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We describe a high-throughput colorimetric assay measuring β-galactosidase activity in three life cycle stages of Trypanosoma cruzi, the causative agent of Chagas disease. This assay can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner.
Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi.
T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission.
This paper describes an assay based on measuring the activity of the cytoplasmic β-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red β-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a β-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.
Chagas disease (ChD), or American trypanosomiasis, is a parasitic disease caused by the flagellated protozoan, Trypanosoma cruzi (T. cruzi). ChD begins with an asymptomatic or oligosymptomatic acute phase that is usually undiagnosed, followed by a lifelong chronic phase. In the chronicity, ~30% of patients manifest-decades after the infection-a variety of debilitating conditions, including myocardiopathy, mega-digestive syndromes, or both, with a mortality rate ranging from 0.2% to 20%1,2,3. Asymptomatic chronic patients may have no clinical signs but remain....
NOTE: An overview of the entire experimental design is depicted in Figure 1.
Figure 1: Overview of the in vitro screening assay of Trypanosoma cruzi Dm28c/pLacZ line using CPRG as a substrate for the colorimetric reaction. The assay consists of seeding the parasites (1), incubati.......
Following the protocol described above, β-galactosidase-expressing Dm28c epimastigotes were incubated with 6 concentrations of BZN (2.5, 5, 10, 20, 40, 80 µM) (or compounds of interest) for 72 h. After this period, CPRG reagent was added along with detergent, which lyses the cells and releases β-galactosidase. CPRG is cleaved by the β-galactosidase to produce chlorophenol red, leading to a change in color from yellow to reddish (Figure 2A). Chlorophenol red was measured b.......
This paper describes an assay based on determining the cytoplasmic β-galactosidase activity released due to membrane lysis of T. cruzi epimastigotes, trypomastigotes, or infected cells with amastigotes in the presence of the substrate CPRG. We used T. cruzi Dm28c/pLacZ parasites, a stable parasite strain obtained after transfection with a β-galactosidase-bearing plasmid constructed by Buckner and co-authors10. This assay has been used to search for antitrypanocidal comp.......
We thank Dr. Buckner for kindly providing the pLacZ plasmid. This work was supported by Agencia Nacional de Promoción Científica y Tecnológica, Ministerio de Ciencia e Innovación Productiva from Argentina (PICT2016-0439, PICT2019-0526, PICT2019-4212), and Research Council United Kingdom [MR/P027989/1]. Servier Medical Art was used to produce Figure 1 (https://smart.servier.com).
....Name | Company | Catalog Number | Comments |
1 L beaker | Schott Duran | 10005227 | |
10 mL serological pipette sterile | Jet Biofil | GSP211010 | |
5 mL serological pipette sterile | Jet Biofil | GSP010005 | |
96-well plates | Corning | 3599 | |
Benznidazole | Sigma Aldrich | 419656 | N-Benzyl-2-nitro-1H-imidazole-1-acetamide |
Biosafty Cabinet | Telstar | Bio II A/P | |
Centrifuge tube 15 mL conical bottom sterile | Tarson | 546021 | |
Centrifuge tube 50 mL conical bottom sterile | Tarson | 546041 | |
CO2 Incubator | Sanyo | MCO-15A | |
CPRG | Roche | 10 884308001 | Chlorophenol Red-β-D-galactopyranoside |
DMEM, High Glucose | Thermo Fisher Cientific | 12100046 | Powder |
DMSO | Sintorgan | SIN-061 | Dimethylsulfoxid |
Fetal Calf Serum | Internegocios SA | FCS FRA 500 | Sterile and heat-inactivated |
G418 disulphate salt solution | Roche | G418-RO | stock concentration: 50 mg/mL |
Glucose D(+) | Cicarelli | 716214 | |
Graduated cylinder | Nalgene | 3663-1000 | |
Hemin | Frontier Scientific | H651-9 | |
KCl | Cicarelli | 867212 | |
Liver Infusion | Difco | 226920 | |
Microcentrifuge tube 1.5 mL | Tarson | 500010-N | |
Microplate Spectrophotometer | Biotek | Synergy HTX | |
Na2HPO4 | Cicarelli | 834214 | |
NaCl | Cicarelli | 750214 | |
Neubauer chamber | Boeco | BOE 01 | |
Nonidet P-40 | Antrace | NIDP40 | 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol |
Prism | Graphpad | Statistical Analysis software | |
Sodium Bicarbonate | Cicarelli | 929211 | NaHCO3 |
Sorvall ST 16 Centrifuge | Thermo Fisher Cientific | 75004380 | |
T-25 flasks | Corning | 430639 | |
Tryptose | Merck | 1106760500 | |
Vero cells | ATCC | CRL-1587 |
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