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.

in-vitro-drug-screening-against-all-life-cycle-stages-trypanosoma

Research

JoVE Journal

Biochemistry

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...