Antiproliferative Plaque Assay for Screening in vitro Bioactive Molecules against Toxoplasma gondii Tachyzoites

Summary

This work aims to describe a simple method of plaque assay to aid in the screening of new bioactive molecules against the tachyzoite stage of the protozoan Toxoplasma gondii.

Abstract

Toxoplasmosis is a parasitic infection commonly related to ocular lesions and neonatal malformations. Since the early 1950s, the first-line treatment of this disease has relied on the combination of sulfadiazine, pyrimethamine, and folinic acid. Over those years, only a few alternative regimens have been introduced. This highlights the need for discovering new bioactive molecules against Toxoplasma gondii. Given that this pathogen is an obligate intracellular parasite, traditional drug screening in the laboratory typically requires expensive materials and equipment, such as assays using fluorescent proteins or β-galactosidase-expressing parasites. Additionally, methods like optical microscopy quantification can be time-consuming. The plaque assay is a method that evaluates the intensity of intracellular pathogen proliferation by measuring the number of regions and area of destruction in a cell monolayer damaged by the parasite's lytic cycle. This work describes an optimized plaque assay protocol designed for screening active molecules against intracellular tachyzoites of T. gondii in vitro. This protocol utilizes inexpensive materials and a straightforward laboratory setup, yielding rapid and reproducible results that facilitate the identification of new active molecules against this parasite by various research groups.

Introduction

Toxoplasmosis is a zoonotic disease with a heterogeneous distribution worldwide, affecting approximately one-third of the global population^1,2. The symptoms of this disease are associated with the degree of host immunity and the replication of the parasite^3,4. The acute phase of the disease, characterized by the replication of the tachyzoite stage, is generally asymptomatic in immunocompetent individuals^5,6. In contrast, immunocompromised individuals present with more frequent symptoms, includi....

Protocol

The manipulation of parasite and cell cultures, as well as waste disposal and destination, were performed in accordance with Brazilian legislation, as required by the National Agency of Sanitary Vigilance (ANVISA) RDC nº 222/2018. All procedures were supervised by the Internal Biosafety Committee (CIBio) from ICB-UFMG. This study does not involve the use of animals, humans, or genetically modified organisms. Grants from CNPq, CAPES, and FAPEMIG supported this work. ESMD (CNPq 308082/2023-0) and RWAV (CNPq 305574/2021-3) are CNPq productivity fellows. The reagents and the equipment used are listed in the Table of Materials.

Results

For evaluating the plaque assay after treatment, both the positive (cells infected with tachyzoites and untreated) and negative (uninfected cells with 0.1% DMSO) controls are crucial. At the end of the experiment, the positive control well should exhibit well-defined empty spaces with remnants of adhered cells (Figure 2). In contrast, the negative control well should display an intact monolayer with no empty spaces. The negative control is crucial for confirming the integrity of the monolaye.......

Discussion

The standardization of new protocols for antiproliferative tests is a fundamental step for advancing scientific research on the identification of future drugs. The protocol presented here offers an alternative approach to in vitro assessing the activity of bioactive molecules against tachyzoites of T. gondii. Given that T.gondii is an obligate intracellular parasite that locomotes by gliding motility, when the parasites egress from a lysed cell, they will invade the cells located in the vicinit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05 % Trypsin-EDTA	Life Technologies	25300-062
10 mL serological pipette	Olen	K17-110
15mL falcon tube centrifuge	Fanem	Model: 206
5 mL serological pipette	Olen	K17-115
6-well culture plate	Sarstedt	833920
Analytical balance	Mettler Toledo	Model: AB204 Serial No: 1116473813
Antibiotic-Antimycotic (100X)	Life Technologies	15240-062
Automatic pipette	Kasvi	K1-AID
Centrifuge	Eppendorf	Model 5415 R Serial No: 0023591
CO2 Incubator	PHC Corporation	Model No : MCO-230AICUVL-PA Serial No: 210360090
Culture bottle 25 cm2	Sarstedt	833910
Dimethyl sulfoxide (DMSO) P.A.	Merck	1,02,95,21,000
Ethanol P.A	Merck	1,00,98,31,000
Fetal Bovine Serum (FBS)	Life Technologies	12657-029
Formaldehyde 37-38%	Panreac	131328
GraphPad Prism	GraphPad Software	-	Version 8.0.1
ImageJ  software	-	-	Version 1.52e
Imaging documentation systems	Bio-Rad	Model:  ChemiDoc MP Imaging System Serial No: 734BR2249
Laminar flow hood	Veco do Brasil	Model: VLFS-09
L-glutamine 200 mM (100X)	Life Technologies	25030-081
Micro tube 0.5 mL	Sarstedt	72,699
Neonatal Normal Human Dermal Fibroblast (NHDF)	Lonza	CC-3132	Generously provided by Dr. Sheila Nardelli, from Fiocruz Paraná, Brazil
Neubauer chamber 0.100 mm 0.0025 mm2	New Optics	7301-1
pHmeter	Micronal	Model: B374 Serial No: 30/37
Pipette 20-200 µL	Kasvi basic	22032893
Pipette 2-20 µL	Labmate Pro	656630226
Pipette  0.2-2 µL	Labmate Pro	556610114
Pipette  1000-5.000 µL	Uniscience	YM4A021482
Pipette  100-1000 µL	Eppendorf Research plus	Q34354C
Potassium Phosphate Monobasic  (KH2PO4) P.A	Synth	01F2002.01.AF
RPMI-1640 medium	Sigma-Aldrich	R0883
Sodium chloride (NaCl) P.A.	Cromoline Química Fina	-
Sodium phosphate dibasic (Na2HPO4) P.A.	CRQ Produtos Quimicos Eireli	R2715920500
Tip 1.000 µL	Sarstedt	7,01,186
Tip 10 µL	Sarstedt	7,03,010
Tip 5.000 µL	Sarstedt	70,11,83,001
Tip  200 µL	Sarstedt	7,03,030
Transfer pipette  (3.5 mL)	Sarstedt	86,11,71,001
Tube 15 mL	Sarstedt	6,25,54,502
Violet Crystal	Merck	101408
Vortex mixer	Phoenix	Model: AP56 Serial No: 6633
Water Bath	Hemoquimica do Brasil	Model: HM1003 Serial No: 700001556