21-July-2023

A genetically engineered Plasmodium falciparum expressing NanoLuc
as an innovative tool for malaria drug discovery.
【Summary】
Plasmodium falciparum infects two types of hosts, human and vector mosquitoes.

We have created a transgenic P. falciparum that expresses two reporter proteins, GFP andNanoLuc, at high levels in both the human- and the vector mosquito stage.

We demonstrated that the generated parasites can be used to evaluate antimalarial compounds inmulti-stage.

Our study provides a robust platform for developing anti-malarial drugs that are effective in multiplestages.

【Abstract】
This study has generated a new genetically modified Plasmodium falciparum reporter line that
could contribute to developing new anti-malarial drugs. This transgenic P. falciparum reporter line
constantly expresses two reporter proteins, GFP and NanoLuc, at high levels in two different hosts,
human and mosquito. By utilising the expression of these two reporter proteins, P. falciparum can be
successfully visualised and quantified with high sensitivity. Furthermore, by using NanoLuc-derivedsignals, we have demonstrated the utility of the reporter line to evaluate the drug efficacy of anti-malarial
compounds that are effective in multiple stages in the parasite life cycle. Using this drug platform, weidentified a new compound, OU0074008, which kills the asexual blood stages of P. falciparum from alibrary of 1920 compounds provided by Osaka University. This study suggests that a drug discovery
platform using P. falciparum expressing NanoLuc will greatly advance the development of anti-malarialdrugs.
The study was carried out by a research team led by Assistant Professor Shinya Miyazaki andResearch Fellow Yukiko Miyazaki (currently Assistant Professor, Department of Protozoology) andAssociate Professor Daniel Ken Inaoka from Nagasaki University, Institute of Tropical Medicine, Shionogi
Global Infectious Diseases Division. The research is also an international collaboration with Leiden
University and TropIQ in the Netherlands, Sorbonne University in France, and Graduate School of
Pharmaceutical Sciences, Osaka University. This study has been published in the journalCommunications Biology (doi: 10.1038/s42003-023-05078-5, Link:
https://www.nature.com/articles/s42003-023-05078-5).

【Introduction】
Malaria is a protozoan infectious disease transmitted by mosquitoes in tropical areas of Africa
and South-East Asia that causes an enormous number of infections and deaths. Malaria symptoms suchas fever and chills are caused by the multiplication of P. falciparum in human red blood cells (asexualblood stage). On the other hand, some portion of the parasites in the red blood cells differentiates to astage called the gametocyte stage and are transferred into the mosquito's midgut by mosquito bloodsucking. The malaria parasites that have multiplied in the mosquito host developed into a stage called
the sporozoite stage, which again enters the human body through the mosquito's bite. The infectedsporozoites first infect the human liver, where they undergo an incubation period before infecting human
red blood cells. Thus, P. falciparum has a very sophisticated parasitic strategy for infecting two hosts,
humans and mosquitoes (Fig.1).
To treat malaria, therapeutic agents have been used to kill P. falciparum in the red blood cells.
However, resistance to existing anti-malarial drugs is emerging, and developing anti-malarial drugs withnew modes of action is desired. P. falciparum is transmitted by mosquitoes, so drugs that prevent thespread of the parasites into the mosquito host (transmission inhibitors) are considered essential for
malaria control. Furthermore, as there is an incubation period of one to two weeks in the liver after
infection of humans by mosquito bites, killing the parasites in the liver can prevent malaria onset.
Therefore, anti-malarial drugs that are effective in multiple stages are expected to contribute to malaria
control as they can be effective as therapeutic, prophylactic or transmission inhibitors (Fig.1).

Fig.1 The whole life cycle of P. falciparum and desired anti-malarial drugs.

【Results】
In this study, we genetically modified P. falciparum to simultaneously express GFP, a fluorescentprotein, and NanoLuc, a luciferase. The life cycle of P. falciparum was recapitulated in the laboratory, andthe expression of GFP was confirmed in the asexual blood-, gametocyte-, oocyst-, sporozoite- and liverstages (Fig. 2). These results indicate that microscopic observation of GFP fluorescence signals
facilitates the observation of Plasmodium morphology and numbers in the multiple stages.

Fig.2 GFP expression in P. falciparum in the multiple stages of the life cycle.
The NanoLuc signal, expressed simultaneously with GFP, was then used to evaluate antimalarial drugs. P. falciparum in the asexual blood stage was seeded onto plates to which antimalarial
compounds had been added, and the NanoLuc signal can be detected to determine the growth-inhibitory
activity of a given compound. We found that as the concentration of the compound increased, the degreeof inhibition of Plasmodium growth also increased (Fig.3). By applying this system to the gametocyte andliver stages and detecting the NanoLuc signal, we show that it is possible to evaluate anti-malarial
activity not only in the asexual blood stage but also in the gametocyte and intrahepatic phases. The
series of experiments suggested that it is possible to evaluate antimalarial compounds at multiple stages
using the NanoLuc-expressing parasites.

Fig.3 Evaluation of anti-malarial compounds using asexual blood stages of NanoLuc line.

【Discussion and future perspectives】
In this study, we established a new reporter, P. falciparum expressing the highly sensitive
NanoLuc in both stages of humans and mosquitoes and a new drug discovery platform. By utilising thesignals of GFP and NanoLuc, which are expressed in multiple stages, it is expected to greatly promote
drug discovery research and basic research on P. falciparum itself. In the future, this study will be applied
to the development of new anti-malarial drugs through high-throughput screening using NanoLucexpressing P. falciparum and evaluation of the anti-malarial effects on P. falciparum in the multiplestages.
The transgenic P. falciparum reporter line developed in this study is available for transfer andcollaboration with academia and drug discovery researchers upon reasonable request. If you areinterested in using this reporter Plasmodium parasites, we would be happy to hear from you with thecontact details at the end of this document.
【Glossary】
Malaria
Malaria infections caused by Plasmodium spp. Several species of Plasmodium are known tocause malaria in humans, this study focused on Plasmodium falciparum, which causes the most severe
symptoms in humans. This parasite is transmitted from humans to mosquitoes by the blood-sucking ofvector mosquitoes, multiplies in the mosquito's body and then infects other humans again by themosquito's bite. Therefore, there is a need for a drug that is effective against both parasites in two hosts,
humans and mosquitoes.
Fluorescent protein
Proteins that emit fluorescence represented by green fluorescent protein (GFP). When afluorescent protein is exposed to light of a specific wavelength, it emits fluorescence of a different
wavelength. The location and amount of fluorescent protein in the cell can be determined using particularmachines to detect the fluorescence.
Luciferase
Proteins that emit luminescence through an enzymatic reaction, as represented by firefly
luciferase. When luciferase reacts with a specific luminescent substance, the enzymatic reaction
proceeds and light of a particular wavelength is emitted. The amount of luciferase can be measured bydetecting the amount of this emitted light with a specific machine. In this study, NanoLuc, a small sizeand highly sensitive luciferase, was used.
High throughput screening
One of the methods of compound discovery (screening) used to find drugs with a particulareffect. It is a step in which thousands to tens of thousands of compounds are evaluated using a specificassay system to identify compounds with the desired effect. In drug discovery research, it is important tofind promising lead compounds at this stage and proceed to further developmental phases.
【Acknowledgement】
We would like to take this opportunity to express our deepest gratitude to all those involved inthis study. This paper is dedicated to the memory of our friend and colleague, Dr. Shahid Khan, whopassed away on the 4th of October, 2019. This work was supported by the European Union's Horizon
2020 Research and Innovation Program under grant agreement No. 733273, and SHIONOGI & Co. Ltd.
The chemical library used in this study was provided by the Platform Project for Supporting Drug
Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life ScienceResearch (BINDS)) from AMED under grant number JP21am0101001(support number 3361).
【Papers】
Journal：Communications Biology
Title：A versatile Plasmodium falciparum reporter line expressing NanoLuc enables highly
sensitive multi-stage drug assays.
Authors
Yukiko Miyazaki1,2*♰, Martijn W. Vos3, Fiona J.A. Geurten2, Pierre Bigeard4, Hans Kroeze2, Shohei
Yoshioka5, Mitsuhiro Arisawa5, Daniel Ken Inaoka1,6,7, Valerie Soulard4, Koen J. Dechering3, BlandineFranke-Fayard2, Shinya Miyazaki2,8*1Department of Molecular Infection Dynamics, Institute of Tropical Medicine (NEKKEN), Nagasaki

University, 852-8523, Nagasaki, Japan.
2Department of Parasitology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands.
3TropIQ Health Sciences, Transistorweg 5, 6534 AT, Nijmegen, The Netherlands.
4Sorbonne Université, Inserm, CNRS, Centre d'Immunologie et des Maladies Infectieuses, CIMI-Paris, F-75013 Paris, France.
5Graduate School of Pharmaceutical Sciences, Osaka University, 565-0871, Osaka, Japan.
6School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki 852-8523, Japan.
7Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
8Department of Cellular Architecture Studies, Institute of Tropical Medicine (NEKKEN), 852-8523,Nagasaki, Nagasaki University, Japan.
♰Present address: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki

University, Nagasaki, Japan.
(doi: 10.1038/s42003-023-05078-5, Link: https://www.nature.com/articles/s42003-023-05078-5)*Corresponding author, E-mail: y.miyazaki@nagasaki-u.ac.jp or smiyazaki@nagasaki-u.ac.jp

【Contact】
Yukiko Miyazaki, Ph.D.
Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University
Assistant Professor
Email: y.miyazaki@nagasaki-u.ac.jp
Shinya Miyazaki, Ph.D.
Department of Cellular Architecture Studies, Institute of Tropical Medicine (NEKKEN), Nagasaki
University
Assistant Professor
Email: smiyazaki@nagasaki-u.ac.jp
Mitsuhiro Arisawa, Ph.D.
Graduate School of Pharmaceutical Sciences, Osaka University
Professor
Email: arisaw@phs.osaka-u.ac.jp