Limited polymorphism in the dihydrofolate reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand
<p><i>Background:</i> The 2022 malaria WHO reported around 4000 <i>P. knowlesi</i> 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 i...
| Main Authors: | , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Elsevier
2023
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| _version_ | 1826311759530557440 |
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| author | Sangsri, R Choowongkomon, K Tuntipaiboontana, R Sugaram, R Boondej, P Sudathip, P Dondorp, AM Imwong, M |
| author_facet | Sangsri, R Choowongkomon, K Tuntipaiboontana, R Sugaram, R Boondej, P Sudathip, P Dondorp, AM Imwong, M |
| author_sort | Sangsri, R |
| collection | OXFORD |
| description | <p><i>Background:</i> The 2022 malaria WHO reported around 4000 <i>P. knowlesi</i> 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 (<i>pkdhfr</i>) and dihydropteroate synthase (<i>pkdhps</i>), putative antimalarial resistance markers for <i>P. knowlesi</i>. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating <i>pkdhps</i> mutations. The haplotype pattern of <i>pkdhfr</i>–<i>pkdhps</i> in Thai isolates was analyzed, and the effects of these <i>pkdhps</i> mutations were predicted by using a computer program.</p>
<p><i>Methods:</i> <i>Pkdhps</i> were amplified and sequenced from 28 <i>P. knowlesi</i> samples collected in 2008 and 2020 from nine provinces across Thailand. Combining <i>pkdhfr</i> sequencing data from previous work with <i>pkdhps</i> data to analyze polymorphisms of <i>pkdhfr</i> and <i>pkdhps</i> 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 <i>P. knowlesi</i> Malaysia isolates.</p>
<p><i>Results:</i> Five nonsynonymous mutations in the <i>pkdhps</i> 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 <i>pkdhfr</i>–<i>pkdhps</i>, the most common one is the R34L <i>pkdhfr</i> mutation and the <i>pkdhps</i> quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in <i>P. knowlesi</i> in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for <i>pkdhfr</i> and <i>pkdhps</i>, 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.</p>
<p><i>Conclusions:</i> A new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (<i>pkdhps</i>) has been developed. The most prevalent <i>pkdhfr</i>-<i>pkdhps</i> haplotypes (53.57%) in southern Thailand are R34L <i>pkdhfr</i> mutation and <i>pkdhps</i> quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.</p> |
| first_indexed | 2024-03-07T08:14:27Z |
| format | Journal article |
| id | oxford-uuid:aa280cf5-5b75-41e0-85f7-d3ceaea668c4 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T08:14:27Z |
| publishDate | 2023 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | oxford-uuid:aa280cf5-5b75-41e0-85f7-d3ceaea668c42023-12-18T17:22:38ZLimited polymorphism in the dihydrofolate reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from ThailandJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:aa280cf5-5b75-41e0-85f7-d3ceaea668c4EnglishSymplectic ElementsElsevier2023Sangsri, RChoowongkomon, KTuntipaiboontana, RSugaram, RBoondej, PSudathip, PDondorp, AMImwong, M<p><i>Background:</i> The 2022 malaria WHO reported around 4000 <i>P. knowlesi</i> 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 (<i>pkdhfr</i>) and dihydropteroate synthase (<i>pkdhps</i>), putative antimalarial resistance markers for <i>P. knowlesi</i>. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating <i>pkdhps</i> mutations. The haplotype pattern of <i>pkdhfr</i>–<i>pkdhps</i> in Thai isolates was analyzed, and the effects of these <i>pkdhps</i> mutations were predicted by using a computer program.</p> <p><i>Methods:</i> <i>Pkdhps</i> were amplified and sequenced from 28 <i>P. knowlesi</i> samples collected in 2008 and 2020 from nine provinces across Thailand. Combining <i>pkdhfr</i> sequencing data from previous work with <i>pkdhps</i> data to analyze polymorphisms of <i>pkdhfr</i> and <i>pkdhps</i> 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 <i>P. knowlesi</i> Malaysia isolates.</p> <p><i>Results:</i> Five nonsynonymous mutations in the <i>pkdhps</i> 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 <i>pkdhfr</i>–<i>pkdhps</i>, the most common one is the R34L <i>pkdhfr</i> mutation and the <i>pkdhps</i> quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in <i>P. knowlesi</i> in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for <i>pkdhfr</i> and <i>pkdhps</i>, 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.</p> <p><i>Conclusions:</i> A new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (<i>pkdhps</i>) has been developed. The most prevalent <i>pkdhfr</i>-<i>pkdhps</i> haplotypes (53.57%) in southern Thailand are R34L <i>pkdhfr</i> mutation and <i>pkdhps</i> quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.</p> |
| spellingShingle | Sangsri, R Choowongkomon, K Tuntipaiboontana, R Sugaram, R Boondej, P Sudathip, P Dondorp, AM Imwong, M Limited polymorphism in the dihydrofolate reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand |
| title | Limited polymorphism in the dihydrofolate reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand |
| title_full | Limited polymorphism in the dihydrofolate reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand |
| title_fullStr | Limited polymorphism in the dihydrofolate reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand |
| title_full_unstemmed | Limited polymorphism in the dihydrofolate reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand |
| title_short | Limited polymorphism in the dihydrofolate reductase (dhfr) and dihydropteroate synthase genes (dhps) of Plasmodium knowlesi isolate from Thailand |
| title_sort | limited polymorphism in the dihydrofolate reductase dhfr and dihydropteroate synthase genes dhps of plasmodium knowlesi isolate from thailand |
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