Name: in vitro drug screening against all life cycle stages of trypanosoma cruzi using parasites expressing &#946;-galactosidase
Uploaded: 2021-11-05T13:00:00
Description: Watch this Scientific Journal Video about In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing Î²-galactosidase at JoVE.com

We thank Dr. Buckner for kindly providing the pLacZ plasmid. This work was supported by Agencia Nacional de Promoci&#243;n Cient&#237;fica y Tecnol&#243;gica, Ministerio de Ciencia e Innovaci&#243;n 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).

NOTE: An overview of the entire experimental design is depicted in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63210/63210fig01.jpg" /> 
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...

<ol>
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This paper describes an assay based on determining the cytoplasmic &#946;-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 &#946;-galactosidase-bearing plasmid constructed by Buckner and co-authors10. This assay has been used to search for antitrypanocidal comp...

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 people6,7. 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 &#946;-galactosidase activity of transfected parasites first described by Bucknet and collaborators10. The &#946;-galactosidase enzyme expressed by the recombinant parasites hydrolyzes the chromogenic substrate, chlorophenol red &#946;-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 &#946;-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 compounds10,11,12,13. This method has already been successfully used to evaluate compounds for activity against T. cruzi in low- and high-throughput screening12,13. Similar approaches have also been used in other protozoan parasites, including Toxoplasma gondii and Leishmania mexicana14,15.
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 &#946;-galactosidase. The assays presented here have been performed with a &#946;-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 L beaker</td>
			<td>Schott Duran</td>
			<td>10005227</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette sterile</td>
			<td>Jet Biofil</td>
			<td>GSP211010</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipette sterile</td>
			<td>Jet Biofil</td>
			<td>GSP010005</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Corning</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Benznidazole</td>
			<td>Sigma Aldrich</td>
			<td>419656</td>
			<td>N-Benzyl-2-nitro-1H-imidazole-1-acetamide</td>
		</tr>
		<tr>
			<td>Biosafty Cabinet</td>
			<td>Telstar</td>
			<td>Bio II A/P</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube 15 mL conical bottom sterile</td>
			<td>Tarson</td>
			<td>546021</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube 50 mL conical bottom sterile</td>
			<td>Tarson</td>
			<td>546041</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Sanyo</td>
			<td>MCO-15A</td>
			<td></td>
		</tr>
		<tr>
			<td>CPRG</td>
			<td>Roche</td>
			<td>10 884308001</td>
			<td>Chlorophenol Red-&#946;-D-galactopyranoside</td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>Thermo Fisher Cientific</td>
			<td>12100046</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sintorgan</td>
			<td>SIN-061</td>
			<td>Dimethylsulfoxid</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Internegocios SA</td>
			<td>FCS FRA 500</td>
			<td>Sterile and heat-inactivated</td>
		</tr>
		<tr>
			<td>G418 disulphate salt solution</td>
			<td>Roche</td>
			<td>G418-RO</td>
			<td>stock concentration: 50 mg/mL</td>
		</tr>
		<tr>
			<td>Glucose D(+)</td>
			<td>Cicarelli</td>
			<td>716214</td>
			<td></td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Nalgene</td>
			<td>3663-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Frontier Scientific</td>
			<td>H651-9</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Cicarelli</td>
			<td>867212</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Infusion</td>
			<td>Difco</td>
			<td>226920</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL</td>
			<td>Tarson</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Spectrophotometer</td>
			<td>Biotek</td>
			<td>Synergy HTX</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Cicarelli</td>
			<td>834214</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Cicarelli</td>
			<td>750214</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Boeco</td>
			<td>BOE 01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P-40</td>
			<td>Antrace</td>
			<td>NIDP40</td>
			<td>2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Statistical Analysis software</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Cicarelli</td>
			<td>929211</td>
			<td>NaHCO3</td>
		</tr>
		<tr>
			<td>Sorvall ST 16 Centrifuge</td>
			<td>Thermo Fisher Cientific</td>
			<td>75004380</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 flasks</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptose</td>
			<td>Merck</td>
			<td>1106760500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>ATCC</td>
			<td>CRL-1587</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro drug screening against all life cycle stages of trypanosoma cruzi using parasites expressing &#946;-galactosidase

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 &#946;-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a &#946;-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.

Following the protocol described above, &#946;-galactosidase-expressing Dm28c epimastigotes were incubated with 6 concentrations of BZN (2.5, 5, 10, 20, 40, 80 &#181;M) (or compounds of interest) for 72 h. After this period, CPRG reagent was added along with detergent, which lyses the cells and releases &#946;-galactosidase. CPRG is cleaved by the &#946;-galactosidase to produce chlorophenol red, leading to a change in color from yellow to reddish (Figure 2A). Chlorophenol red was measured b...

Watch this Scientific Journal Video about In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing Î²-galactosidase at JoVE.com

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

We describe a high-throughput colorimetric assay measuring &#946;-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.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many ...

This protocol describes a high throughput colorimetric assay in the three lifecycle stages of Trypanosoma cruzi to identify new drug candidates for Chagas disease. This can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner. Demonstrating the procedure will be Romina Manarin, a lab cell culture technician from the laboratory of Pamela crib.Begin by culturing beta galactosidase expressing Trypanosoma cruzi epimastigotes in a T-25 tissue culture flask and incubate at 28 degrees Celsius for growing them axenically. Using a Neubauer chamber, quantify parasite growth before subculturing. For maintaining the cultures in a log phase, subculture every 48 to 72 hours in five milliliters of LIT medium supplemented with 10%FCS and geneticin in sulfate at a final concentration of 200 micrograms per milliliters.Then securely close the cap before incubating at 28 degrees Celsius in a vertical position. From the log phase culture, make a suspension of epimastigote in LIT medium supplemented with geneticin and sulfate at the final concentration of 200, 000 cells per milliliter epimastigote per milliliter. After dispensing 100 microliters of suspension per well of a 96 well plate, makeup final volume of well 200 microliters with medium.Make the Vero cell suspension in DMEM supplemented with 2%FCS at the final concentration of 100, 000 cells per milliliter. Seed 100 microliters of the suspension per well in a 96 well tissue culture plate. Incubate the cells overnight for adherence and the next day, observe the cells under the microscope.The next day, rinse the Vero cell monolayer three times with 100 microliters of sterile PBS. Add previously obtained metacyclic trypomastigotes. Add a multiplicity of infection or MOI of 10-1 in 100 microliters of DMEM supplemented with 2%FCS per well and incubate for six hours as demonstrated previously.At the end of the incubation, wash the plate twice with PBS and add 100 microliters of DMEM without phenol red supplemented with 2%FCS. After making the Vero cells suspension in DMEM add the final concentration of 1 million cells per milliliter. Seed 800, 000 cells in five milliliters of the medium in T-25 flasks and incubate overnight for cell adherence.Once the flask is rinsed with PBS, add trypomastigotes, add an MOI of 10-1 in five milliliters of DMEM supplemented with 2%FCS and incubate overnight as demonstrated previously. At the end of incubation, wash the flask twice with PBS. Add five milliliters of fresh DMEM supplemented with 2%FCS and incubate for four days.At the end of the incubation, after observing the supernatant for trypomastigotes presence under an optic microscope. Quantify the trypomastigotes using a Neubauer chamber. Collect the supernatant in a 15 milliliter tube and centrifuge at 7, 000 times G for 10 minutes at room temperature.Then discard the supernatant and resuspend the pellet in DMEM without phenol red supplemented with 2%FCS to obtain a concentration of 1 million trypomastigotes per milliliter. Seed 100 microliters of the trypomastigote suspension per well in a 96 well plate. Add benznidazole solution to epimastigotes, Vero cells with amastigotes, or trypomastigotes as described in the text manuscript.Then incubate the epimastigotes, set 28 degrees Celsius for 72 hours and the trypomastigotes or infected Vero cells with amastigotes for 24 hours at 37 degrees Celsius and 5%carbon dioxide. For the colormetric assay, set up the blank wells by adding 100 microliters of PBS or corresponding medium. Next, add 40 microliters of CPRG substrate solution and 10 microliters of the detergent solution to each well for obtaining a final concentration of 200 micromolar CPRG and 0.1%detergent in a final volume of 250 microliters in each well.After incubation, at 37 degrees Celsius for two hours, measure absorbance at 595 nanometers using a microplate spectrophotometer. In the software, create a new protocol by selecting absorbance as the detection method and end point as the read type, then click okay. Next, add a read step.Type the selected wavelength and click okay. In the plate layout section mark the wells to be read, click okay, and click on read plate, wait for the values to appear on the screen and export them to a spreadsheet to analyze the results and calculate the absorbance values using subtraction as described in the manuscript. In statistical analysis software, plot the concentration of BZN versus the absorbance at 595 nanometers in the XY table.Transform the BZN concentrations to logarithmic values by clicking analyze, selecting the transform option, and clicking okay. For obtaining IC50 values, click analyze, and from the XY analysis list select nonlinear regression curve fit, then click okay. Next, go to the parameters window, in the model tab, go to the dose response inhibition group of built-in equations, select log inhibitor versus response variable slope for parameters as the dose response method leave all other tabs at default values and then click, okay.Click the results section to find the IC50 value, the SD, and the goodness of the fit. Click the graph section to find the XY graph of the logarithmic concentration of the drug versus the absorbance values and ensure that the curve fit is also graphed in a different color. In the present study, the IC50 value obtained for epimastigotes was approximately 20 micromolar, similar to previous studies.In addition, the results indicated liner beta galactosidase activity of the epimastigotes over time. It is very important to perform at least replicates of each condition tested and minimized errors in volume management.

Research

JoVE Journal

Biochemistry

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