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 seropositive throughout their life.

Estimations suggest that ~7 million people are infected worldwide, mostly from Latin America, where ChD is endemic. In these countries, T. cruzi is mainly transmitted through infected blood-sucking triatomine bugs (vector-borne transmission) and less frequently by oral transmission through the ingestion of food contaminated with triatomine feces containing the parasites². Additionally, the parasite can be transmitted via the placenta from chagasic mothers to newborns, through blood transfusions, or during organ transplantation. These vector-independent ways of acquiring the infection and human migration have contributed to the worldwide spread of the disease, evidenced by an increasing number of cases in North America, Europe, and some African, Eastern Mediterranean, and Western Pacific countries⁴. ChD is considered a neglected disease as vector-borne transmission is closely associated with poverty and is a leading public health issue, especially in Latin American low-income countries. Although there are available treatments, mortality due to ChD in Latin America is the highest among parasitic diseases, including malaria².

There are two registered drugs for ChD treatment introduced in the late 1960s and early 1970s: nifurtimox and benznidazole⁵. Both drugs are effective in the acute phase of the disease in adults, children, and congenitally infected newborns, as well as in children with chronic infection, where cure is usually achieved. However, only a few people are diagnosed early enough to be treated in time. According to the latest clinical trials, both drugs have important limitations in adults and were ineffective in reducing symptoms in people with chronic disease; hence, their use in this stage is controversial. Other drawbacks are the prolonged treatment periods required (60-90 days) and the frequent, severe adverse effects observed, which lead to discontinuation of therapy in a proportion of infected people^6,7. It is estimated that fewer than 10% of the people with ChD have been diagnosed, and even fewer have access to treatment, as many affected individuals live in rural areas with no or scarce access to healthcare⁸. These facts highlight the urgent need to find new drugs against T. cruzi to allow for more efficient, safe, and applicable-to-the-field treatments, especially for the chronic phase. In this regard, another challenge in the development of more efficacious compounds is the limitation of systems for assessing drug efficacy in vitro and in vivo⁹.

Although chemical biology and genomic approaches for the identification of potential drug targets have been used in kinetoplastid parasites, the available genomic tools in T. cruzi are limited in contrast to T. brucei or Leishmania. Thus, the screening of compounds with trypanocidal activity is still the most used approach in the search for new chemotherapeutic drug candidates against ChD. Usually, drug discovery in T. cruzi must start with testing the effects of a new drug in an in vitro assay against the epimastigote stage. For decades, the only way for measuring the inhibitory effects of candidate compounds on T. cruzi was manual microscopic counting, which is laborious, time-consuming, and operator-dependent. Moreover, this approach is suitable for assaying a small number of compounds but is unacceptable for high-throughput screening of large compound libraries. Nowadays, many investigations begin with the analysis of a vast number of compounds from different origins that are assayed in vitro, testing their capacity for inhibiting parasite growth. Both colorimetric and fluorometric methods have been developed to increase throughput in these assays, improving the objectivity of the screening and making the whole process less tedious⁹.

One of the most widely used colorimetric methods is based on the β-galactosidase activity of transfected parasites first described by Bucknet and collaborators¹⁰. The β-galactosidase enzyme expressed by the recombinant parasites hydrolyzes the chromogenic substrate, chlorophenol red β-D-galactopyranoside (CPRG), to chlorophenol red, which can be easily measured colorimetrically using a microplate spectrophotometer. Thus, parasite growth in the presence of a variety of compounds can be simultaneously evaluated and quantitated in microtiter plates. This method has been applied to test drugs in epimastigote forms (present in the insect vector), trypomastigotes, and intracellular amastigotes, the mammalian stages of the parasite. Further, several recombinant T. cruzi strains transfected with the pBS:CL-Neo-01/BC-X-10 plasmid (pLacZ)¹⁰ to express the Escherichia coli β-galactosidase enzyme are already available (and new ones can be constructed), which allows the evaluation of parasites from different discrete typing units (DTUs) that may not behave equally toward the same compounds^10,11,12,13. This method has already been successfully used to evaluate compounds for activity against T. cruzi in low- and high-throughput screening^12,13. Similar approaches have also been used in other protozoan parasites, including Toxoplasma gondii and Leishmania mexicana^14,15.

This paper describes and shows a detailed method for an in vitro drug screening against all life cycle stages of T. cruzi using parasites expressing β-galactosidase. The assays presented here have been performed with a β-galactosidase-expressing T. cruzi line obtained by transfection of T. cruzi Dm28c strain from DTU I¹³ with pLacZ plasmid (Dm28c/pLacZ). Additionally, the same protocol could be easily adapted to other strains to compare the performance between compounds and between T. cruzi strains or DTUs.

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), incubati…

Representative 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

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