Deficient metabolic utilization of hydrogen peroxide in <i>Trypanosoma cruzi</i>

Boveris, A; Sies, Helmut; Martino, Elba E.; Docampo, Roberto; Turrens, Julio F.; Stoppani, A. O. M.

doi:10.1042/bj1880643

Cited by 196 publications

(93 citation statements)

References 23 publications

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“…These enzymes have been located in all oxy-radical-generating compartments in plants, with multiple isoforms identified (14). In T. cruzi, significant levels of ascorbate and its oxidized form, dehydroascorbate (DHA), have been detected and previous studies have identified a microsomal-associated ascorbate-dependent peroxidase activity (2,15,16). Here we show that T. cruzi expresses an unusual plant-like ascorbate-dependent hemoperoxidase whose activity is linked to the trypanothione system.…”

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confidence: 80%

“…In contrast to most eukaryotes, T. cruzi lacks catalase and selenium-dependent glutathione peroxidases, enzymes capable of rapidly metabolizing high levels of H 2 O 2 (2,4,5). Instead it has an array of enzyme-mediated mechanisms in which the trypanosomatid-specific thiol trypanothione (N 1 ,N 8 -bisglutathionylspermidine) plays a pivotal role.…”

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confidence: 99%

“…With no prospect of a vaccine, the search for parasite-specific traits exploitable in terms of chemotherapy is a priority (1). It has been reported that trypanosomatids have a limited ability to deal with reactive oxygen species (2,3). In the parasite these are generated by endogenous processes and as a result of external influences such as host immune responses and drug metabolism.…”

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confidence: 99%

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Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Wilkinson

Obado

Maurício

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

133

116

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In most aerobic organisms hemoperoxidases play a major role in H 2O2-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis-Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H 2O2. To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.

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confidence: 80%

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confidence: 99%

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confidence: 99%

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Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Wilkinson

Obado

Maurício

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

133

116

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“…Trypanosomes and leishmania are much more sensitive towards oxidative stress than their respective hosts which may be due to the lack of glutathione peroxidase and catalase (Boveris et al, 1980;Carnieri et al, 1993). In trypanosomatids glutathione reductase is replaced by trypanothione reductase (Fairlamb and Cerami, 1992;Schirmer et al, 1995).…”

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confidence: 99%

Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi

Schöneck

Billaut-Mulot

Numrich

et al. 1997

European Journal of Biochemistry

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This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50066. Comparison of the deduced amino acid sequence of LipDH from 7: crzizi with that of Trypanosoma brucei and man shows identities of 81 % and 50%, respectively.An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpdl of 7: cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for AsnlO which represents the N-terminus of the mature protei-n. This system allowed the synthesis of 1000 U 7: cruzi LipDH/l bacterial cell culture. The recombinant protein was purified to homogeneity by (NH,),SO,-precipitation and affinity chromatography on 5' AMP-Sepharose. The K,,, values for NAD', NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite.LipDH is present in all major developmental stages of 7: cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite.In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason 7: cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of 7: cruzi LipDH in drug activation and the crystallization of the protein.

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“…It has been shown that the parasite antioxidant systems are essential for parasite survival and establishment of the infection in the vertebrate host (4)(5)(6)(7). In contrast to most eukaryotes, T. cruzi lacks catalase and selenium-dependent glutathione peroxidases, which are enzymes capable of rapidly metabolizing high levels of H 2 O 2 (8,9). Instead, it expresses an array of complex enzyme-mediated mechanisms in which the trypanosomatid-specific thiol trypanothione ([T(SH) 2 ], N 1 ,N 8 -bisglutathionylspermidine) plays a central role in the funneling of reducing equivalents to the different peroxidase antioxidant systems (10).…”

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confidence: 99%

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

Hugo

Martínez

Trujillo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H 2 O 2 (k = 2.9 × 10 7 M −1 ·s −1 ) and catalytically decomposes H 2 O 2 using Cc as the reducing substrate with higher efficiency than Asc (k cat /K m = 2.1 × 10 5 versus 3.5 × 10 4 M −1 ·s −1 , respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H 2 O 2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp 233•+ ). Mutation of Trp 233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp 233•+ , a Cys 222 -derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H 2 O 2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp 233 and Cys 222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.Trypanosoma cruzi | heme peroxidase | oxidants | virulence | kinetics

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Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi

Cited by 196 publications

References 23 publications

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi

Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase ( Tc APx-CcP)

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