Fatty acid oxidation participates in resistance to nutrient-depleted environments in the insect stages of Trypanosoma cruzi.

Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis.

Abnormal sterol-induced cell wall glucan deficiency in yeast is due to impaired glucan synthase transport to the plasma membrane.

Abnormal sterols disrupt cellular functions through yet unclear mechanisms. In Saccharomyces cerevisiae, accumulation of Δ8-sterols, the same type of sterols observed in patients of Conradi-Hünermann-Happle syndrome or in fungi after amine fungicide treatment, leads to cell wall weakness. We have studied the influence of Δ8-sterols on the activity of glucan synthase I, the protein synthetizing the main polymer in fungal cell walls, its regulation by the Cell Wall Integrity (CWI) pathway, and its transport from the endoplasmic reticulum to the plasma membrane. We ascertained that the catalytic characteristics were mostly unaffected by the presence of abnormal sterols but the enzyme was partially retained in the endoplasmic reticulum, leading to glucan deficit at the cell wall. Furthermore, we observed that glucan synthase I traveled through an unconventional exocytic route to the plasma membrane that is associated with low density intracellular membranes. Also, we found out that the CWI pathway remained inactive despite low glucan levels at the cell wall. Taken together, these data suggest that Δ8-sterols affect cell walls by inhibiting unconventional secretion of proteins leading to retention and degradation of glucan synthase I, while the compensatory CWI pathway is unable to activate. These results could be instrumental to understand defects of bone development in cholesterol biosynthesis disorders and fungicide mechanisms of action.

Trypanosoma cruzi synthesizes proline via a Δ1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools.

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 µM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.

Dual inhibition of glutaminase and carnitine palmitoyltransferase decreases growth and migration of glutaminase inhibition-resistant triple-negative breast cancer cells.

Triple-negative breast cancers (TNBCs) lack progesterone and estrogen receptors and do not have amplified human epidermal growth factor receptor 2, the main therapeutic targets for managing breast cancer. TNBCs have an altered metabolism, including an increased Warburg effect and glutamine dependence, making the glutaminase inhibitor CB-839 therapeutically promising for this tumor type. Accordingly, CB-839 is currently in phase I/II clinical trials. However, not all TNBCs respond to CB-839 treatment, and the tumor resistance mechanism is not yet fully understood. Here we classified cell lines as CB-839-sensitive or -resistant according to their growth responses to CB-839. Compared with sensitive cells, resistant cells were less glutaminolytic and, upon CB-839 treatment, exhibited a smaller decrease in ATP content and less mitochondrial fragmentation, an indicator of poor mitochondrial health. Transcriptional analyses revealed that the expression levels of genes linked to lipid metabolism were altered between sensitive and resistant cells and between breast cancer tissues (available from The Cancer Genome Atlas project) with low versus high glutaminase (GLS) gene expression. Of note, CB-839-resistant TNBC cells had increased carnitine palmitoyltransferase 2 (CPT2) protein and CPT1 activity levels. In agreement, CB-839-resistant TNBC cells mobilized more fatty acids into mitochondria for oxidation, which responded to AMP-activated protein kinase and acetyl-CoA carboxylase signaling. Moreover, chemical inhibition of both glutaminase and CPT1 decreased cell proliferation and migration of CB-839-resistant cells compared with single inhibition of each enzyme. We propose that dual targeting of glutaminase and CPT1 activities may have therapeutic relevance for managing CB-839-resistant tumors.

A Fluorinated Phenylbenzothiazole Arrests the Trypanosoma cruzi Cell Cycle and Diminishes the Infection of Mammalian Host Cells.

Chagas disease (CD) is a human infection caused by Trypanosoma cruzi CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side effects, and its effectiveness in the chronic phase of the disease is controversial. In this contribution, substituted phenylbenzothiazole derivatives were synthesized and biologically evaluated as trypanocidal agents against Trypanosoma cruzi The trypanocidal activities of the most promising compounds were determined through systematic in vitro screening, and their modes of action were determined as well. The physicochemical-structural characteristics responsible for the trypanocidal effects were identified, and their possible therapeutic application in Chagas disease is discussed. Our results show that the fluorinated compound 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] phenol (BT10) has the ability to inhibit the proliferation of epimastigotes [IC50(Epi) = 23.1 ± 1.75 µM] and intracellular forms of trypomastigotes [IC50(Tryp) = 8.5 ± 2.9 µM] and diminishes the infection index by more than 80%. In addition, BT10 has the ability to selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus DNA. The mode of action for BT10 on T. cruzi suggests that the development of fluorinated phenylbenzothiazole with electron-withdrawing substituent is a promising strategy for the design of trypanocidal drugs.

Glutamine Analogues Impair Cell Proliferation, the Intracellular Cycle and Metacyclogenesis in Trypanosoma cruzi.

Trypanosoma cruzi is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, T. cruzi passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to T. cruzi survival. Recently, we described that T. cruzi can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for T. cruzi survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.

In Vitro Cellular Division of Trypanosoma abeli Reveals Two Pathways for Organelle Replication.

Since the observation of the great pleomorphism of fish trypanosomes, in vitro culture has become an important tool to support taxonomic studies investigating the biology of cultured parasites, such as their structure, growth dynamics, and cellular cycle. Relative to their biology, ex vivo and in vitro studies have shown that these parasites, during the multiplication process, duplicate and segregate the kinetoplast before nucleus replication and division. However, the inverse sequence (the nucleus divides before the kinetoplast) has only been documented for a species of marine fish trypanosomes on a single occasion. Now, this previously rare event was observed in Trypanosoma abeli, a freshwater fish trypanosome. Specifically, from 376 cultured parasites in the multiplication process, we determined the sequence of organelle division for 111 forms; 39% exhibited nucleus duplication prior to kinetoplast replication. Thus, our results suggest that nucleus division before the kinetoplast may not represent an accidental or erroneous event occurring in the main pathway of parasite reproduction, but instead could be a species-specific process of cell biology in trypanosomes, such as previously noticed for Leishmania. This "alternative" pathway for organelle replication is a new field to be explored concerning the biology of marine and freshwater fish trypanosomes.

Diminished Prolinemia in Chronic Chagasic Patients: A New Clue for Disease Pathology?

Trypanosoma cruzi, the etiological agent of Chagas disease, is dependent on proline for a variety of processes, such as energy metabolism, host cell invasion, differentiation, and resistance to osmotic, metabolic, and oxidative stress. On this basis, we investigated a possible relationship between prolinemia and severity of T. cruzi infection in chronic patients, as reported here. The study population consisted of 112 subjects, separated into 83 chronically T. cruzi-infected patients and 29 age-matched healthy volunteers (control) of both sexes, recruited at the Chagas Disease Service from the Department of Cardiology, Hospital Provincial del Centenario de Rosario (Rosario, Argentina). Chagasic patients were separated into three groups: chronic asymptomatic, mild/moderate, and severe chronic chagasic cardiomyopathy (CCC) subjects. We observed a significant decrease of 11.7% in prolinemia in chagasic patients when compared to controls. Further analysis within the three groups of chagasic patients also revealed a statistically significant decrease of prolinemia in severe CCC patients compared to controls, showing a relative difference of 13.6% in proline concentrations. These data point to the possibility that collagen-which participates in the healing process of cardiac tissue-and proline metabolism in the myocardium could constitute new factors affecting the evolution of Chagas disease.

Metabolomic profiling reveals a finely tuned, starvation-induced metabolic switch in Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi, the etiological agent of Chagas disease, is a protozoan parasite with a complex life cycle involving a triatomine insect and mammals. Throughout its life cycle, the T. cruzi parasite faces several alternating events of cell division and cell differentiation in which exponential and stationary growth phases play key biological roles. It is well accepted that arrest of the cell division in the epimastigote stage, both in the midgut of the triatomine insect and in vitro, is required for metacyclogenesis, and it has been previously shown that the parasites change the expression profile of several proteins when entering this quiescent stage. However, little is known about the metabolic changes that epimastigotes undergo before they develop into the metacyclic trypomastigote stage. We applied targeted metabolomics to measure the metabolic intermediates in the most relevant pathways for energy metabolism and oxidative imbalance in exponentially growing and stationary growth-arrested epimastigote parasites. We show for the first time that T. cruzi epimastigotes transitioning from the exponential to the stationary phase exhibit a finely tuned adaptive metabolic mechanism that enables switching from glucose to amino acid consumption, which is more abundant in the stationary phase. This metabolic plasticity appears to be crucial for survival of the T. cruzi parasite in the myriad different environmental conditions to which it is exposed during its life cycle.

The Non-Canonical Substrates of Trypanosoma cruzi Tyrosine and Aspartate Aminotransferases: Branched-Chain Amino Acids.

Trypanosoma cruzi, the etiological agent of Chagas disease, lacks genes that encode canonical branched-chain aminotransferases. However, early studies showed that when epimastigotes were grown in the presence of 14 C1 -DL-leucine, the label was incorporated into various intermediates. More recently, our studies provided evidence that T. cruzi epimastigotes display a single ATP-dependent and saturable transport system that enables epimastigotes to uptake branched-chain amino acids (BCAAs) from the culture media. To extend our knowledge of the first step of BCAA catabolism, the ability of this parasite's noncanonical broad specificity aminotransferases, such as tyrosine aminotransferase (TAT) and aspartate aminotransferase (ASAT), to transaminate these amino acids was investigated. Indeed, our results show that TAT and ASAT utilize BCAAs as substrates; however, both enzymes differ in their catalytic competence in utilizing these amino donors. For instance, ASAT transaminates isoleucine nearly 10-fold more efficiently than does TAT. This unique characteristic of TAT and ASAT allows to explain how BCAAs can be oxidized in the absence of a BCAA transaminase in T. cruzi.

Development and in vitro/in vivo evaluation of a novel benznidazole liquid dosage form using a quality-by-design approach.

OBJECTIVES: To develop an alcohol-free solution suitable for children of benznidazole, the drug of choice for treatment of Chagas disease. METHODS: In a quality-by-design approach, a systematic optimisation procedure was carried out to estimate the values of the factors leading to the maximum drug concentration. The formulations were analysed in terms of chemical and physical stability and drug content. The final preparation was subjected to an in vivo palatability assay. Mice were infected and treated orally in a murine model. RESULTS: The results showed that benznidazole solubility increased up to 18.38 mg/ml in the optimised co-solvent system. The final formulation remained stable at all three temperatures tested, with suitable drug content and no significant variability. Palatability of the preparation was improved by taste masking of BZL. In vivo studies showed that both parasitaemia and mortality diminished, particularly at a dose of 40 mg/kg/day. CONCLUSION: Quality by design was a suitable approach to formulate a co-solvent system of benznidazole. The in vivo studies confirmed the suitability of the optimised such solutions to diminish both parasitaemia and mortality. Thus, this novel alternative should be taken into account for further clinical evaluation in all age ranges.

An Aromatic Diamidine That Targets Kinetoplast DNA, Impairs the Cell Cycle in Trypanosoma cruzi, and Diminishes Trypomastigote Release from Infected Mammalian Host Cells.

Trypanosoma cruzi is the etiological agent of Chagas disease, affecting approximately 10 million people in the Americas and with some 40 million people at risk. The objective of this study was to evaluate the anti-T. cruzi activity of three new diamidines that have a 3,4-ethylenedioxy extension of the thiophene core, designated MB17, MB19, and MB38. All three diamidines exhibited dose-dependent inhibition of epimastigote replication. The mechanisms of action of these diamidines were investigated. Unlike MB17 and MB19, MB38 exhibited a significant increase in the number of annexin-propidium iodide double-labeled cells compared to levels in control parasites. As MB17 had shown a lower 50% inhibitory concentration (IC50) against epimastigote growth, the mechanism of action of this drug was studied in more detail. MB17 triggered a decrease in the intracellular ATP levels. As a consequence, MB17 affected the genomic DNA and kinetoplast DNA (kDNA) and impaired the parasite cell cycle. Moreover, MB17 caused DNA fragmentation, with a more severe effect on kDNA than on nuclear DNA, resulting in dyskinetoplastic cells. MB17 was tested for toxicity and effectiveness for the treatment of infected CHO-K1 cells, exhibiting a 50% cytotoxic concentration (CC50) of 13.47 ± 0.37 µM and an IC50 of 0.14 ± 0.12 µM against trypomastigote release. MB17 also diminished the infection index by 60% at 0.5 µM. In conclusion, despite belonging to the same family, these diamidines have different efficiencies. To summarize, MB17 was the most potent of these diamidines against epimastigotes, producing DNA damage preferentially in kDNA, impairing the parasite cell cycle, and decreasing the infection index and trypomastigote release from infected mammalian host cells, with a high selectivity index (SI) (<90). These data suggest that MB17 could be an interesting lead compound against T. cruzi.

The Uptake of GABA in Trypanosoma cruzi.

Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.

Branched-chain amino acids modulate the proteomic profile of Trypanosoma cruzi metacyclogenesis induced by proline.

Trypanosoma cruzi, the causative agent of Chagas disease, has a complex life cycle that involves triatomine insects as vectors and mammals as hosts. The differentiation of epimastigote forms into metacyclic trypomastigotes within the insect vector is crucial for the parasite's life cycle progression. Factors influencing this process, including temperature, pH, and nutritional stress, along with specific metabolite availability, play a pivotal role. Amino acids like proline, histidine, and glutamine support cell differentiation, while branched-chain amino acids (BCAAs) inhibit it. Interestingly, combining the pro-metacyclogenic amino acid proline with one of the anti-metacyclogenic BCAAs results in viable metacyclics with significantly reduced infectivity. To explore the characteristics of metacyclic parasites differentiated in the presence of BCAAs, proteomics analyses were conducted. Metacyclics obtained in triatomine artificial urine (TAU) supplemented with proline alone and in combination with leucine, isoleucine, or valine were compared. The analyses revealed differential regulation of 40 proteins in TAU-Pro-Leu, 131 in TAU-Pro-Ile, and 179 in TAU-Pro-Val, as compared to metacyclics from TAU-Pro. Among these, 22%, 11%, and 13% of the proteins were associated with metabolic processes, respectively. Notably, enzymes related to glycolysis and the tricarboxylic acid (TCA) cycle were reduced in metacyclics with Pro-BCAAs, while enzymes involved in amino acid and purine metabolic pathways were increased. Furthermore, metacyclics with Pro-Ile and Pro-Val exhibited elevated enzymes linked to lipid and redox metabolism. The results revealed five proteins that were increased and four that were decreased in common in the presence of Pro+BCAAs, indicating their possible participation in key processes related to metacyclogenesis. These findings suggest that the presence of BCAAs can reshape the metabolism of metacyclics, contributing to the observed reduction in infectivity in these parasites.

Cysteine synthase: multiple structures of a key enzyme in cysteine synthesis and a potential drug target for Chagas disease and leishmaniasis.

Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Šfor TcCS, 1.75 Šfor LiCS and 2.75 Šfor TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein-protein inhibitors.

Navigating the boundaries between metabolism and epigenetics in trypanosomes.

Epigenetic marks enable cells to acquire new biological features that favor their adaptation to environmental changes. These marks are chemical modifications on chromatin-associated proteins and nucleic acids that lead to changes in the chromatin landscape and may eventually affect gene expression. The chemical tags of these epigenetic marks are comprised of intermediate cellular metabolites. The number of discovered associations between metabolism and epigenetics has increased, revealing how environment influences gene regulation and phenotype diversity. This connection is relevant to all organisms but underappreciated in digenetic parasites, which must adapt to different environments as they progress through their life cycles. This review speculates and proposes associations between epigenetics and metabolism in trypanosomes, which are protozoan parasites that cause human and livestock diseases.

Assignment of putative functions to membrane "hypothetical proteins" from the Trypanosoma cruzi genome.

Protozoan parasites cause thousands of deaths each year in developing countries. The genome projects of these parasites opened a new era in the identification of therapeutic targets. However, the putative function could be predicted for fewer than half of the protein-coding genes. In this work, all Trypanosoma cruzi proteins containing predicted transmembrane spans were processed through an automated computational routine and further analyzed in order to assign the most probable function. The analysis consisted of dissecting the whole predicted protein in different regions. More than 5,000 sequences were processed, and the predicted biological functions were grouped into 19 categories according to the hits obtained after analysis. One focus of interest, due to the scarce information available on trypanosomatids, is the proteins involved in signal-transduction processes. In the present work, we identified 54 proteins belonging to this group, which were individually analyzed. The results show that by means of a simple pipeline it was possible to attribute probable functions to sequences annotated as coding for "hypothetical proteins." Also, we successfully identified the majority of candidates participating in the signal-transduction pathways in T. cruzi.

Proteome and morphological analysis show unexpected differences between promastigotes of Leishmania amazonensis PH8 and LV79 strains.

BACKGROUND: Leishmaniases are diseases caused by Leishmania protozoans that affect around 12 million people. Leishmania promastigotes are transmitted to vertebrates by female phlebotomine flies during their blood meal. Parasites attach to phagocytic cells, are phagocytosed and differentiate into amastigotes. We previously showed that PH8 and LV79 strains of Leishmania amazonensis have different virulence in mice and that their amastigotes differ in their proteomes. In this work, we compare promastigotes' infectivity in macrophages, their proteomes and morphologies. METHODS/PRINCIPAL FINDINGS: Phagocytosis assays showed that promastigotes adhesion to and phagocytosis by macrophages is higher in PH8 than LV79. To identify proteins that differ between the two strains and that may eventually contribute for these differences we used a label-free proteomic approach to compare promastigote´s membrane-enriched fractions. Proteomic analysis enabled precise discrimination of PH8 and LV79 protein profiles and the identification of several differentially abundant proteins. The proteins more abundant in LV79 promastigotes participate mainly in translation and amino acid and nucleotide metabolism, while the more abundant in PH8 are involved in carbohydrate metabolism, cytoskeleton composition and vesicle/membrane trafficking. Interestingly, although the virulence factor GP63 was more abundant in the less virulent LV79 strain, zymography suggests a higher protease activity in PH8. Enolase, which may be related to virulence, was more abundant in PH8 promastigotes. Unexpectedly, flow cytometry and morphometric analysis indicate higher abundance of metacyclics in LV79. CONCLUSIONS/SIGNIFICANCE: Proteome comparison of PH8 and LV79 promastigotes generated a list of differential proteins, some of which may be further prospected to affect the infectivity of promastigotes. Although proteomic profile of PH8 includes more proteins characteristic of metacyclics, flow cytometry and morphometric analysis indicate a higher abundance of metacyclics in LV79 cultures. These results shed light to the gaps in our knowledge of metacyclogenesis in L. amazonensis, and to proteins that should be studied in the context of infection by this species.

An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection.

Trypanosoma cruzi is a hemoflagellated parasite causing Chagas disease, which affects 6-8 million people in the Americas. More than one hundred years after the description of this disease, the available drugs for treating the T. cruzi infection remain largely unsatisfactory. Chloroquinoline and arylamidine moieties are separately found in various compounds reported for their anti-trypanosoma activities. In this work we evaluate the anti-T. cruzi activity of a collection of 26 "chimeric" molecules combining choroquinoline and amidine structures. In a first screening using epimastigote forms of the parasite as a proxy for the clinically relevant stages, we selected the compound 7-chloro-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]quinoline (named here as A6) that performed better as an anti-T. cruzi compound (IC50 of 2.2 ± 0.3 µM) and showed a low toxicity for the mammalian cell CHO-K1 (CC50 of 137.9 ± 17.3 µM). We initially investigated the mechanism of death associated to the selected compound. The A6 did not trigger phosphatidylserine exposure or plasma membrane permeabilization. Further investigation led us to observe that under short-term incubations (until 6 hours), no alterations of mitochondrial function were observed. However, at longer incubation times (4 days), A6 was able to decrease the intracellular Ca2+, to diminish the intracellular ATP levels, and to collapse mitochondrial inner membrane potential. After analysing the cell cycle, we found as well that A6 produced an arrest in the S phase that impairs the parasite proliferation. Finally, A6 was effective against the infective forms of the parasite during the infection of the mammalian host cells at a nanomolar concentration (IC50(tryps) = 26.7 ± 3.7 nM), exhibiting a selectivity index (SI) of 5,170. Our data suggest that A6 is a promising hit against T. cruzi.

Dichloroacetate and Pyruvate Metabolism: Pyruvate Dehydrogenase Kinases as Targets Worth Investigating for Effective Therapy of Toxoplasmosis.

Toxoplasmosis, a protozoan infection caused by Toxoplasma gondii, is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment. New therapies should be developed by exploring peculiarities of the parasite's metabolic pathways, similarly to what has been well described in cancer cell metabolism. An example is the switch in the metabolism of cancer that blocks the conversion of pyruvate into acetyl coenzyme A in mitochondria. In this context, dichloroacetate (DCA) is an anticancer drug that reverts the tumor proliferation by inhibiting the enzymes responsible for this switch: the pyruvate dehydrogenase kinases (PDKs). DCA has also been used in the treatment of certain symptoms of malaria; however, there is no evidence of how this drug affects apicomplexan species. In this paper, we studied the metabolism of T. gondii and demonstrate that DCA also inhibits T. gondii's in vitro infection with no toxic effects on host cells. DCA caused an increase in the activity of pyruvate dehydrogenase followed by an unbalanced mitochondrial activity. We also observed morphological alterations frequently in mitochondria and in a few apicoplasts, essential organelles for parasite survival. To date, the kinases that potentially regulate the activity of pyruvate metabolism in both organelles have never been described. Here, we confirmed the presence in the genome of two putative kinases (T. gondii PDK [TgPDK] and T. gondii branched-chain α-keto acid dehydrogenase kinase [TgBCKDK]), verified their cellular localization in the mitochondrion, and provided in silico data suggesting that they are potential targets of DCA.IMPORTANCE Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely. We report here that the treatment of T. gondii with an anticancer drug, dichloroacetate (DCA), was effective in decreasing in vitro infection without toxicity to human cells. It is known that PDK is the main target of DCA in mammals, and this inactivation increases the conversion of pyruvate into acetyl coenzyme A and reverts the proliferation of tumor cells. Moreover, we verified the mitochondrial localization of two kinases that possibly regulate the activity of pyruvate metabolism in T. gondii, which has never been studied. DCA increased pyruvate dehydrogenase (PDH) activity in T. gondii, followed by an unbalanced mitochondrial activity, in a manner similar to what was previously observed in cancer cells. Thus, we propose the conserved kinases as potential regulators of pyruvate metabolism and interesting targets for new therapies.