Glucose metabolism sustains heme-induced Trypanosoma cruzi epimastigote growth in vitro.

Chagas disease is caused by the protozoan parasite, Trypanosoma cruzi. This parasite alternates between an insect vector and a mammalian host. T. cruzi epimastigotes reside in the insect vector and coexist with the blood components of the vertebrate host. The metabolic profile of T. cruzi has been extensively studied; however, changes in its metabolism in response to signaling molecules present in the vector are poorly understood. Heme acts as a physiological oxidant that triggers intense epimastigote proliferation and upregulates the expression of genes related to glycolysis and aerobic fermentation in vitro. Here, heme-cultured epimastigotes increased D-glucose consumption. In fact, heme-cultured parasites secreted more succinate (the end product of the so-called succinic fermentation) followed by glucose intake. Increased succinate levels reduced the extracellular pH, leading to acidification of the supernatant. However, the acidification and proliferation stimulated by heme was impaired when glycolysis was inhibited. Otherwise, when glucose amount is enhanced in supernatant, heme-cultured parasites increased its growth whereas the glucose depletion caused a delay in proliferation. Heme supplementation increased epimastigote electron transport system-related O2 consumption rates, while glucose addition reduced both the electron transport system-related O2 consumption rates and spare respiratory capacity, indicating a Crabtree-like effect. These results show that glycolysis predominated in heme-cultured epimastigotes over oxidative phosphorylation for energy supply when glucose is present to sustain its high proliferation in vitro. Furthermore, it provided an insight into the parasite biology in the vector environment that supply glucose and the digestion of blood generates free heme that can lead to the growth of T. cruzi epimastigotes.

Hypoxia Effects on Trypanosoma cruzi Epimastigotes Proliferation, Differentiation, and Energy Metabolism.

Trypanosoma cruzi, the causative agent of Chagas disease, faces changes in redox status and nutritional availability during its life cycle. However, the influence of oxygen fluctuation upon the biology of T. cruzi is unclear. The present work investigated the response of T. cruzi epimastigotes to hypoxia. The parasites showed an adaptation to the hypoxic condition, presenting an increase in proliferation and a reduction in metacyclogenesis. Additionally, parasites cultured in hypoxia produced more reactive oxygen species (ROS) compared to parasites cultured in normoxia. The analyses of the mitochondrial physiology demonstrated that hypoxic condition induced a decrease in both oxidative phosphorylation and mitochondrial membrane potential (ΔΨm) in epimastigotes. In spite of that, ATP levels of parasites cultivated in hypoxia increased. The hypoxic condition also increased the expression of the hexokinase and NADH fumarate reductase genes and reduced NAD(P)H, suggesting that this increase in ATP levels of hypoxia-challenged parasites was a consequence of increased glycolysis and fermentation pathways. Taken together, our results suggest that decreased oxygen levels trigger a shift in the bioenergetic metabolism of T. cruzi epimastigotes, favoring ROS production and fermentation to sustain ATP production, allowing the parasite to survive and proliferate in the insect vector.

The Screen of a Phage Display Library Identifies a Peptide That Binds to the Surface of Trypanosoma cruzi Trypomastigotes and Impairs Their Infection of Mammalian Cells.

Background: Chagas is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. On the order of seven million people are infected worldwide and current therapies are limited, highlighting the urgent need for new interventions. T. cruzi trypomastigotes can infect a variety of mammalian cells, recognition and adhesion to the host cell being critical for parasite entry. This study focuses on trypomastigote surface ligands involved in cell invasion. Methods: Three selection rounds of a phage peptide display library for isolation of phages that bind to trypomastigotes, resulted in the identification of the N3 dodecapeptide. N3 peptide binding to T. cruzi developmental forms (trypomastigotes, amastigotes and epimastigotes) was evaluated by flow cytometry and immunofluorescence assays. Parasite invasion of Vero cells was assessed by flow cytometry and immunofluorescence assays. Results: Phage display screening identified the N3 peptide that binds preferentially to the surface of the trypomastigote and amastigote infective forms as opposed to non-infective epimastigotes. Importantly, the N3 peptide, but not a control scrambled peptide, inhibits trypomastigote invasion of Vero cells by 50%. Conclusion: The N3 peptide specifically binds to T. cruzi, and by doing so, inhibits Vero cell infection. Follow-up studies will identify the molecule on the parasite surface to which the N3 peptide binds. This putative T. cruzi ligand may advance chemotherapy design and vaccine development.

The Potent Trypanocidal Effect of LQB303, a Novel Redox-Active Phenyl-Tert-Butyl-Nitrone Derivate That Causes Mitochondrial Collapse in Trypanosoma cruzi.

Chagas disease, which is caused by Trypanosoma cruzi, establishes lifelong infections in humans and other mammals that lead to severe cardiac and gastrointestinal complications despite the competent immune response of the hosts. Furthermore, it is a neglected disease that affects 8 million people worldwide. The scenario is even more frustrating since the main chemotherapy is based on benznidazole, a drug that presents severe side effects and low efficacy in the chronic phase of the disease. Thus, the search for new therapeutic alternatives is urgent. In the present study, we investigated the activity of a novel phenyl-tert-butyl-nitrone (PBN) derivate, LQB303, against T. cruzi. LQB303 presented trypanocidal effect against intracellular [IC50/48 h = 2.6 µM] and extracellular amastigotes [IC50/24 h = 3.3 µM] in vitro, leading to parasite lysis; however, it does not present any toxicity to host cells. Despite emerging evidence that mitochondrial metabolism is essential for amastigotes to grow inside mammalian cells, the mechanism of redox-active molecules that target T. cruzi mitochondrion is still poorly explored. Therefore, we investigated if LQB303 trypanocidal activity was related to the impairment of the mitochondrial function of amastigotes. The investigation showed there was a significant decrease compared to the baseline oxygen consumption rate (OCR) of LQB303-treated extracellular amastigotes of T. cruzi, as well as reduction of "proton leak" (the depletion of proton motive force by the inhibition of F1Fo ATP synthase) and "ETS" (maximal oxygen consumption after uncoupling) oxygen consumption rates. Interestingly, the residual respiration ("ROX") enhanced about three times in LQB303-treated amastigotes. The spare respiratory capacity ratio (SRC: cell ability to meet new energy demands) and the ATP-linked OCR were also impaired by LQB303 treatment, correlating the trypanocidal activity of LQB303 with the impairment of mitochondrial redox metabolism of amastigotes. Flow cytometric analysis demonstrated a significant reduction of the ΔΨm of treated amastigotes. LQB303 had no significant influence on the OCR of treated mammalian cells, evidencing its specificity against T. cruzi mitochondrial metabolism. Our results suggest a promising trypanocidal activity of LQB303, associated with parasite bioenergetic inefficiency, with no influence on the host energy metabolism, a fact that may point to an attractive alternative therapy for Chagas disease.

New perspectives for hydrogen peroxide in the amastigogenesis of Trypanosoma cruzi in vitro.

Trypanosoma cruzi has a complex life cycle involving four life stages: the replicative epimastigotes and metacyclic trypomastigotes in the invertebrate host digestive tract, and intracellular amastigotes and bloodstream trypomastigotes in the mammalian host. Trypomastigotes can invade any nucleated cell, including macrophages, which produce ROS that enhance intracellular infection. However, how ROS modulate T. cruzi infection in the mammalian cell remains unclear. Therefore, the present work aimed to investigate the role of ROS during the stimulation of amastigogenesis in vitro. Our results showed that H2O2 improves the differentiation process in vitro and that it was impaired by Peg-Catalase. However, the antioxidants GSH and NAC had no influence on induced amastigogenesis, which suggests the specificity of H2O2 to increase intracellular differentiation. Amastigogenesis physiologically occurs in low pH, thus we investigated whether parasites are able to produce ROS during amastigogenesis. Interestingly, after 60 min of differentiation induction in vitro, we observed an increase in H2O2 production, which was inhibited by the mitochondrial-targeted antioxidant, mitoTEMPO and Cyclosporine A (a mitochondrial permeability transition pore -mPTP- inhibitor), suggesting mitochondrion as a H2O2 source. Indeed, quantitative real time (qPCR) showed an increase of the mitochondrial superoxide dismutase (FeSODA) gene expression after 60 min of induced amastigogenesis, reinforcing the hypothesis of mitochondrial ROS induction during intracellular differentiation of T. cruzi. The reduction of cellular respiration and the decreased ΔΨm observed during amastigogenesis can explain the increased mitochondrial ROS through mPTP opening. In conclusion, our results suggest that H2O2 is involved in the amastigogenesis of T. cruzi.

Oleic acid and hydroxytyrosol present in olive oil promote ROS and inflammatory response in normal cultures of murine dermal fibroblasts through the NF-κB and NRF2 pathways.

Few studies have evaluated the effects of olive oil on normal tissues like skin and its components. Hence, we investigated whether olive oil could increase the production of ROS and oxidative damage in murine dermal fibroblast cultures in a short-term exposition. In addition, we evaluated the role of oleic acid and hydroxytyrosol, which are the two most important components of olive oil, in the associated mechanisms of action, and the metabolism of long-chain fatty acids from olive oil. To study this, neonatal murine dermal fibroblasts (NMDF) were incubated with olive oil, oleic acid, or hydroxytyrosol for 24 or 72 h. The NMDF incubated with olive oil or oleic acid showed an increase in the production of ROS after 24 h, lipid peroxidation, and protein carbonylation after 72 h, as well as increased expression of nuclear factor-kappa B (NF-κB) p65 and cyclooxygenase-2 (COX-2) after 72 h. However, NMDF treated with olive oil or hydroxytyrosol demonstrated an increase in the expression of nuclear factor-erythroid2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) after 72 h. In addition, NMDF treated with olive oil also showed an increase in the protein expression of diacylglycerol acyltransferase1 (DGAT1), which promotes triacylglycerol synthesis, and in the levels of triacylglycerols. The microscopic analysis showed Nile red-positive lipid droplets inside olive oil-treated NMDF after 72 h. Moreover, gas chromatography-mass spectrometry demonstrated high levels of oleic acid in the olive oil-treated NMDF after 72 h. In conclusion, oleic acid present in the olive oil promotes the production of ROS and oxidative damage in murine dermal fibroblasts, which leads to NF-κB p65 and COX-2 expression, while hydroxytyrosol promotes NRF2 and HO-1 expression. In addition, NMDF area capable of absorbing long-chain fatty acids derived from olive oil, which promotes the synthesis and the accumulation of triacylglycerols into cytoplasm of NMDF through DGAT1 activation.

Gene expression profiling of Trypanosoma cruzi in the presence of heme points to glycosomal metabolic adaptation of epimastigotes inside the vector.

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi, and is transmitted by triatomine insects during its blood meal. Proliferative epimastigotes forms thrive inside the insects in the presence of heme (iron protoporphyrin IX), an abundant product of blood digestion, however little is known about the metabolic outcome of this signaling molecule in the parasite. Trypanosomatids exhibit unusual gene transcription employing a polycistronic transcription mechanism through trans-splicing that regulates its life cycle. Using the Deep Seq transcriptome sequencing we characterized the heme induced transcriptome of epimastigotes and determined that most of the upregulated genes were related to glucose metabolism inside the glycosomes. These results were supported by the upregulation of glycosomal isoforms of PEPCK and fumarate reductase of heme-treated parasites, implying that the fermentation process was favored. Moreover, the downregulation of mitochondrial gene enzymes in the presence of heme also supported the hypothesis that heme shifts the parasite glycosomal glucose metabolism towards aerobic fermentation. These results are examples of the environmental metabolic plasticity inside the vector supporting ATP production, promoting epimastigotes proliferation and survival.

Novel nitroimidazole derivatives evaluated for their trypanocidal, cytotoxic, and genotoxic activities.

The current treatment of Chagas disease is based on the use of two drugs, nifurtimox (Nfx) and benznidazole (Bnz), both of which present limited efficacy in the chronic stage of the disease and toxic side effects. Thus, the discovery of novel compounds is urgently required. Herein, we report the successful synthesis of 4-nitroimidazole analogs of Bnz via nucleophilic aromatic substitution or cycloaddition reactions. The analogs were biologically evaluated, and compound 4 (4-cyclopropyl-1-(1-methyl-4-nitro-1H-imidazole-5-yl)-1H-1,2,3-triazole) was identified as the most potent against both the trypomastigote (IC50 = 5.4 µM) and amastigote (IC50 = 12.0 µM) forms of T. cruzi, showing activity in the same range as Bnz (IC50 = 8.8 and 8.7 µM, respectively). The cytotoxic and genotoxic activities of compounds 5, 4 and 11 were assessed. These three compounds were cytotoxic and genotoxic to RAW and HepG2 cells and mutagenic to Salmonella enterica strains. However, 4 exhibited toxic effects only at concentrations higher than those needed for trypanocidal activity. Molecular docking of 4 showed the importance of the size and π-π interactions between the nitroimidazole and the cofactor (flavin mononucleotide) of T.cruzi-nitroreductase (TcNTR). Moreover, the residues His503 and Tyr545 are relevant for binding to TcNTR. Our design strategy was capable of generating novel and active Bnz analogs.

Heme crystallization in a Chagas disease vector acts as a redox-protective mechanism to allow insect reproduction and parasite infection.

Heme crystallization as hemozoin represents the dominant mechanism of heme disposal in blood feeding triatomine insect vectors of the Chagas disease. The absence of drugs or vaccine for the Chagas disease causative agent, the parasite Trypanosoma cruzi, makes the control of vector population the best available strategy to limit disease spread. Although heme and redox homeostasis regulation is critical for both triatomine insects and T. cruzi, the physiological relevance of hemozoin for these organisms remains unknown. Here, we demonstrate that selective blockage of heme crystallization in vivo by the antimalarial drug quinidine, caused systemic heme overload and redox imbalance in distinct insect tissues, assessed by spectrophotometry and fluorescence microscopy. Quinidine treatment activated compensatory defensive heme-scavenging mechanisms to cope with excessive heme, as revealed by biochemical hemolymph analyses, and fat body gene expression. Importantly, egg production, oviposition, and total T. cruzi parasite counts in R. prolixus were significantly reduced by quinidine treatment. These effects were reverted by oral supplementation with the major insect antioxidant urate. Altogether, these data underscore the importance of heme crystallization as the main redox regulator for triatomine vectors, indicating the dual role of hemozoin as a protective mechanism to allow insect fertility, and T. cruzi life-cycle. Thus, targeting heme crystallization in insect vectors represents an innovative way for Chagas disease control, by reducing simultaneously triatomine reproduction and T. cruzi transmission.

Modulation of mitochondrial metabolism as a biochemical trait in blood feeding organisms: the redox vampire hypothesis redux.

Hematophagous organisms undergo remarkable metabolic changes during the blood digestion process, increasing fermentative glucose metabolism, and reducing respiratory rates, both consequence of functional mitochondrial remodeling. Here, we review the pathways involved in energy metabolism and mitochondrial functionality in a comparative framework across different hematophagous species, and consider how these processes regulate redox homeostasis during blood digestion. The trend across distinct species indicate that a switch in energy metabolism might represent an important defensive mechanism to avoid the potential harmful interaction of oxidants generated from aerobic energy metabolism with products derived from blood digestion. Indeed, in insect vectors, blood feeding transiently reduces respiratory rates and oxidant production, irrespective of tissue and insect model. On the other hand, a different scenario is observed in several unrelated parasite species when exposed to blood digestion products, as respiratory rates reduce and mitochondrial oxidant production increase. The emerging picture indicates that re-wiring of energy metabolism, through reduced mitochondrial function, culminates in improved tolerance to redox insults and seems to represent a key step for hematophagous organisms to cope with the overwhelming and potentially toxic blood meal.

Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production.

Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation.

Mutagenic and Cytotoxicity LQB 123 Profile: Safety and Tripanocidal Effect of a Phenyl-t-Butylnitrone Derivative.

The therapeutic options for Chagas disease are limited and its treatment presents a number of drawbacks including toxicity, drug resistance, and insufficient effectiveness against the chronic stage of the disease. Therefore, new therapeutical options are mandatory. In the present work, we evaluated the effect of a phenyl-tert-butylnitrone (PBN) derivate, LQB 123, against Trypanosoma cruzi forms. LQB 123 presented a trypanocidal effect against bloodstream trypomastigotes (IC50 = 259.4 ± 6.1 µM) and intracellular amastigotes infecting peritoneal macrophages (IC50 = 188.2 ± 47.5 µM), with no harmful effects upon the mammalian cells (CC50 values greater than 4 mM), resulting in a high selectivity index (CC50/IC50 > 20). Additionally, metacyclic trypomastigotes submitted to LQB 123 presented an IC50 of about 191.8 ± 10.5 µM and epimastigotes forms incubated with different concentrations of LQB 123 presented an inhibition of parasite growth with an IC50 of 255.1 ± 3.6 µM. Finally, we investigated the mutagenic potential of the nitrone by the Salmonella/microsome assay and observed no induction of mutagenicity even in concentrations as high as 33000 µM. Taken together, these results present a nonmutagenic compound, with trypanocidal activity against all relevant forms of T. cruzi, offering new insights into CD treatment suggesting additional in vivo tests.

CALCIUM-INDUCED LIPID PEROXIDATION IS MEDIATED BY RHODNIUS HEME-BINDING PROTEIN (RHBP) AND PREVENTED BY VITELLIN.

Lipid peroxidation is promoted by the quasi-lipoxygenase (QL) activity of heme proteins and enhanced by the presence of free calcium. Unlike mammalian plasma, the hemolymph of Rhodnius prolixus, a vector of Chagas disease, contains both a free heme-binding protein (RHBP) and circulating lipoproteins. RHBP binds and prevents the heme groups of the proteins from participating in lipid peroxidation reactions. Herein, we show that despite being bound to RHBP, heme groups promote lipid peroxidation through a calcium-dependent QL reaction. This reaction is readily inhibited by the presence of ethylene glycol tetraacetic acid (EGTA), the antioxidant butylated hydroxytoluene or micromolar levels of the main yolk phosphoprotein vitellin (Vt). The inhibition of lipid peroxidation is eliminated by the in vitro dephosphorylation of Vt, indicating that this reaction depends on the interaction of free calcium ions with negatively charged phosphoamino acids. Our results demonstrate that calcium chelation mediated by phosphoproteins occurs via an antioxidant mechanism that protects living organisms from lipid peroxidation.

Proliferation and differentiation of Trypanosoma cruzi inside its vector have a new trigger: redox status.

Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H2O2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. ß-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.

The effect of thiamine deficiency on inflammation, oxidative stress and cellular migration in an experimental model of sepsis.

BACKGROUND: Sepsis is a prevalent condition in critically ill patients and may be associated with thiamine deficiency (TD). The aim of this study was to evaluate the effect of TD on inflammation, oxidative stress and cellular recruitment in a sepsis model. METHODS: The experimental sepsis model, cecal ligation and puncture (CLP), was utilized on mice in comparison with a sham procedure. The following four groups were compared against each other: SHAM with AIN93G complete chow, SHAM with thiamine deficient (TD) chow, CLP with AIN93G complete chow, and CLP with TD chow. Thiamine pyrophosphate (TPP) blood concentrations were determined, and blood and peritoneal fluid were evaluated for differences in TNF-alpha, IL-1, IL-6, KC and MCP-1/CCL2 levels. In addition, the levels of 4-HNE adducts in liver proteins were evaluated by Western Blot. RESULTS: The mean TPP blood concentration from the mice fed with the complete chow was 303.3 ± 42.6 nmol/L, and TD occurred within 10 days. TNF-α and MCP-1 concentrations in the peritoneal fluid were significantly greater in the CLP with TD chow group when compared with the other groups. The blood IL-1ß level, however, was lower in the CLP with TD chow group. Liver 4-HNE levels were highest in the TD chow groups. Blood mononuclear cell numbers, as well as peritoneal total leukocyte, mononuclear cell and neutrophil numbers were greater in the CLP with TD chow group. Peritoneal bacterial colony forming units (CFU) were significantly lower in the CLP with TD chow group. CONCLUSION: TD was associated with greater bacterial clearance, oxidative stress and inflammatory response changes.

Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease.

It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10(-5)M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10(-6)M ShPI-I, in the same periods of time, reduced parasite viability about 91-95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents.

Terpenic fraction of Pterodon pubescens inhibits nuclear factor kappa B and extracellular signal-regulated protein kinase 1/2 activation and deregulates gene expression in leukemia cells.

BACKGROUND: Plant derived compounds have been shown to be important sources of several anti-cancer agents. As cell cycle deregulation and tumor growth are intimately linked, the discovery of new substances targeting events in this biochemical pathway would be of great value. The anti-leukemic effect of an ethanolic extract of Pterodon pubescens seeds (EEPp) has been previously demonstrated and now we show that a terpenic subfraction (SF5) of EEPp containing farnesol, geranylgeraniol and vouacapan derivatives induces apoptosis in the human chronic myelogenous leukemia cell line K562. This work addresses SF5's antiproliferative mechanisms in these cells since they are still unclear. METHODS: DNA synthesis in K562 cells was assessed by [3H]-methyl-thymidine incorporation and cell cycle status by flow cytometry. The expression of cyclins D1 and E2, of the cell cycle inhibitor p21 and of the proto-oncogene c-myc was evaluated by semi-quantitative RT-PCR. Extracellular-signal-regulated kinases (ERK) 1/2 and nuclear factor kappa B (NF-κB) activation was evaluated by western blotting. RESULTS: In K562 cells, SF5 treatment induced a higher inhibition of DNA synthesis and cell growth than the original EEPp hexanic fraction from which SF5 originated, and also arrested the cell cycle in G1. Exposure of these cells to SF5 led to a decrease in cyclin E2 and c-myc expression while p21 mRNA levels were increased. Furthermore, SF5 inhibited the activation of mitogen-activated protein kinase (MAPK) ERK 1/2 and NF-κB. CONCLUSIONS: This work suggests that the anti-leukemic action of SF5 is linked to the inhibition of ERKs, NF-κB and c-myc signaling pathways resulting in reduced cyclin E2 mRNA expression and cell cycle arrest in the G1 phase.

The Role of Heme and Reactive Oxygen Species in Proliferation and Survival of Trypanosoma cruzi.

Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS) which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology.

Heme-induced ROS in Trypanosoma cruzi activates CaMKII-like that triggers epimastigote proliferation. One helpful effect of ROS.

Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time- and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism.

The heme uptake process in Trypanosoma cruzi epimastigotes is inhibited by heme analogues and by inhibitors of ABC transporters.

Heme (iron protoporphyrin IX) is an important molecule involved in many biological reactions, including oxygen transport, respiration, photosynthesis and drug detoxification. Trypanosoma cruzi parasites, the etiological agent of Chagas' disease, take up heme from the environment to supply their nutritional needs because they do not synthesize this cofactor. However, the mechanisms involved in heme transport across biological membranes are poorly understood. Indeed, in T. cruzi, no heme transporter has yet been characterized. In the present work, we evaluate the heme uptake processes by T. cruzi epimastigotes using fluorescent heme-analogues. Heme uptake decreased significantly when cells were pretreated with different concentrations of SnPPIX, PdMPIX or ZnMPIX, this observed competition suggests that they are taken up by the same transport system. We studied the growth behavior of epimastigotes using the same heme-analogues and the treatments with SnPPIX or PdMPIX impaired cell growth but when heme was added to the culture medium the observed inhibition was partially reversed. In addition, we tested how the heme uptake processes are affected by the presence of different transporter inhibitors. When the cells were treated with inhibitors and then incubated with heme, heme uptake decreased significantly for all treatments. These results constitute a strong indication for the existence of a protein associated with porphyrin transport in T. cruzi, possibly ATP-binding cassette transporters (ABC-transporter).