Trypanosomatid essential metabolic pathway: New approaches about heme fate in Trypanosoma cruzi

doi:10.1016/j.bbrc.2014.05.004

Biochemical and Biophysical Research Communications

Volume 449, Issue 2, 27 June 2014, Pages 216-221

https://doi.org/10.1016/j.bbrc.2014.05.004 Get rights and content

Highlights

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An active heme detoxification mechanism is suggested in this work.
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For the first time, biliverdin, a heme degradation intermediate, was identified in this parasite.
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A putative HO-like protein increases in the presence of heme in T. cruzi.

Abstract

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and depends on hosts for its nutritional needs. Our group has investigated heme (Fe-protoporphyrin IX) internalization and the effects on parasite growth, following the fate of this porphyrin in the parasite. Here, we show that epimastigotes cultivated with heme yielded the compounds α-meso-hydroxyheme, verdoheme and biliverdin (as determined by HPLC), suggesting an active heme degradation pathway in this parasite. Furthermore, through immunoprecipitation and immunoblotting assays of epimastigote extracts, we observed recognition by an antibody against mammalian HO-1. We also detected the localization of the HO-1-like protein in the parasite using immunocytochemistry, with antibody staining primarily in the cytoplasm. Although HO has not been described in the parasite’s genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets.

Introduction

Chagas disease or American trypanosomiasis is caused by the parasite Trypanosoma cruzi, which is transmitted to a vertebrate host by triatomine insects during feeding [1]. This disease is recognized by the World Health Organization as one of the neglected global diseases that infects an estimated 10 million people, primarily in Latin America, where the disease is endemic, and the migration of populations between countries has created new epidemiological, economic, social and political challenges [2]. More than 100 years after its discovery, Chagas disease does not have an efficient chemotherapy and new drugs and therapies should be sought for the treatment of Chagas disease.

T. cruzi has a complex life cycle within different hosts, including humans, which supply the nutritional requirements lacking in the parasite [3]. Among these nutrients, heme is crucial to the proliferation of parasite epimastigotes [4], [5] and is an essential supplement for the culture of these cells [6], [7]. Heme catalyzes many of the oxidation processes in biological systems and is important in cellular function and organismal homeostasis. Furthermore it is also a regulatory molecule, and the absolute intracellular concentration must be tightly regulated [8], [9].

Although it has been reported that T. cruzi lacks a complete pathway for heme biosynthesis [10], [11], these species contains hemeprotein-like cytochromes involved in essential metabolic pathways. Buchensky et al. [12] published work showing the first functional characterization of T. cruzi ORFs that encode enzymes involved in heme A biosynthesis (TcCox10 and TcCox15), the prosthetic group of the mitochondrial cytochrome c oxidase and several bacterial terminal oxidases. Moreover, the heme porphyrin is important in T. cruzi epimastigote biology and must therefore be scavenged from the host. Heme uptake may occur via a specific porphyrin transporter, possibly a member of the ABC-transporter family [5], [13].

However, free heme in solution is a potent free radical producer and is extremely harmful to biological systems [8], [14], [15], therefore its control is essential to the survival of the organism. One heme detoxification pathway occurs via the enzyme heme oxygenase (HO), an enzyme that is physiologically important, in part, because of the biological properties of its organic reaction products. In mammals, HO catalyzes heme degradation in an oxygen-dependent reaction and produces biliverdin (BV), carbon monoxide (CO) and iron (Fe⁺²). BV is reduced by biliverdin reductase to bilirubin, which is excreted as a glucuronic acid conjugate [16], [17]. Three HO isoforms have been characterized in several mammals, HO-1, HO-2 and HO-3, products of three different genes [18].

As the system for the enzymatic degradation of heme in T. cruzi is unknown, the object of this work was to study heme catabolism in this parasite. We identified heme degradation products and HO expression, a result that suggested the action of a heme oxygenase-like enzyme involved in T. cruzi heme homeostasis, underscoring its high potential as a chemotherapeutic target.

Section snippets

Parasites

The T. cruzi strain Dm 28c (CT-IOC-010) was obtained from the Trypanosomatid Collection at the Oswaldo Cruz Institute, FIOCRUZ, Brazil. The epimastigotes were grown at 28 °C for 7 days in brain–heart infusion medium (BHI) and supplemented with 30 μM heme and 10% fetal calf serum (FCS). The cultures were maintained in 100-mL bottles with an initial density of 20–30 × 10⁶ cells/mL in 30 mL of medium. Growth was monitored by counting the cells in a Neubauer chamber.

Extraction of heme and metabolites from T. cruzi epimastigotes

The epimastigote cells were maintained

Results and discussion

Although it is well known that trypanosomatids must obtain extracellular heme from their hosts as a nutritional cofactor [24], it remains unclear how heme is catabolized in these parasites. To study the heme degradation products of T. cruzi, we adopted a liquid-chromatography-method from Paiva et al. [25] using heme (Fe-protoporphyrin IX) and biliverdin IXα as standards to analyze the production of heme metabolites (Fig. 1A).

T. cruzi epimastigotes were treated as described in the Material and

References (37)

F.A. Lara et al.
Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes
Biochem. Biophys. Res. Commun.
(2007)
S.W. Ryter et al.
The heme synthesis and degradation pathways: role in oxidant sensitivity: heme oxygenase has both pro and antioxidant properties
Free Radic. Biol. Med.
(2000)
M.E. Lombardo et al.
5-Aminolevulinic acid synthesis in epimastigotes of Trypanosoma cruzi
Int. J. Biochem. Cell Biol.
(2003)
M.P. Cupello et al.
The heme uptake process in Trypanosoma cruzi epimastigotes is inhibited by heme analogues
Act. Trop.
(2011)
L.J. Deterding et al.
Identification of free radicals on hemoglobin from its self-peroxidation using mass spectrometry and immunospin trapping: observation of a histidinyl radical
J. Biol. Chem.
(2004)
R. Tenhunen et al.
Microsomal heme oxygenase. Characterization of the enzyme
J. Biol. Chem.
(1969)
G.R.C. Braz et al.
A missing pathway in the cattle tick boophilus microplus
Curr. Biol.
(1999)
O.H. Lowry et al.
Protein measurement with the folin phenol reagent
J. Biol. Chem.
(1951)
T. Yoshida et al.
Mechanism of heme degradation by heme oxygenase
J. Inorg. Biochem.
(2000)
K.M. Matera et al.
Oxygen and one reducing equivalent are both required for the conversion of alpha-hydroxyhemin to verdoheme in heme oxygenase
J. Biol. Chem.
(1996)

J.C. Lagarias

The structure of verdohemochrome and its implications for the mechanism of heme catabolism

Biochim. Biophys. Acta

(1982)

A. Wilks et al.

Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine

J. Biol. Chem.

(2000)

M.B. Arnao et al.

A kinetic study on the suicide inactivation of peroxidase by hydrogen peroxide

Biochim. Biophys. Acta

(1990)

S.A. Adediran

Kinetic of the formation of p-670 and the decay of compound III of horseradish peroxidase

Arch. Biochem. Biophys.

(1996)

P. Andreoletti et al.

Verdoheme formation in Proteus mirabilis catalase

Biochim. Biphys. Acta

(2009)

K. Okada

The novel heme oxygenase-like protein from Plasmodium falciparum converts heme to bilirubin IXa in the apicoplast

FEBS Lett.

(2009)

P.A. Sigala et al.

Direct tests of enzymatic degradation by the malaria parasite Plasmodium falciparum

J. Biol. Chem.

(2012)

C. Chagas

Nova tripanomíase humana. Estudos sobre a morfologia e o ciclo evolutivo do Schizotrypanum cruzi, n. gen., agente etiológico de nova entidade mórbida do homem

Mem. Inst. Oswaldo Cruz

(1909)

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A new model for Trypanosoma cruzi heme homeostasis depends on modulation of TcHTE protein expression
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Citation Excerpt :
These results strongly suggest that heme requirement could modulate TcHTE protein level (and mRNA) in the parasite, supporting a direct relation between TcHTE and heme transport activity. We cannot exclude heme degradation as part of a mechanism to control intracellular heme, but genes encoding enzymes with heme oxygenase homology have not been identified in the genome of T. cruzi (16). Presumably, heme degradation via heme oxygenase like activity reported previously (16) would not be enough to get rid of any excess of heme when epimastigotes were exposed at high concentration of hemin.
Heme is an essential cofactor for many biological processes in aerobic organisms, which can synthesize it de novo through a conserved pathway. Trypanosoma cruzi, the etiological agent of Chagas disease, as well as other trypanosomatids relevant to human health, are heme auxotrophs, meaning they must import it from their mammalian hosts or insect vectors. However, how these species import and regulate heme levels is not fully defined yet. It is known that the membrane protein TcHTE is involved in T. cruzi heme transport, although its specific role remains unclear. In the present work, we studied endogenous TcHTE in the different life cycle stages of the parasite to gain insight into its function in heme transport and homeostasis. We have confirmed that TcHTE is predominantly detected in replicative stages (epimastigote and amastigote), in which heme transport activity was previously validated. We also showed that in epimastigotes, TcHTE protein and mRNA levels decrease in response to increments in heme concentration, confirming it as a member of the heme response gene family. Finally, we demonstrated that T. cruzi epimastigotes can sense intracellular heme by an unknown mechanism and regulate heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity by adjusting the expression of TcHTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of a critical pathway for T. cruzi biology allowing the identification of novel and essential proteins.
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So far only one publication exists describing iron liberation from heme via an HO activity [25]. In trypanosomatids the formation of verdoheme and biliverdin was identified in epimastigotes [61], suggesting a heme degradation pathway via an HO-like protein. Hemin was shown to be an essential growth factor in LtP cultures [62].
Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC₅₀ values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC₅₀ value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λ_max 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.
Interactions between 4-aminoquinoline and heme: Promising mechanism against Trypanosoma cruzi
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Thus, association of quinoline drugs with heme, which seems to be essential for chloroquine uptake (Bray et al., 1999), inhibits the hemozoin formation in the digestive vacuole. Interestingly, the detection of a heme oxygenase-like enzyme in T. cruzi (Cupello et al., 2014) indicates a different heme catabolism pathway compared to the Plasmodium spp. Although the complete pathway is still unknown, results showed a rapid uptake of heme via the ABC transporter (Cupello et al., 2011), that reaches the reservosome, an acid compartment that stores lipids and proteases (Cunha-e-Silva et al., 2006).
Chagas disease is a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. The current drugs used to treat this disease have limited efficacy and produce severe side effects. Quinolines, nitrogen heterocycle compounds that form complexes with heme, have a broad spectrum of antiprotozoal activity and are a promising class of new compounds for Chagas disease chemotherapy. In this study, we evaluated the activity of a series of 4-arylaminoquinoline-3-carbonitrile derivatives against all forms of Trypanosoma cruzi in vitro. Compound 1g showed promising activity against epimastigote forms when combined with hemin (IC50<1 μM), with better performance than benznidazole, the reference drug. This compound also inhibited the viability of trypomastigotes and intracellular amastigotes. The potency of 1g in combination with heme was enhanced against epimastigotes and trypomastigotes, suggesting a similar mechanism of action that occurs in Plasmodium spp. The addition of hemin to the culture medium increased trypanocidal activity of analog 1g without changing the cytotoxicity of the host cell, reaching an IC50 of 11.7 μM for trypomastigotes. The mechanism of action was demonstrated by the interaction of compound 1g with hemin in solution and prevention of heme peroxidation. Compound 1g and heme treatment induced alterations of the mitochondrion-kinetoplast complex in epimastigotes and trypomastigotes and also, accumulation of electron-dense deposits in amastigotes as visualized by transmission electron microscopy. The trypanocidal activity of 4-aminoquinolines and the elucidation of the mechanism involving interaction with heme is a neglected field of research, given the parasite's lack of heme biosynthetic pathway and the importance of this cofactor for parasite survival and growth. The results of this study can improve and guide rational drug development and combination treatment strategies.
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¹: These authors contributed equally to this work.

²: Present address: Laboratório de Biologia Estrutural, IOC, FIOCRUZ, Rio de Janeiro, Brazil.

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Trypanosomatid essential metabolic pathway: New approaches about heme fate in Trypanosoma cruzi

Highlights

Abstract

Introduction

Section snippets

Parasites

Extraction of heme and metabolites from T. cruzi epimastigotes

Results and discussion

Biochem. Biophys. Res. Commun.

Free Radic. Biol. Med.

Int. J. Biochem. Cell Biol.

Act. Trop.

J. Biol. Chem.

J. Biol. Chem.

Curr. Biol.

J. Biol. Chem.

J. Inorg. Biochem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Biochim. Biphys. Acta

FEBS Lett.

J. Biol. Chem.

Nova tripanomíase humana. Estudos sobre a morfologia e o ciclo evolutivo do Schizotrypanum cruzi, n. gen., agente etiológico de nova entidade mórbida do homem

Mem. Inst. Oswaldo Cruz