Life science
Global health

Development of the method for identifying drug
resistance gene from Malaria parasites
Department of Molecular protozoology, Research Institute for Microbial Diseases
Professor Shiroh Iwanaga
https://researchmap.jp/shirohiwanaga?lang=en

Life science

Abstract
Drug resistance is one of the biggest threats to malaria control.
The identification of drug resistance genes not only assists for elucidating the mechanism of resistance, but also is useful for monitoring the spread of resistance. We developed a method for rapid
identification of drug resistance genes by functional screening
using Plasmodium falciparum artificial chromosome technology.
We further succeeded in identifying a new mefloquine resistancegene from the drug-resistant parasite isolated from malaria patientsin the border area between Thai-Myanmar by using the developed
method, demonstrating its feasibility.

▍ Background & Results
Malaria patients are currently treated using artemisinin combination therapy (ACT), in which artemisinin is used as a first-line
drug along with partner drugs. ACT has greatly contributed to thedecrease in malaria deaths over the past two decades; however,since 2009, treatment failure caused by resistance to the drugs
used during ACT has been reported in endemic areas includingSouth-East Asia and Africa. Therefore, there is concern that thetherapeutic effects of ACT may be decreasing.
Identification of drug-resistance genes contributes to not only
the elucidation of molecular mechanisms of resistance but also thedevelopment of molecular markers for surveillance; thus, it offers
one potential solution to the problem of drug resistance. However,current method for identifying drug resistance gene using next
generation sequencing technology is laborious and time consuming. To solve this technical limitation, we developed a method toidentify resistance genes by functional screening; a high-coverage
genomic library of a drug-resistant strain is directly generated ina drug-sensitive strain, and the resistance gene is then identified
from this library using drug screening (Fig. 1). In this method, aparasite clone, in which a drug-resistant gene from a resistantparasite is introduced, acquires newly resistance and can survive
during screening with drug. In contrast, other clones, in whichgenes unrelated to resistance are introduced, remains susceptible
and is killed by screening. Indeed, this method was applied to mefloquine-resistant Plasmodium parasites with unknown resistancegenes, and a novel transporter gene (multidrug resistance protein 7,PfMDR7) was successfully identified as a resistance gene, demonstrating its utility (Fig. 2).

!"#$%"&'(')*+),*"*'()&%-.&//012!"#$%"&'(')*+)%$&+&!"#AB>&+)"0;&"&,=>?8@4

345%67%!89%:"*$;&+)'-,*")(*/%<($&')(0+2?/&C)"0,0"*)(0+!"#$%'&+'()(@&,*"*'()&D*"*'()&%*CE#("(+$%<"#$%"&'(')*+C&-<"#$%"&'(')*+)%,*"*'()&F%.&//012$%&'()*+&<"#$%'C"&&+(+$G#"@(@(+$%,*"*'()&'

H&C0@&"&<%,=>?8@4%:"0;%,*"*'()&'%'&/&C)&<%7.%:#+C)(0+*/%'C"&&+(+$Figure 1: Schematic of the method for identification of drug resistance genes fromP. falciparum.
Yellow and blue indicate drug-resistant and -susceptible parasites, respectively.
In the presence of drug, the parasite (yellow), which acquires resistance newly by
being introduced drug resistance gene, can survived. The drug resistance genecan be identified by analyzing the insert DNA fragment of artificial chromosome
recovered from those survived parasites.

▍ Significance of the research and Future perspective
The method developed is totally different in principle from thecurrent method using next-generation sequencing technology. The
rapidity of the method is particularly noteworthy; drug resistancegenes can be identified within only 2 to 3 months. In addition, only
one strain of drug resistant parasite from a patient is enogh to forgene identification, and it is expected to enable the monitoring andcontainment of resistance before it spreads globally. In the future, itwill be essential to monitor the emergence of resistance along withthe development of new drugs, which is expected to make a significant contribution to the advancement of malaria control.

Figure 2: Identification of new mefloquine resistance genes.
Four independent DNA fragments were identified as a result of mefloquinescreening, and all encoded the specific region on chromosome 12. Pf MDR7(yellow) was identified from the common region in those four fragments.

P a t e n t Japanese Patent No.5773447
Iwanaga, Shiroh; Kubota, Rie; Nishi, Tsubasa et al. Genome-wide functional screening of drug-resistance genes in Plasmodium falciparum. Nat Commun.
Tr e a t i s e
2022,13(1):6163. doi: 10.1038/s41467-022-33804-w
URL
K e y w o r d malaria, drug resistance gene, artificial chromosome