In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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), incubating them with BZN (2 and 3), and then adding the colorimetric substrate (4). When parasites are lysed, β-ga....

Results

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.......

Discussion

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.......

Materials

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