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Summary: Rafael Radi is testing the hypothesis that during mammalian infection with Trypanosoma cruzi, the parasite that causes Chagas disease, the interplay between the metabolism of the amino acid L-arginine and oxidation-reduction processes of both the parasite and host target cells determine cell survival or death. He is testing the hypothesis using biochemical, cellular, and in vivo model systems. The finding may permit development of novel and effective infection control strategies.

Trypanosoma cruzi, the parasite that causes Chagas disease (or American trypanosomiasis), infects approximately 18 million people throughout South America, and puts 60 million at risk. We are studying the redox biology of T. cruzi and the mammalian host cell with the aims of understanding the mechanisms of infection, immune evasion, and control and of obtaining relevant information to define metabolic pathways in T. cruzi that are likely to be drug target candidates and thus contribute to drug design and evaluation.

We are testing the hypothesis that, during mammalian infection with T. cruzi, the interplay of redox processes of the parasite and host target cells determines cell survival or death. To do this, we are using biochemical, cell, and in vivo model systems. The findings may permit development of novel and effective infection control strategies.

Mammalian macrophages are the first line of defense during the invasion of T. cruzi. Under appropriate stimulation conditions, macrophages can produce cytotoxic free radicals such as the superoxide radical anion (O₂^•−) and nitric oxide (^•NO) as well as oxidants such as hydrogen peroxide (H₂O₂) and the peroxynitrite anion (ONOO⁻). The interaction of these reactive species with T. cruzi inside the phagolysosome can cause severe cell damage to the parasite, compromising its vital functions and metabolism and eventually leading to its death. A particularly toxic macrophage-derived oxidant against T. cruzi is the peroxynitrite anion, the product of the diffusion-controlled reaction between superoxide and nitric oxide radicals. Peroxynitrite promotes a series of oxidation and nitration reactions in key biomolecules, including proteins, membrane lipids, and DNA. Peroxynitrite not only participates as a toxic mediator in cellular immune responses but, as we and others have shown, is also a central pathogenic mediator in a variety of disease conditions, including cardiovascular pathology, inflammation, and neurodegenerative processes. In this regard, we have extensively characterized the reaction chemistry and kinetics of peroxynitrite interactions with cell components. Given that peroxynitrite and related oxidants are transient, short-lived species (biological half-lives less than 10 milliseconds), their detection relies on specialized instrumentation and the detection of reaction “footprints” or products. To achieve these objectives we have developed methodologies that include high-performance liquid chromatography (HPLC)–based methods, mass spectrometry techniques for product identification and quantitation, and fast and ultrafast kinetic techniques such as stopped-flow spectrophotometry and pulse radiolysis for determination of rate constants. We have detected free radical intermediates with electron paramagnetic resonance (EPR)–based techniques.

While macrophage-derived reactive species attack T. cruzi, the parasite, in turn, has potent antioxidant defense mechanisms to cope with host cell–derived oxidants. In this regard, we have characterized the capacity of T. cruzi peroxiredoxins (fast-reacting thiol-containing enzymes) to rapidly and catalytically decompose peroxynitrite to nitrite in a two-electron reduction process fueled by trypanothione-linked reactions. (Trypanothione is a low-molecular-weight compound with two thiol groups per molecule; it is unique to trypanosomatids and is synthesized by the enzyme trypanothione syntethase through the ATP-dependent conjugation of two molecules of glutathione with spermidine, an l-arginine–derived polyamine.) The activities of mitochondrial and cytosolic T. cruzi peroxiredoxins are emerging as key components for evading the macrophage-dependent cellular immune response. Experiments with genetically engineered T. cruzi that overexpress variants of peroxiredoxins demonstrated that parasites become virulent and are capable of bypassing the action of macrophage-derived oxidants both in vitro and in vivo. Given that trypanothione is a central molecule in oxidant detoxification reactions, we have developed HPLC-based analytical methods to determine the levels of trypanothione and precursors and products under various cellular stress conditions. Moreover, the enzyme trypanothione syntethase is not present in the human genome and has been validated as a drug target in Trypanosoma brucei; therefore, we are exploring a series of compounds that, serving as trypanothione syntethase inhibitors, may facilitate T. cruzi death once the cellular immune response is orchestrated.

Another area of work involves the characterization and biological significance of programmed cell death (PCD) in T. cruzi. Although PCD for a long time was believed to occur exclusively in mammalian cells, data from several groups, including our own, have unambiguously demonstrated that, under appropriate stimulation, PCD can occur in trypanosomatids including Leishmania, T. brucei, and T. cruzi. Host mediators as well as drugs can trigger PCD in these unicellular eukaryotic cells; however, information remains scarce, and the mechanisms of PCD in trypanosomatids remain poorly understood. In this context, we have clarified biochemical characteristics of PCD in T. cruzi. Using a combination of methodologies that include inactivation of the oxidant-sensitive iron–sulfur-containing enzyme aconitase, EPR spin trapping and immune spin trapping, oxidation of redox-sensitive fluorescence probes, and augmentation of the glucose flux through the pentose phosphate pathway, we established that cellular oxidative stress in T. cruzi results in PCD. Enhanced superoxide generation in mitochondria can be explained by inhibition of mitochondrial respiration and electron flux due to the action of cell death mediators in the mitochondrial inner membrane. Notably, overexpression of the mitochondrial antioxidant enzyme superoxide dismutase (FeSOD A; its function is to eliminate superoxide radicals) resulted in substantial protection of T. cruzi from cell death, confirming the key role of mitochondrially produced superoxide in apoptotic signaling in T. cruzi. PCD in T. cruzi requires proteolytic processing, but this process is downstream of enhanced mitochondrial superoxide formation. The proteases participating in T. cruzi PCD remain to be identified.

Overall, the data obtained with overexpression of peroxiredoxins and FeSOD in T. cruzi, together with other data in the literature demonstrating that antioxidant mechanisms in T. cruzi are upregulated during the transformation of noninfective to infective forms as well as during development of drug resistance, clearly point to T. cruzi antioxidant enzymes as emerging virulence factors. Future experiments in animal models of disease with various T. cruzi strains of different pathogenic capacity and genetically modified parasites will provide further information to verify this hypothesis.

Last updated September 2008