Summary

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

Published: July 22, 2022
doi:

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 HTS1,2,3. 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 dogs10,11 and abortion in cows12,13. Further, Neospora caninum exhibits morphological and phylogenetical close similarities of Toxoplasma gondii9,14. 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 reported21,22. 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 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 r…

Representative Results

A principle of the assay is summarized in Supplementary Figure 1 and based on a previous report12,18. 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 assay15,22. Additionally, omitting the step of stopping the enzyme reaction with an acid …

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

This work was partly supported by the Platform for Drug Discovery, Informatics and Structural Life Science, a Grant-in-Aid for Scientific Research (C) from Japan Society for Promotion of Science (JSPS-21K06566). The authors sincerely thank Asai (Keio University School of medicine) and Harada (Kyoto Institute of Technology) and Stephen Stratton for gifting recombinant two NTPase active mutants and his contribution in the preparation of this manuscript, respectively.

Materials

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

Referencias

  1. Bianca, C. B., et al. A robotic platform to screen aqueous two-phase systems for overcoming inhibition in enzymatic reactions. Bioresource Technology. 280, 37-50 (2019).
  2. Aliaksei, V., Jan, D. B. Robot-scientists will lead tomorrow’s biomaterials discovery. Current Opinion in Biomedical Engineering. 6, 74-80 (2018).
  3. Mandeep, D., Kusum, P., Dharini, P., Nikolaos, E. L., Pratyoosh, S. Robotics for enzyme technology: innovations and technological perspectives. Applied Microbiology and Biotechnology. 105, 4089-4097 (2021).
  4. Dubey, J. P. . Toxoplasmosis of Animals and Man. , (1988).
  5. Michael, C., Sneller, H., Clifford, L. . Infections in the Immunocompromised Host in Clinical Immunology., 3rd ed. , (2008).
  6. Schäfer, G., et al. Immediate versus deferred antiretroviral therapy in HIV-infected patients presenting with acute AIDS-defining events (toxoplasmosis, Pneumocystis jirovecii-pneumonia): A prospective, randomized, open-label multicenter study (IDEAL-study). AIDS Research and Therapy. 16, 34 (2019).
  7. Ramanan, P., et al. Toxoplasmosis in non-cardiac solid organ transplant recipients: A case series and review of literature. Transplant Infectious Disease. 26, 13218 (2019).
  8. Rivera, E. M., et al. Toxoplasma gondii seropositivity associated to peri-urban living places in pregnant women in a rural area of Buenos Aires province, Argentina. Parasite Epidemiology and Control. 7, 00121 (2019).
  9. Donahoe, S. L., Lindsay, S. A., Krockenberger, M., Phalen, D., Šlapeta, J. A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife. International Journal of Parasitology: Parasites and Wildlife. 4, 216-238 (2015).
  10. Crookshanks, J. L., Taylor, S. M., Haines, D. M., Shelton, G. D. Treatment of canine pediatric Neospora caninum myositis following immunohistochemicalidentification of tachyzoites in muscle biopsies. TheCanadian Veterinary Journal. 48, 506-508 (2007).
  11. Bartner, L. R., et al. Testing for Bartonella ssp. DNA in cerebrospinal fluid of dogs with inflammatory central nervous system disease. Journal of Veterinary Internal Medicine. 32, 1983-1988 (2018).
  12. Changoluisa, D., Rivera-Olivero, I. A., Echeverria, G., Garcia-Bereguiain, M. A., de Waard, J. H. Working group "Applied Microbiology" of the School of Biological Sciences and Engineering at Yachay Tech University. Serology for Neosporosis, Q fever and Brucellosis to assess the cause of abortion in two dairy cattleherds in Ecuador. BMC Veterinary Research. 15, 194 (2019).
  13. Serrano-Martínez, M. E., et al. Evaluation of abortions spontaneously induced by Neospora caninum and risk factors in dairy cattle from Lima, Peru. Revista Brasileira de Parasitologia Veterinaria. 28, 215-220 (2019).
  14. Matoba, K., et al. Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii. Acta Crystallographica Section F Structural Biology Communications. 66, 1445-1448 (2010).
  15. Nakaar, V., Beckers, C. J., Polotsky, V., Joiner, K. A. Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase. Molecular and Biochemical Parasitology. 97, 209-220 (1998).
  16. Pastor-Fernández, I., et al. The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granuleprotein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress. Parasites and Vectors. 9, 352 (2016).
  17. Silverman, J. A., et al. Induced activation of the Toxoplasma gondii nucleoside triphosphate hydrolase leads to depletion of host cell ATP levels and rapid exit of intracellular parasites from infected cells. Journal of Biological Chemistry. 273, 12352-12359 (1998).
  18. Olias, P., Sibley, L. D. Functional analysis of the role of Toxoplasma gondii nucleoside triphosphate hydrolases I and II in acute mouse virulence and immune suppression. Infection and Immunity. 84, 1994-2001 (2016).
  19. Leineweber, M., et al. First Characterization of the Neospora caninum Dense Granule Protein GRA9. BioMed Research International. 2017, 6746437 (2017).
  20. Krug, U., Zebisch, M., Krauss, M., Sträter, N. Structural insight into activation mechanism of Toxoplasma gondii nucleoside triphosphatediphosphohydrolases by disulfide reduction. Journal of Biological Chemistry. 287, 3051-3066 (2012).
  21. Kumagai, K., Kojima, H., Okabe, T., Nagano, T. Development of a highly sensitive, high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection. Analytical Biochemistry. , 146-155 (2014).
  22. Harada, M., et al. Establishment of novel high-standard chemiluminescent assay for NTPase in two protozoans and its high-throughput screening. Marine Drugs. 18, 161 (2020).
  23. Kurata, R., et al. Establishment of novel reporter cells stably maintaining transcription factor-driven human secreted alkaline phosphatase expression. Current Pharmaceutical Biotechnology. 19, 224-231 (2018).
  24. Zhang, J. H., Chung, T. D. Y., Oldenburg, K. R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening. 4, 67-73 (1999).
  25. Nakajima-Nakano, K., Makioka, A., Yamashita, N., Matsuo, N., Asai, T. Evaluation of serodiagnosis of toxoplasmosis by using the recombinant nucleoside triphosphate hydrolase isoforms expressed in Escherichia coli. Parasitology International. 48, 215-222 (2000).
This article has been published
Video Coming Soon
Keep me updated:

.

Citar este artículo
Kurata, R., Harada, M., Nagai, J., Cui, X., Isagawa, T., Semba, H., Yoshida, Y., Takeda, N., Maemura, K., Yonezawa, T. Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm. J. Vis. Exp. (185), e62874, doi:10.3791/62874 (2022).

View Video