Hydrogen peroxide metabolism in Trypanosoma brucei

Contrary to previous reports in the literature, bloodstream forms of the haemoflagellate protozoan Trypanosoma brucei brucei are not deficient in their ability to metabolize hydrogen peroxide, although they either lack or only possess the normal enzymes for H2O2 detoxification, catalase (EC 1.11.1.6) and glutathione peroxidase (EC 1.11.1.9), at extremely low levels. The hydrogen peroxide which is consumed appears to be reduced by NADPH derived from glucose via the pentose phosphate pathway. This process requires the newly discovered cofactor trypanothione.     

Peroxidases of trypanosomatids

This article provides an overview about the recent advances in the dissection of the peroxide metabolism of Trypanosomatidae. This family of protozoan organisms comprises the medically relevant parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. Over the past 10 years, three major families of peroxidases have been identified in these organisms: (a) 2-cysteine peroxiredoxins, (b) nonselenium glutathione peroxidases, and (c) ascorbate peroxidases. In trypanosomatids, these enzymes display the unique feature of using reducing equivalents derived from trypanothione, a dithiol found exclusively in these protozoa. The electron transfer between trypanothione and the peroxidases is mediated by a redox shuttle, which can either be tryparedoxin, ascorbate, or even glutathione. The preference for the intermediate molecule differs among each peroxidase and so does the specificity for the peroxide substrate. These observations, added to the fact that these peroxidases are distributed throughout different subcellular compartments, point to the existence of an elaborate peroxide metabolism in trypanosomatids. With the completion of the trypanosomatids genome, other molecules displaying peroxidase activity might be added to this list in the future.     

Cloning, expression and functional characterisation of a peroxiredoxin from the potato cyst nematode Globodera rostochiensis

We report the cloning, expression and functional characterisation of a peroxidase belonging to the peroxiredoxin family from the potato cyst nematode Globodera rostochiensis, the first molecule of this type from any nematode parasitic on plants. The G. rostochiensis peroxiredoxin catalyses the breakdown of hydrogen peroxide, but not cumene or t-butyl hydroperoxide, in a trypanosomatid reducing system comprising trypanothione reductase, trypanothione and tryparedoxin. In common with its homologues from Onchocerca volvulus and Brugia malayi, the G. rostochiensis enzyme is present on the surface of invasive and post-infective juveniles despite the apparent lack of a cleavable N-terminal signal peptide. The possibility that the G. rostochiensis peroxiredoxin plays a role in protection of the parasite from plant defence responses is discussed.     

Heterologous priming-boosting with DNA and modified vaccinia virus Ankara expressing tryparedoxin peroxidase promotes long-term memory against Leishmania major in susceptible BALB/c Mice

Leishmaniasis affects 12 million people, but there are no vaccines in routine clinical use. Th1 polarizing vaccines that elicit long-term protection are required to prevent disease in susceptible populations. We recently showed that heterologous priming-boosting with tryparedoxin peroxidase (TRYP) DNA followed by TRYP-modified vaccinia virus Ankara (TRYP MVA) protected susceptible BALB/c mice from Leishmania major. Here we compared treatment with TRYP DNA with treatment with TRYP DNA/TRYP MVA. We found that equivalent levels of protection during the postvaccination effector phase correlated with equivalent levels of serum immunoglobulin G2a and gamma interferon (IFN-gamma) in draining lymph nodes. In contrast, challenge infection during the memory phase revealed that there was enhanced clinical efficacy with TRYP DNA/TRYP MVA. This correlated with higher levels of effector phase splenic IFN-gamma, sustained prechallenge levels of memory phase IFN-gamma, and a more polarized post-L. major challenge Th1 response compared to the Th2/T(reg) response. Thus, TRYP DNA/TRYP MVA, but not TRYP DNA alone, provides long-term protection against murine leishmaniasis.     

Trypanothione biosynthesis in Leishmania major

Trypanothione plays a crucial role in regulation of intracellular thiol redox balance and in defence against chemical and oxidant stress. Crithidia fasciculata requires two enzymes for the formation of trypanothione, namely glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and a glutathionylspermidine-dependent trypanothione synthetase (TryS; EC 6.3.1.9), whereas Trypanosoma cruzi and Trypanosoma brucei use a broad-specificity trypanothione synthetase to make trypanothione from glutathione (GSH) and spermidine. Here, we report the identification of two genes in Leishmania major with similarity to previously identified GSPS and TRYS. GSPS is an apparent pseudogene containing two frame shift mutations and two stop codons, whereas TRYS is in a single open-reading frame. The enzyme encoded by TRYS was expressed and found to catalyse formation of trypanothione with GSH and either spermidine or glutathionylspermidine. When GSH is varied as substrate the enzyme displays substrate inhibition (apparent Km=89 microM, Ki(s)=1mM, k(cat)=2s-1). At a fixed GSH concentration, the enzyme obeys simple hyperbolic kinetics with the other substrates with apparent Km values for spermidine, glutathionylspermidine and MgATP of 940, 40 and 63 microM, respectively. Immunofluorescence and sub-cellular fractionation studies indicate that TryS localises to the cytosol of L. major promastigotes. Phylogenetic analysis of the GspS and TryS amino acid sequences suggest that in the trypanosomatids, TryS has evolved to replace the GspS/TryS complex in C. fasciculata. It also appears that the L. major still harbours a redundant GSPS pseudogene that may be currently in the process of being lost from its genome.     

Two linked genes of Leishmania infantum encode tryparedoxins localised to cytosol and mitochondrion

Tryparedoxins are components of the hydroperoxide detoxification cascades of Kinetoplastida, where they mediate electron transfer between trypanothione and a peroxiredoxin, which reduces hydroperoxides and possibly peroxynitrite. Tryparedoxins may also be involved in DNA synthesis, by their capacity to reduce ribonucleotide reductase. Here we report on the isolation of two tryparedoxin genes from Leishmania infantum, Li7XN1 and LiTXN2, which share the same genetic locus. These genes are both single copy and code for two active tryparedoxin enzymes, LiTXN1 and LiTXN2, with different biochemical and biological features. LiTXN1 is located to the cytosol and is upregulated in the infectious forms of the parasite, strongly suggesting that it might play an important role during infection. LiTXN2 is the first mitochondrial tryparedoxin described in Kinetoplastida. Biochemical assays performed on the purified recombinant proteins have shown that LiTXN1 preferentially reduces the cytosolic L. infantum peroxiredoxins, LicTXNPx1 and LicTXNPx2, whereas LiTXN2 has a higher specific activity for a mitochondrial peroxiredoxin, LimTXNPx. Kinetically, the two tryparedoxins follow a ping-pong mechanism and show no saturation. We suggest that LiTXN1 and LiTXN2 are part of two distinct antioxidant machineries, one cytosolic, the other mitochondrial, that complement each other to ensure effective defence from several sources of oxidants throughout the development of L. infantum.     

Trypanosomal antioxidants and emerging aspects of redox regulation in the trypanosomatids

Leishmania and Trypanosoma are two genera of the protozoal Order Kinetoplastida that cause widespread diseases of humans and their livestock. The production of reactive oxygen and nitrogen intermediates by the host plays an important role in the control of infections by these organisms. Signal transduction and its redox regulation have not been studied in any depth in trypanosomatids, but homologs of the redox-sensitive signal transduction machinery of other eukaryotes have been recognized. These include homologs of activator protein-1, human apurinic endonuclease 1 (Ref-1) endonuclease, iron-responsive protein, protein kinases, and phosphatases. The detoxification of peroxide is catalyzed by a trypanothione-dependent system that has no counterpart in mammals, and thus ranks as one of the biochemical peculiarities of trypanosomatids. There is substantial evidence that trypanothione is essential for the survival of Trypanosoma brucei and for the virulence of Leishmania spp. Apart from trypanothione and its precursors, trypanosomatids also possess significant amounts of N(1)-methyl-4-mercaptohistidine or ovothiol A, but its function in the trypanosomatids is not presently understood. The biosynthesis of ovothiol A in Crithidia fasciculata proceeds by addition of sulfur from cysteine to histidine to form 4-mercaptohistidine. S-(4'-L-Histidyl)-L-cysteine sulfoxide is the transsulfuration intermediate. 4-Mercaptohistidine is subsequently methylated with S-adenosylmethionine as the likely methyl donor.     

In vivo effects of difluoromethylornithine on trypanothione and polyamine levels in bloodstream forms of Trypanosoma brucei

The effect of D,L-alpha-difluoromethylornithine (DFMO) on thiol and polyamine levels in Trypanosoma brucei was investigated by isolating trypanosomes from infected rats treated with DFMO for 12-48 h. Concentrations of thiols, polyamines and other amino-compounds were measured by an automated high-performance liquid chromatography method. The levels of DFMO in rat plasma (0.02-1.34 mM) is similar to that found in the parasites (0.27-0.99 mM), concentrations which exceed the Ki of DFMO for T. brucei ornithine decarboxylase. Treatment with DFMO increases intracellular levels of ornithine, S-adenosylmethionine and decarboxylated S-adenosylmethionine and decreases putrescine and spermidine. Putrescine is undetectable after 12 h treatment with DFMO and after 48 h spermidine is decreased by 76%. By 48 h, the spermidine-glutathione conjugates glutathionylspermidine and dihydrotrypanothione (bis(glutathionyl)spermidine) are also decreased by 41 and 66%, respectively. In contrast, levels of glutathione show a slight increase. These changes in metabolite levels are consistent with the biosynthetic pathway proposed for Crithidia fasciculata, where trypanothione is synthesized from spermidine and glutathione via the intermediates N1- and N8-glutathionyl-spermidine. Trypanothione is thought to have two important roles in trypanosomatid metabolism: the maintenance of intracellular thiols in the correct redox state and in the removal of hydrogen peroxide and other hydroperoxides. Thus, it is proposed that depletion of this metabolite may be an important contributory factor to the selective toxic effect of DFMO, particularly in its synergistic effect with other trypanocidal drugs.     

Characterisation of melarsen-resistant Trypanosoma brucei brucei with respect to cross-resistance to other drugs and trypanothione metabolism.

An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (greater than 200-fold resistance to melarsen oxide and greater than 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.     

The biosynthesis of trypanothione and N1-glutathionylspermidine in Crithidia fasciculata

Studies on the biosynthesis of trypanothione [N1,N8-bis(glutathionyl)-spermidine] in the insect trypanosomatid Crithidia fasciculata have led to the discovery of an additional sulfur-containing peptide conjugated to spermidine. Labelling studies with [3H]spermidine show that 50% of the total intracellular spermidine is incorporated into peptide conjugates, the major component being N1-glutathionylspermidine. This compound has previously been identified in Escherichia coli, as the principal low molecular weight thiol in stationary phase, but not the logarithmic phase of growth. In contrast, in C. fasciculata, this compound is present in all phases of growth. In the presence of glutathione and ATP, extracts of C. fasciculata can catalyse conversion of spermidine to N1-glutathionylspermidine and trypanothione. Both N1- and N8-regioisomers of glutathionylspermidine will replace spermidine in the reaction, suggesting they may be intermediates in the biosynthetic pathway to trypanothione. The antiprotozoal drugs berenil, pentamidine, ethidium bromide, imidocarb, methylglyoxal-bis(guanylhydrazone) and 1,3-diacetylbenzene-bis(guanylhydrazone) had no effect on the synthesis of N1-glutathionylspermidine or trypanothione in vitro.     

Possible role of the NADH-fumarate reductase in superoxide anion and hydrogen peroxide production in Trypanosoma brucei

Mitochondrial membranes from Trypanosoma brucei procyclic trypomastigotes generated superoxide anion and hydrogen peroxide in a 2:1 ratio when supplemented with NADH. Fumarate inhibited hydrogen peroxide formation (Ki = 16 microM) with the same affinity as it stimulated NADH-fumarate reductase activity. Superoxide anion production was also 65% inhibited by fumarate (Ki = 20 microM). The KM for NADH of the NADH-fumarate reductase (60 microM) was also similar to that for hydrogen peroxide generation in the absence of fumarate (30 microM). These results suggest that the NADH-fumarate reductase is involved as a source for free radical generation in T. brucei mitochondria.     

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.     

Cloning and characterization of the subunits comprising the catalytic core of the Trypanosoma brucei mitochondrial ATP synthase

The Trypanosoma brucei mitochondrial F(1)-ATPase has been previously isolated and characterized. It is composed of five subunits of molecular weights 55000, 42000, 32000, 22000, and 17000 [1]. We have identified the alpha and beta subunits of the T. brucei F(1)-ATPase by N-terminal sequence determination together with analysis of cDNA and genomic clones. The genes for both subunits are homologous to the same subunits from other organisms. They contain the Walker A and B boxes of homology and a putative mitochondrial import sequence. The isolated T. brucei alpha subunit is unusually small at 42 kDa. The alpha cDNA clone encodes a protein of predicted size 59 kDa with a mitochondrial import presequence at the N-terminus. The predicted size was confirmed by expression of a 59 kDa protein from the cDNA clone in vitro. These results suggest that the alpha subunit may have an unusually large mitochondrial presequence of 159 amino acids. In contrast, the estimated size of the native beta subunit (55 kDa) correlates well with the size predicted from the cDNA clone, 57 kDa, from which a 21 amino acid presequence has been removed in vivo. The size of the beta subunit was confirmed by expression in an in vitro and an Escherichia coli expression system. The purified recombinant beta subunit, like the native F(1)-ATPase, can be labeled by the photoaffinity nucleotide analogue 8-azido ATP. Binding of the 8-azido ATP probe is best competed by the natural substrate ATP, and is significantly reduced by pretreatment with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazide as has been shown with beta subunits of other organisms. The differential binding of this photoaffinity analogue was used to resolve the identities of the alpha and beta subunits of the ATP synthase from T. brucei. These results are in contrast to results previously obtained for a related trypanosomatid Crithidia fasciculata.     

The 6-phosphogluconate dehydrogenase from Trypanosoma cruzi: the absence of two inter-subunit salt bridges as a reason for enzyme instability

The third enzyme of the pentose phosphate pathway (PPP), 6-phosphogluconate dehydrogenase (6PGDH), is present in the four major stages of Trypanosoma cruzi, CL Brener clone. The enzyme was too unstable to be purified from epimastigote cell-free extracts. Two genes encoding 6PGDH were cloned and sequenced; the predicted amino acid sequences differ only in five non-essential residues. Since Southern blots suggested the presence of a single copy per haploid genome, the two genes found are probably alleles. One of these genes, encoding a protein with 78.6% identity with the Trypanosoma brucei 6PGDH, was expressed in Escherichia coli as an active recombinant enzyme, which was as unstable as the native 6PGDH. Modeling of the T. cruzi enzyme using the three-dimensional structure of the T. brucei 6PGDH as template suggested the lack of two out of five salt bridges proposed to strengthen subunit interactions in the active dimer. Restoring of these bridges by site-directed mutagenesis resulted in a more stable recombinant T. cruzi 6PGDH, which was used to determine the kinetic parameters. The K(m) value for 6-phosphogluconate (22.2+/-0.4 microM) was identical to the values reported for 6PGDHs from mammals, but the K(m) for NADP (5.9+/-0.2 microM) was significantly lower than the value reported for the human enzyme, and closer to that for the T. brucei enzyme. This suggests the possibility that inhibitors of the T. brucei 6PGDH, under development as potential drugs against African Trypanosomiasis, might also be successful for the chemotherapy of Chagas disease.     

十二指肠钩虫谷胱甘肽转移酶AduGST-1基因的克隆和重组表达

目的克隆十二指肠钩虫谷胱甘肽转移酶（GST）AduGST-1基因,并在大肠埃希菌中表达获得重组AduGST-1。方法设计特异引物,以十二肠钩虫成虫cDNA为模板,通过PCR扩增AduGST-1基因。将获得的AduGST-1编码序列克隆至原核表达载体pETHF,构建重组表达质粒pETHF/AduGST-1。重组质粒转化至大肠埃希菌BL21（DE3）,用IPTG诱导表达、Ni亲和层析分离纯化重组AduGST-1,SDS-PAGE分析重组蛋白表达及纯化情况。结果成功扩增到AduGST-1全长编码序列,并登记到GenBank（accession no.JQ812812）。AduGST-1编码序列长度为624bp,编码307个氨基酸残基。成功构建了重组表达质粒pETHF/AduGST-1,在BL21（DE3）中表达并纯化了重组AduGST-1。结论首次报道从十二指肠钩虫中分离到GST基因,该基因可在大肠埃希菌中高效表达,并分离纯化了重组GST蛋白,为进一步研究AduGST-1功能与应用奠定了基础。     

The enigmatic physiological roles of AhpCF,Gpx, Npr and Kat in peroxide stress response of Enterococcus faecium

In this work, the physiological roles of the primary peroxide scavenging activities of Enterococcus faecium AUS0004 strain were analysed. This healthcare-associated pathogen harbours genes encoding putative NADH peroxidase (Npr), alkyl hydroperoxide reductase (AhpCF), glutathione peroxidase (Gpx) and manganese-dependent catalase (Mn-Kat). Gene expression analyses showed that npr and kat genes are especially and significantly induced in cells treated with hydrogen peroxide (H₂O₂) and cumene hydroperoxide (CuOOH), which suggested an important function of these enzymes to protect E. faecium against peroxide stress. Mutants affected in one or several predicted anti-oxidative activities mentioned above showed that neither the peroxidases nor the catalase are implicated in the defence against peroxide challenges. However, our investigations allowed us to show that Npr is responsible for the degradation of approximately 45% of metabolically derived H₂O₂ which avoids accumulation of the peroxide to lethal concentrations.     

牛AsiaⅠ型口蹄疫病毒重组蛋白疫苗的构建及功能研究

目的为了取代有安全隐患的牛AsiaⅠ型口蹄疫病毒灭活疫苗,我们构建了基因工程重组蛋白疫苗。方法应用PCR方法合成含有表位的基因片段,经过克隆连接获得重组基因,在大肠杆菌中表达后经镍亲和层析纯化重组蛋白。重组蛋白免疫豚鼠后,分别经ELISA和乳鼠中和实验检测血清中抗FMDV抗体水平。结果构建了牛AsiaⅠ型口蹄疫病毒重组蛋白疫苗的结构基因,并成功表达和纯化了该重组蛋白。功能实验表明,该蛋白在豚鼠体内诱生了高水平的抗牛AsiaⅠ型口蹄疫病毒的中和性抗体。结论该重组蛋白为制备牛AsiaⅠ型口蹄疫病毒新型疫苗提供了有价值的线索。