TY - JOUR
T1 - Limited Polymorphism in the Dihydrofolate Reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand
AU - Sangsri, Raweewan
AU - Choowongkomon, Kiattawee
AU - Tuntipaiboontana, Runch
AU - Sugaram, Rungniran
AU - Boondej, Patcharida
AU - Sudathip, Prayuth
AU - Dondorp, Arjen M.
AU - Imwong, Mallika
N1 - Funding Information: Ethical approval for the study was obtained from the ethical review committees of Faculty of Tropical Medicine, Mahidol University (MUTM2020-082-03 and MUTM2023-015-01). We thank Ms. Watcharee Pagornrat, Ms. Wanassanan Madmanee, Ms. Kanokon Suwannasin, and Ms. Jindarat Kouhathog for their help throughout the project. This research project was supported by Mahidol University, Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (NSRF) to MI, and part of the Mahidol-University Oxford Tropical Medicine Research Programme funded by the Wellcome Trust of the UK (core grant 106698/B/14/Z) and Wellcome OA statement. This research was funded in whole, or in part, by the Wellcome Trust [220211]. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. The funding sources did not participate in data analysis or the final decision to publish the manuscript. Funding Information: This research project was supported by Mahidol University, Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (NSRF) to MI, and part of the Mahidol-University Oxford Tropical Medicine Research Programme funded by the Wellcome Trust of the UK (core grant 106698/B/14/Z ) and Wellcome OA statement. This research was funded in whole, or in part, by the Wellcome Trust [220211]. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. The funding sources did not participate in data analysis or the final decision to publish the manuscript. Publisher Copyright: © 2023
PY - 2023/12/1
Y1 - 2023/12/1
N2 - Background: The 2022 malaria WHO reported around 4000 P. knowlesi infections in the South-East Asia region. In the same period, 72 positive cases were reported by the Department of Disease Control in Thailand, suggesting a persistent infection. Little is known about dihydrofolate reductase (pkdhfr) and dihydropteroate synthase (pkdhps), putative antimalarial resistance markers for P. knowlesi. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating pkdhps mutations. The haplotype pattern of pkdhfr–pkdhps in Thai isolates was analyzed, and the effects of these pkdhps mutations were predicted by using a computer program. Methods: Pkdhps were amplified and sequenced from 28 P. knowlesi samples collected in 2008 and 2020 from nine provinces across Thailand. Combining pkdhfr sequencing data from previous work with pkdhps data to analyze polymorphisms of pkdhfr and pkdhps haplotype. Protein modeling and molecular docking were constructed using two inhibitors, sulfadoxine and sulfamethoxazole, and further details were obtained through analyses of protein–ligand interactions by using the Genetic Optimisation for Ligand Docking program. A phylogenetic tree cluster analysis was reconstructed to compare the P. knowlesi Malaysia isolates. Results: Five nonsynonymous mutations in the pkdhps were detected outside the equivalence of the binding pocket sites to sulfadoxine and sulfamethoxazole, which are at N391S, E421G, I425R, A449S, and N517S. Based on the modeling and molecular docking analyses, the N391S and N517S mutations located close to the enzyme-binding pocket demonstrated a different docking score and protein–ligand interaction in loop 2 of the enzyme. These findings indicated that it was less likely to induce drug resistance. Of the four haplotypes of pkdhfr–pkdhps, the most common one is the R34L pkdhfr mutation and the pkdhps quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in P. knowlesi in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for pkdhfr and pkdhps, the samples isolated from eastern Thailand displayed a close relationship with Cambodia isolates, while southern Thailand isolates showed a long branch separated from the Malaysian isolates. Conclusions: A new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (pkdhps) has been developed. The most prevalent pkdhfr-pkdhps haplotypes (53.57%) in southern Thailand are R34L pkdhfr mutation and pkdhps quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.
AB - Background: The 2022 malaria WHO reported around 4000 P. knowlesi infections in the South-East Asia region. In the same period, 72 positive cases were reported by the Department of Disease Control in Thailand, suggesting a persistent infection. Little is known about dihydrofolate reductase (pkdhfr) and dihydropteroate synthase (pkdhps), putative antimalarial resistance markers for P. knowlesi. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating pkdhps mutations. The haplotype pattern of pkdhfr–pkdhps in Thai isolates was analyzed, and the effects of these pkdhps mutations were predicted by using a computer program. Methods: Pkdhps were amplified and sequenced from 28 P. knowlesi samples collected in 2008 and 2020 from nine provinces across Thailand. Combining pkdhfr sequencing data from previous work with pkdhps data to analyze polymorphisms of pkdhfr and pkdhps haplotype. Protein modeling and molecular docking were constructed using two inhibitors, sulfadoxine and sulfamethoxazole, and further details were obtained through analyses of protein–ligand interactions by using the Genetic Optimisation for Ligand Docking program. A phylogenetic tree cluster analysis was reconstructed to compare the P. knowlesi Malaysia isolates. Results: Five nonsynonymous mutations in the pkdhps were detected outside the equivalence of the binding pocket sites to sulfadoxine and sulfamethoxazole, which are at N391S, E421G, I425R, A449S, and N517S. Based on the modeling and molecular docking analyses, the N391S and N517S mutations located close to the enzyme-binding pocket demonstrated a different docking score and protein–ligand interaction in loop 2 of the enzyme. These findings indicated that it was less likely to induce drug resistance. Of the four haplotypes of pkdhfr–pkdhps, the most common one is the R34L pkdhfr mutation and the pkdhps quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in P. knowlesi in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for pkdhfr and pkdhps, the samples isolated from eastern Thailand displayed a close relationship with Cambodia isolates, while southern Thailand isolates showed a long branch separated from the Malaysian isolates. Conclusions: A new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (pkdhps) has been developed. The most prevalent pkdhfr-pkdhps haplotypes (53.57%) in southern Thailand are R34L pkdhfr mutation and pkdhps quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.
KW - Plasmodium knowlesi
KW - pkdhfr
KW - pkdhps
UR - http://www.scopus.com/inward/record.url?scp=85171453632&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.actatropica.2023.107016
DO - https://doi.org/10.1016/j.actatropica.2023.107016
M3 - Article
C2 - 37683820
SN - 0001-706X
VL - 248
JO - Acta tropica
JF - Acta tropica
M1 - 107016
ER -