Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Summary

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Abstract

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Introduction

Robotics have been established as sophisticated and powerful tools for achieving significant breakthroughs in various fields beyond industry and fabrication engineering, such as biochemistry, molecular biology, and clinical research, and notably HTS^1,2,3. Toxoplasma gondii is a major parasite and a single-cell parasitic eukaryote⁴ that causes serious health issues in humans⁵ and many homeothermic animals⁴, resulting in infections leading to Toxoplasmosis, a particularly severe condition in AIDS patients⁶, organ transplant recipients⁷, and pregnant women⁸. Neospora caninum belonging to Phylum Apicomplexa⁹ mainly infects dogs and cows⁶, which results in Encephalomyelitis and Myositis-Polyradiculitis in dogs^10,11 and abortion in cows^12,13. Further, Neospora caninum exhibits morphological and phylogenetical close similarities of Toxoplasma gondii^9,14. Additionally, they have a nucleoside triphosphate hydrolase (NTPase; EC3.6.1.15)¹⁴. The enzymes are quite different from conventional ecto-ATPase¹⁴. These parasites generate a considerable amount of NTPase proteins, 2%-8% of the total protein and play an important role as dormant enzymes in their tachyzoite stage¹⁵. It should be noted that in dense secretory granules, these are condensed¹⁶ and secreted into the parasitophorous vacuole¹⁶. As a biochemical enzymatic character, NTPase is activated by dithiothreitol¹⁷. It is predicted that the inducers such as the dithiol compound, an unidentified enzyme such as dithiol-disulfide oxidoreductase, and another exhibit the same nature. They have not yet been identified in parasites. However, the enzyme does play an important role in releasing tachyzoite from infected host cells¹⁷.

Toxoplasma gondii has two NTPase isoforms¹⁸: type I enzyme TgNTPase-I, and type II enzyme TgNTPase-II¹⁸. The former preferentially utilizes triphosphate nucleosides as a substrate¹⁸. The latter hydrolyzes both triphosphate and diphosphate nucleosides¹⁸. The homology is 97% in amino acid levels¹⁸. Neospora caninum also has an orthologue of TgNTPase-I named NcNTPase¹⁹. The homology is 73% in amino acid levels¹⁹. Prof. Asai and Prof. Harada generated recombinants of both the NTPase using E. coli. and changed the constitutively active mutants of these as previously reported²⁰. They kindly gifted the two active mutants. Both enzymes can convert ATP to ADP in vitro²⁰. Very recently, we measured the activity of NTPase using ADP content hydrolyzed by the enzymes. Finally, we succeeded in establishing the high-standard assay through the process of determining ADP content with a combination of fluorescence and enzymatic reaction as previously reported^21,22. We also did high-throughput screening (HTS)²².

This study introduces detailed procedures of a novel high-accuracy and dynamic-range assay²¹ and a detailed explanation on how to prepare reagents to measure the enzyme activity and develop fluorescent intensity using a robot arm for HTS.

Protocol

1. Expression and purification of recombinant TgNTPase-I and NcNTPase

1. Prepare the expression plasmid and introduce it to the E. coli. strain BL21.
   ​NOTE: Detailed information on constructs and procedures is shown in a previous report¹⁴. In this study, both TgNTPase-I and NcNTPase constitutively active mutants were kindly gifted by Prof. Asai and Prof. Harada.

2. Preparation and placement of biofluorescent reaction solution onto the plate

1. Prepare 2x biofluorescent reaction solution as described in Table 1 for 2 mL of master mix for a 400-well assay in 384-well format.
2. Prepare stock solutions using distilled water (DW) for each indicated concentration with the exception of Resazurin and N-ethylmaleimide. Dissolve the remaining two reagents with DMSO in the appropriate concentration. Then, store the reagents at -30 °C until the time to conduct the experiments.
3. Add 15 µL of 2x biofluorescent reaction solution to each of the 368 wells (384 well format).
   ​NOTE: The first plate serves as a reservoir to have enough of the reagent to conduct experiments twice.

3. Put test compounds or extracts onto the bottom of each well in the assay plate

1. Put 0.5 µL of each compound onto the bottom of the plate using a robot arm from the library mother plate, including test compounds.
2. Add 0.5 µL of DMSO to both negative and positive control.

4. Preparation of enzyme reaction mixture and the beginning of the reaction

1. Prepare enzyme reaction solution as given in Table 2.
2. Add NTPase (Final concentration is 0.0002 µg/mL) to 50 mL of the enzyme reaction mixture, mix quickly and transfer to a plastic reservoir.
3. Proceed to simultaneously inject 4.5 µL into each well except for the negative control line using the robot arm.
4. Add the same amount of the enzyme reaction mixture with PBS instead of the enzyme preceded by the simultaneous injection.
5. Place the plate in an incubator at 37 °C for 10 min.

5. Conduct real-time measurement of enzymatic activities using a microplate reader

1. After 10 min of incubation, simultaneously add 5 µL of 2x biofluorescent reaction solution to each well immediately using the robot arm.
2. Temporarily stop the robot arm to hold the solution in each tip over the location where the plate is to be placed.
3. Once the assay plate is in place, restart the robot arm.
   NOTE: To avoid saturation of the generated ADP, immediately measure the fluorescent intensity.
4. Quickly spin down (200 x g for 1 min) the plate, and place the plate in the plate reader.
5. Start the real-time measurement of fluorescence at 540 nm/590 nm (excitation/emission) every minute for 1 h.

6. Analysis of results

1. Calculate values as the mean ± standard error of the mean (SEM) from the indicated and replicated samples in each experimental group; replicate experiments to ensure consistency.
2. Perform statistical significance using a Student's t-test.
3. Calculate values as statistically significant if the P values are P < 0.05.
4. Calculate Signal-to-Background ratio (S/B), Signal-to-Noise ratio (S/N) and Z'-factor using the following formulae:
   S/B = Average ligand/Average vehicle
   S/N = (Average ligand - Average vehicle)/Standard deviation vehicle
   Z'-factor = 1 - (3 x Standard deviation ligand + Standard deviation vehicle)/(Average ligand - Average vehicle)
5. Ensure that S/B, S/N, and Z'-factor are more than 3, 10 and 0.5, respectively.
   NOTE: Confirm whether each experiment meets the general criterion sufficient to do HTS as stated in previous reports^23,24.

Results

A principle of the assay is summarized in Supplementary Figure 1 and based on a previous report^12,18. The assay was designed in a 384-well format, as shown in Figure 1. The far-right and left lines were avoided on the plate. The two lines next to the far left and right lines were then used as negative control and positive control with or without the enzyme, respectively (n = 16). This allowed for the 320 compounds on...

Discussion

We succeeded in establishing a novel high-dynamic range and -accuracy assay with a combination of a classical enzyme assay and a fluorescent assay for ADP, which is the end product through ATPase, including Tg and Nc ATPase²². In order to carry out HTS, it is important that the assay has better values of S/B, S/N, and Z' factor than a classical enzyme assay^15,22. Additionally, omitting the step of stopping the enzyme reaction with an acid ...

Materials

Name	Company	Catalog Number	Comments
12 stage-workstation EDR-384 SX	Biotec Co., Ltd.	EDR-384SX	Robot arm Pippeting system
384 well tips	Biotech Co., Ltd.		Custom made
ADP-hexokinase	Asahi Kasei Pharma Co., Ltd.	T-92
ATP	Oriental Yeast, Co, Ltd.	45140000
BSA	Wako Pure Chemical Industries, Ltd.	011-15144
Diaphorase-I	Unitika Ltd.	Di-1
DMSO	Nacalai Tesch, Inc.	13406-55
G6P dehydrogenase	Oriental Yeast, Co, Ltd.	306-50143
Glucose	Wako Pure Chemical Industries, Ltd.	049-31165
Greiner 384 well micro-plate non-binding shallow well Black		#784900
HEPES	Wako Pure Chemical Industries, Ltd.	342-01375
Mg(CH3COO)2	Wako Pure Chemical Industries, Ltd.	130-00095
NADP	Oriental Yeast, Co, Ltd.	44332000
N-ethylmaleimide	Wako Pure Chemical Industries, Ltd.	056-02062
PHERAstar FS	BMG LABTECH JAPAN L.t.d.	PHERAstar FS	Multimode microplate reader
Resazurin	Wako Pure Chemical Industries, Ltd.	191-07581
Seahorse Labware 384 Well Low profile reservoirs		S30022 25/CS
TrisHCl	Wako Pure Chemical Industries, Ltd.	W01COBQE-4120
Triton X-100	Nacalai Tesch, Inc.	35501-02