scholarly journals Bioluminescent:fluorescent Trypanosoma cruzi Reporter Strains as Tools for Exploring Chagas Disease Pathogenesis and Drug Activity

2020 ◽  
Vol 26 ◽  
Author(s):  
Martin C. Taylor ◽  
Alexander I. Ward ◽  
Francisco Olmo ◽  
Amanda F. Francisco ◽  
Shiromani Jayawardhana ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Parasite Burden ◽  
New Drugs ◽  
Host Cells ◽  
Complex Nature ◽  
Disease Pathogenesis ◽  
Drug Activity ◽  
Cellular Environment

: Chagas disease results from infection with the trypanosomatid parasite Trypanosoma cruzi. Progress in developing new drugs has been hampered by the long term and complex nature of the condition and by our limited understanding of parasite biology. Technical difficulties in assessing the parasite burden during the chronic stage of infection have also proved to be a particular challenge. In this context, the development of non-invasive, highly sensitive bioluminescence imaging procedures, based on parasites that express a red-shifted luciferase, has greatly enhanced our ability to monitor infections in experimental models. Applications of this methodology have led to new insights into tissue tropism and infection dynamics, and have been a major driver in drug development. The system has been further modified by the generation of parasite reporter lines that express bioluminescent:fluorescent fusion proteins, an advance that has allowed chronic infections in mice to be examined at a cellular level. By exploiting bioluminescence to identify the rare sites of tissue infection, and fluorescence to detect T. cruzi at the level of individual host cells in histological sections, it has been possible to investigate the replication and differentiation status of parasites in vivo and to examine the cellular environment of infection foci. In combination, these data are providing a framework for the detailed dissection of disease pathogenesis and drug activity.

scholarly journals Repurposing Carvedilol as a Novel Inhibitor of the Trypanosoma cruzi Autophagy Flux That Affects Parasite Replication and Survival

2021 ◽  
Vol 11 ◽  
Author(s):  
Cynthia Vanesa Rivero ◽  
Santiago José Martínez ◽  
Paul Novick ◽  
Juan Agustín Cueto ◽  
Betiana Nebaí Salassa ◽  
...  
Keyword(s):  
Chagas Disease ◽  
Parasite Burden ◽  
Drug Repurposing ◽  
Parasite Load ◽  
New Drugs ◽  
Host Cells ◽  
Whole Body ◽  
Parasite Replication

T. cruzi, the causal agent of Chagas disease, is a parasite able to infect different types of host cells and to persist chronically in the tissues of human and animal hosts. These qualities and the lack of an effective treatment for the chronic stage of the disease have contributed to the durability and the spread of the disease around the world. There is an urgent necessity to find new therapies for Chagas disease. Drug repurposing is a promising and cost-saving strategy for finding new drugs for different illnesses. In this work we describe the effect of carvedilol on T. cruzi. This compound, selected by virtual screening, increased the accumulation of immature autophagosomes characterized by lower acidity and hydrolytic properties. As a consequence of this action, the survival of trypomastigotes and the replication of epimastigotes and amastigotes were impaired, resulting in a significant reduction of infection and parasite load. Furthermore, carvedilol reduced the whole-body parasite burden peak in infected mice. In summary, in this work we present a repurposed drug with a significant in vitro and in vivo activity against T. cruzi. These data in addition to other pharmacological properties make carvedilol an attractive lead for Chagas disease treatment.

scholarly journals Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 4081-4087 ◽  
Author(s):  
Craig Weinkauf ◽  
Ryan Salvador ◽  
Mercio PereiraPerrin
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Neuronal Cell ◽  
Host Cells ◽  
Neurotrophin Receptor ◽  
Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

scholarly journals In Vitro and In Vivo Activities of E5700 and ER-119884, Two Novel Orally Active Squalene Synthase Inhibitors, against Trypanosoma cruzi

2004 ◽  
Vol 48 (7) ◽  
pp. 2379-2387 ◽  
Author(s):  
Julio A. Urbina ◽  
Juan Luis Concepcion ◽  
Aura Caldera ◽  
Gilberto Payares ◽  
Cristina Sanoja ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Public Health Problem ◽  
Squalene Synthase ◽  
Host Cells ◽  
In Vivo Studies ◽  
Inorganic Pyrophosphate ◽  
Orally Active

ABSTRACT Chagas' disease is a serious public health problem in Latin America, and no treatment is available for the prevalent chronic stage. Its causative agent, Trypanosoma cruzi, requires specific endogenous sterols for survival, and we have recently demonstrated that squalene synthase (SQS) is a promising target for antiparasitic chemotherapy. E5700 and ER-119884 are quinuclidine-based inhibitors of mammalian SQS that are currently in development as cholesterol- and triglyceride-lowering agents in humans. These compounds were found to be potent noncompetitive or mixed-type inhibitors of T. cruzi SQS with K i values in the low nanomolar to subnanomolar range in the absence or presence of 20 μM inorganic pyrophosphate. The antiproliferative 50% inhibitory concentrations of the compounds against extracellular epimastigotes and intracellular amastigotes were ca. 10 nM and 0.4 to 1.6 nM, respectively, with no effects on host cells. When treated with these compounds at the MIC, all of the parasite's sterols disappeared from the parasite cells. In vivo studies indicated that E5700 was able to provide full protection against death and completely arrested the development of parasitemia when given at a concentration of 50 mg/kg of body weight/day for 30 days, while ER-119884 provided only partial protection. This is the first report of an orally active SQS inhibitor that is capable of providing complete protection against fulminant, acute Chagas' disease.

scholarly journals Limited Ability of Posaconazole To Cure both Acute and Chronic Trypanosoma cruzi Infections Revealed by Highly SensitiveIn VivoImaging

2015 ◽  
Vol 59 (8) ◽  
pp. 4653-4661 ◽  
Author(s):  
Amanda Fortes Francisco ◽  
Michael D. Lewis ◽  
Shiromani Jayawardhana ◽  
Martin C. Taylor ◽  
Eric Chatelain ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Imaging System ◽  
Ex Vivo ◽  
Parasite Burden ◽  
Significant Activity ◽  
Content Type ◽  
Chronic Stage

ABSTRACTThe antifungal drug posaconazole has shown significant activity againstTrypanosoma cruziin vitroand in experimental murine models. Despite this, in a recent clinical trial it displayed limited curative potential. Drug testing is problematic in experimental Chagas disease because of difficulties in demonstrating sterile cure, particularly during the chronic stage of infection when parasite burden is extremely low and tissue distribution is ill defined. To better assess posaconazole efficacy against acute and chronic Chagas disease, we have exploited a highly sensitive bioluminescence imaging system which generates data with greater accuracy than other methods, including PCR-based approaches. Mice inoculated with bioluminescentT. cruziwere assessed byin vivoandex vivoimaging, with cyclophosphamide-induced immunosuppression used to enhance the detection of relapse. Posaconazole was found to be significantly inferior to benznidazole as a treatment for both acute and chronicT. cruziinfections. Whereas 20 days treatment with benznidazole was 100% successful in achieving sterile cure, posaconazole failed in almost all cases. Treatment of chronic infections with posaconazole did however significantly reduce infection-induced splenomegaly, even in the absence of parasitological cure. The imaging-based screening system also revealed that adipose tissue is a major site of recrudescence in mice treated with posaconazole in the acute, but not the chronic stage of infection. Thisin vivoscreening model for Chagas disease is predictive, reproducible and adaptable to diverse treatment schedules. It should provide greater assurance that drugs are not advanced prematurely into clinical trial.

scholarly journals Amaryllidaceae alkaloids with anti-Trypanosoma cruzi activity

2020 ◽  
Author(s):  
Nieves Martinez-Peinado ◽  
Nuria Cortes-Serra ◽  
Laura Torras-Claveria ◽  
Maria-Jesus Pinazo ◽  
Joaquim Gascon ◽  
...  
Keyword(s):  
Natural Products ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
New Drugs ◽  
Host Cells ◽  
Amaryllidaceae Alkaloids ◽  
Cytotoxicity Assays ◽  
Limited Efficacy ◽  
Single Compound ◽  
New Compounds

Abstract Background: Chagas disease, caused by the protozoan Trypanosoma cruzi, is a neglected disease that affects ~7 million people worldwide. Development of new drugs to treat the infection remains a priority since those currently available have frequent side effects and limited efficacy at the chronic stage. Natural products provide a pool of diversity structures to lead the chemical synthesis of novel molecules for this purpose. Herein we analyzed the anti- T. cruzi activity of 9 alkaloids derived from plants of the Amaryllidaceae family. Methods: the activity of each alkaloid was assessed by means of a newly developed anti- T. cruzi phenotypic assay. We further evaluated the compounds that inhibited the parasite growth on two distinct cytotoxicity assays to discard those that were toxic to host cells and assure parasite selectivity. Results: we identified a single compound (hippeastrine 2 ) that was selectively active against the parasite yielding selectivity indexes of 12.7 and 35.2 against Vero and HepG2 cells, respectively. Conclusions: results reported here suggest that natural products are an interesting source of new compounds for the development of drugs against Chagas disease.

scholarly journals Amaryllidaceae alkaloids with anti-Trypanosoma cruzi activity

2020 ◽  
Author(s):  
Nieves Martinez-Peinado ◽  
Nuria Cortes-Serra ◽  
Laura Torras-Claveria ◽  
Maria-Jesus Pinazo ◽  
Joaquim Gascon ◽  
...  
Keyword(s):  
Natural Products ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Specific Activity ◽  
New Drugs ◽  
Host Cells ◽  
Amastigote Stage ◽  
Limited Efficacy ◽  
Single Compound ◽  
New Compounds

Abstract Background: Chagas disease, caused by the protozoan Trypanosoma cruzi, is a neglected disease that affects ~7 million people worldwide. Development of new drugs to treat the infection remains a priority since those currently available have frequent side effects and limited efficacy at the chronic stage. Natural products provide a pool of diversity structures to lead the chemical synthesis of novel molecules for this purpose. Herein we analyzed the anti- T. cruzi activity of 9 alkaloids derived from plants of the Amaryllidaceae family. Methods: the activity of each alkaloid was assessed by means of an anti- T. cruzi phenotypic assay. We further evaluated the compounds that inhibited the parasite growth on two distinct cytotoxicity assays to discard those that were toxic to host cells and assure parasite selectivity. Results: we identified a single compound (hippeastrine 2 ) that was selectively active against the parasite yielding selectivity indexes of 12.7 and 35.2 against Vero and HepG2 cells, respectively. Moreover, it showed specific activity against the amastigote stage (IC 50 = 3.31 μM). Conclusions: results reported here suggest that natural products are an interesting source of new compounds for the development of drugs against Chagas disease.

scholarly journals Amaryllidaceae alkaloids with anti-Trypanosoma cruzi activity

2020 ◽  
Author(s):  
Nieves Martinez-Peinado ◽  
Nuria Cortes-Serra ◽  
Laura Torras-Claveria ◽  
Maria-Jesus Pinazo ◽  
Joaquim Gascon ◽  
...  
Keyword(s):  
Natural Products ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Specific Activity ◽  
New Drugs ◽  
Host Cells ◽  
Amastigote Stage ◽  
Limited Efficacy ◽  
Single Compound ◽  
New Compounds

Abstract Background: Chagas disease, caused by the protozoan Trypanosoma cruzi, is a neglected disease that affects ~7 million people worldwide. Development of new drugs to treat the infection remains a priority since those currently available have frequent side effects and limited efficacy at the chronic stage. Natural products provide a pool of diversity structures to lead the chemical synthesis of novel molecules for this purpose. Herein we analyzed the anti- T. cruzi activity of 9 alkaloids derived from plants of the Amaryllidaceae family. Methods: the activity of each alkaloid was assessed by means of an anti- T. cruzi phenotypic assay. We further evaluated the compounds that inhibited the parasite growth on two distinct cytotoxicity assays to discard those that were toxic to host cells and assure parasite selectivity. Results: we identified a single compound (hippeastrine 2 ) that was selectively active against the parasite yielding selectivity indexes of 12.7 and 35.2 against Vero and HepG2 cells, respectively. Moreover, it showed specific activity against the amastigote stage (IC 50 = 3.31 μM). Conclusions: results reported here suggest that natural products are an interesting source of new compounds for the development of drugs against Chagas disease.

scholarly journals Pep5, a Fragment of Cyclin D2, Shows Antiparasitic Effects in Different Stages of the Trypanosoma cruzi Life Cycle and Blocks Parasite Infectivity

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Christiane Bezerra de Araujo ◽  
Loyze Paola de Lima ◽  
Simone Guedes Calderano ◽  
Flávia Silva Damasceno ◽  
Ariel M. Silber ◽  
...  
Keyword(s):  
Cell Death ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Fold Increase ◽  
Host Cells ◽  
Public Health Issue ◽  
Mammalian Host ◽  
Cyclin D2 ◽  
Content Type

ABSTRACT Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cell-penetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells. Additionally, Pep5-cpp reduces the in vivo tumor volume of rat glioblastoma. Chagas disease, which is caused by the flagellated parasite Trypanosoma cruzi, is a neglected disease that occurs mainly in the Americas, where it is considered an important public health issue. Given that there are only two options for treating the disease, it is exceptionally crucial to search for new molecules with potential pharmacological action against the parasites. In this study, we demonstrate that Pep5-cpp induces cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi. The Pep5-cpp peptide was also able to decrease the percentage of infected cells without causing any detectable toxic effects in mammalian host cells. The infective, i.e., trypomastigote form of T. cruzi pretreated with Pep5-cpp was unable to infect LLC-MK2 monkey kidney cells. Also, Pep5-binding proteins were identified by mass spectrometry, including calmodulin-ubiquitin-associated protein, which is related to the virulence and parasitemia of T. cruzi. Taken together, these data suggest that Pep5 can be used as a novel alternative for the treatment of Chagas disease.

scholarly journals Intracellular DNA replication and differentiation of Trypanosoma cruzi is asynchronous within individual host cells in vivo at all stages of infection

2019 ◽  
Author(s):  
Martin. C. Taylor ◽  
Alexander Ward ◽  
Francisco Olmo ◽  
Shiromani Jayawardhana ◽  
Amanda F. Francisco ◽  
...  
Keyword(s):  
Dna Replication ◽  
Trypanosoma Cruzi ◽  
Genetically Modified ◽  
Host Cells ◽  
Mammalian Host ◽  
Disease Pathogenesis ◽  
Individual Host ◽  
Infected Cells ◽  
Parasite Replication

ABSTRACTInvestigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.AUTHOR SUMMARYChagas disease, caused by the protozoan parasite Trypanosoma cruzi, is becoming an emerging threat in non-endemic countries and establishing new foci in endemic countries. The treatment available has not changed significantly in over 40 years. Therefore, there is an urgent need for a greater understanding of parasite biology and disease pathogenesis to identify new therapeutic targets and to maximise the efficient use of existing drugs. We have used genetically modified strains of T. cruzi carrying a bioluminescence/fluorescence dual reporter fusion gene to monitor parasite replication in vivo during both acute and chronic infections in a mouse model. Utilising TUNEL assays for mitochondrial DNA replication and EdU incorporation for total DNA replication, we have found that parasite division within infected cells is asynchronous in all phases of infection. Differentiation also appears to be uncoordinated, with replicating amastigotes co-existing with non-dividing trypomastigotes in the same host cell.

scholarly journals In VitroandIn VivoTrypanosomicidal Action of Novel Arylimidamides against Trypanosoma cruzi

2016 ◽  
Vol 60 (4) ◽  
pp. 2425-2434 ◽  
Author(s):  
F. H. Guedes-da-Silva ◽  
D. G. J. Batista ◽  
M. B. Meuser ◽  
K. C. Demarque ◽  
T. O. Fulco ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Parasite Load ◽  
Host Cells ◽  
Content Type ◽  
Days Of Therapy ◽  
Different Strains ◽  
Dose Dependent

ABSTRACTArylimidamides (AIAs) have been shown to have considerable biological activity against intracellular pathogens, includingTrypanosoma cruzi, which causes Chagas disease. In the present study, the activities of 12 novel bis-AIAs and 2 mono-AIAs against different strains ofT. cruziin vitroandin vivowere analyzed. The most active wasm-terphenyl bis-AIA (35DAP073), which had a 50% effective concentration (EC50) of 0.5 μM for trypomastigotes (Y strain), which made it 26-fold more effective than benznidazole (Bz; 13 μM). It was also active against the Colombiana strain (EC50= 3.8 μM). Analysis of the activity against intracellular forms of the Tulahuen strain showed that this bis-AIA (EC50= 0.04 μM) was about 100-fold more active than Bz (2 μM). The trypanocidal effect was dissociated from the ability to trigger intracellular lipid bodies within host cells, detected by oil red labeling. Both an active compound (35DAP073) and an inactive compound (26SMB060) displayed similar activation profiles. Due to their high selectivity indexes, two AIAs (35DAP073 and 35DAP081) were moved toin vivostudies, but because of the results of acute toxicity assays, 35DAP081 was excluded from the subsequent tests. The findings obtained with 35DAP073 treatment of infections caused by the Y strain revealed that 2 days of therapy induced a dose-dependent action, leading to 96 to 46% reductions in the level of parasitemia. However, the administration of 10 daily doses in animals infected with the Colombiana strain resulted in toxicity, preventing longer periods of treatment. The activity of the combination of 0.5 mg/kg of body weight/day 35DAP073 with 100 mg/kg/day Bz for 10 consecutive days was then assayed. Treatment with the combination resulted in the suppression of parasitemia, the elimination of neurological toxic effects, and survival of 100% of the animals. Quantitative PCR showed a considerable reduction in the parasite load (60%) compared to that achieved with Bz or the amidine alone. Our results support further investigations of this class with the aim of developing novel alternatives for the treatment of Chagas disease.

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