTO‐1‐promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C. elegans. Deletion analysis identified an ~300‐nucleotide sequence upstream of the ATG start site necessary for GSTO‐1 expression. Site‐specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt‐2 indicated the involvement of this gut‐specific transcription factor in GSTO‐1 expression. Transcriptional up‐regulation under stress conditions of GSTO‐1 was confirmed by analyzing promoter‐reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO‐1 in vivo, transgenic animals overexpressing GSTO‐1 were generated exhibiting an increased resistance to juglone‐, paraquat‐, and cumene hydroperoxide‐induced oxidative stress. Specific silencing of the GSTO‐1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress‐responsive GSTO‐1 plays a key role in counteracting environmental stress.—Burmeister, C., Lüersen, K., Heinick, A., Hussein, A., Domagalski, M., Walter, R. D., Liebau, E. Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO‐1). FASEB J. 22, 343–354 (2008)

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.

Gene structure, activity and localization of a catalase from intracellular bacteria in Onchocerca volvulus.

Within the context of studies on the antioxidant enzymes in Onchocerca volvulus, DNA clones encoding catalase (CAT) were isolated from an O. volvulus adult lambda zapII cDNA library. Analysis of their nucleotide and encoded amino acid sequences revealed that they derive from intracellular bacteria, rather than the O. volvulus nuclear genome. The endobacterial CAT gene was found to lie in a gene cluster, followed by a ferritin gene and an excinuclease gene. The endobacterial CAT gene encodes a functional enzyme capable of detoxifying H2O2, demonstrated by producing an active recombinant protein in an E. coli expression system. The purified 54 kDa protein has CAT activity over a broad pH range, with a specific activity of 103,000 +/- 3000 U mg(-1). The optical spectrum of the endobacterial CAT shows that it is a ferric haem-containing protein with a Soret band at 405 nm. To investigate the phylogeny of the intracellular bacterium in O. volvulus, a segment of the 16S rRNA gene was amplified from total genomic DNA by a polymerase chain reaction using universal eubacterial primers. A phylogenetic analysis of the O. volvulus-derived 16S rRNA sequence revealed that the endobacterium belongs to a distinct Wolbachia clade of the order Rickettsiales. Onchocercomata and biopsies containing different onchocercal species were immunohistochemically stained using polyclonal antibodies raised against the recombinant endobacterial CAT. CAT was detected in the endobacteria in the hypodermis of adult male and female O. volvulus, O. ochengi, O. gibsoni and O. fasciata. The endobacterial enzyme was also detected in onchocercal oocytes and all embryonic stages including intrauterine microfilariae as well as skin microfilariae. O. volvulus thus harbours Wolbachia-like endosymbionts which are transovarially transmitted and show particular affinity for the hypodermal tissues of the lateral chords.

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

Identification and characterization of the functional amino acids at the active site of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system, composed of the pyridine nucleotide-disulfide oxidoreductase thioredoxin reductase, the small peptide thioredoxin, and NADPH as a reducing cofactor, is one of the major thiol-reducing systems of the cell. Recent studies revealed thatPlasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, called large thioredoxin reductases. The large thioredoxin reductases are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. The putative essential amino acids at the catalytic center of large thioredoxin reductase from P. falciparumwere determined by using site-directed mutagenesis techniques. To analyze the putative active site cysteines (Cys88 and Cys93) three mutant proteins were constructed substituting alanine or serine residues for cysteine residues. Further, to evaluate the function of His509 as a putative proton donor/acceptor of large thioredoxin reductase this residue was replaced by either glutamine or alanine. All mutants were expressed in the E. coli system and characterized. Steady state kinetic analysis revealed that the replacement of Cys88 by either alanine or serine and Cys93 by alanine resulted in a total loss of enzymatic activity. These results clearly identify Cys88and Cys93 as the active site thiols of large thioredoxin reductase. The replacement of His509 by glutamine yielded in a 95% loss of thioredoxin reductase activity; replacement by alanine provoked a loss of 97% of enzymatic activity. These results identify His509 as active site base, but imply that its function can be substituted, although inefficiently, by an alternative proton donor, similar to glutathione reductase. Spectral analysis of wild-type P. falciparum thioredoxin reductase revealed a 550-nm absorption band upon reduction which resembles the EH2 form of glutathione reductase and lipoamide dehydrogenase. This spectral feature, recently also reported for the human placenta protein (Arscott, L. D., Gromer, S., Schirmer, R. H., Becker K., and Williams, C. H., Jr. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3621–3626), further illustrates the similarity between large thioredoxin reductases and glutathione reductases and stresses the profound differences to smallE. coli thioredoxin reductase.

Assessing the polyamine metabolism of Plasmodium falciparum as chemotherapeutic target

More than 30 years ago the potent ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) was designed as new anticancer drug. Its efficacy was not as expected since the polyamine metabolism in mammalian cells seemed to be far more complex. However when DFMO was applied to African trypanosomes its effect on this protozoan parasite was highly convincing. Thenceforward many researchers tested DFMO and also other polyamine synthesis inhibitors against different parasites among them the causative agent of malaria Plasmodium. This review recapitulates the different attempts to interfere chemically with the plasmodial polyamine metabolism, the impact on the disease as well as its biochemical and molecular background. It will show that this fast proliferating organism depends for growth on high amounts of polyamines and that Plasmodium has its own and unique polyamine synthesis, differing highly from the mammalian one mainly in the arrangement of the key enzymes, S-adenosylmethionine decarboxylase and ornithine decarboxylase (AdoMetDC/ODC), on a bifunctional protein.

Glutathione synthetase from Plasmodium falciparum

GSH is the major low-molecular-mass thiol in most organisms. The tripeptide maintains a reduced intracellular environment and protects cellular components from damaging oxidation. GSH is synthesized by the action of two ATP-dependent enzymic steps, in which gamma-glutamylcysteine synthetase (gamma-GCS) catalyses the ligation of glutamate and cysteine and subsequently glutathione synthetase (GS) adds glycine to the dipeptide. Recently it was shown that the synthesis of gamma-glutamylcysteine is crucial for the survival of the erythrocytic stages of the malaria parasite Plasmodium falciparum by using the specific gamma-GCS inhibitor buthionine sulphoximine. In order to investigate further the synthetic pathway of the tripeptide in the parasite, GS was cloned and expressed recombinantly. The deduced amino acid sequence of P. falciparum GS shares only a moderate degree of identity with other known GSs, but the residues responsible for substrate and co-factor binding are almost all conserved, with the exception of the ones involved in gamma-glutamylcysteine binding. The protein is active as a dimer, with a subunit molecular mass of 77 kDa, and the addition of reducing reagents such as dithiothreitol is essential in maintaining enzymic activity, indicating that thiol groups are important for stability and enzymic activity. The K(app)(m) values for gamma-glutamyl-alpha-aminobutyrate, ATP and glycine were determined to be 107.1 microM, 59.1 microM and 5.04 mM, respectively, and the V(max) of 5.24 +/- 0.7 micromol.min(-1).mg(-1) was in the same range as that of the mammalian enzymes. However, the negative co-operativity observed for gamma-glutamylcysteine binding to the rat enzyme was not found for the parasite protein. This may be due to the alteration of several amino acids in the gamma-glutamylcysteine-binding site.