Skip to main content
Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Genetic Variability of Plasmodium malariae dihydropteroate synthase (dhps) in Four Asian Countries

  • Naowarat Tanomsing,

    Affiliation Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

  • Mayfong Mayxay,

    Affiliations Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR, Faculty of Postgraduate Studies, University of Health Sciences, Vientiane, Lao PDR, Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

  • Paul N. Newton,

    Affiliations Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU), Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao PDR, Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

  • Francois Nosten,

    Affiliations Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Bangkok, Thailand

  • Christiane Dolecek,

    Affiliations Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam

  • Tran Tinh Hien,

    Affiliations Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam

  • Nicholas J. White,

    Affiliations Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

  • Nicholas P. J. Day,

    Affiliations Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

  • Arjen M. Dondorp,

    Affiliations Centre for Tropical Medicine, Nuffield Department of Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

  • Mallika Imwong

    noi@tropmedres.ac

    Affiliation Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

Abstract

The dihydropteroate synthase (dhps) genes of 44 P. malariae strains from four Asian countries were isolated. Only a limited number of polymorphisms were observed. Comparison with homologous mutations in other Plasmodium species showed that these polymorphisms are unlikely to be associated with sulfadoxine resistance.

Introduction

Molecular characterizations of antifolate resistance in Plasmodium falciparum and P. vivax have revealed stepwise increase in antifolate resistance with additional mutations within the dihydrotheorate synthase (dhps) and dihydrofolate reductase (dhfr) genes [1], [2], [3], [4]. Although patients infected with P. malariae are usually not treated with antifolate drugs, exposure is still likely in endemic areas where sulfadoxine - pyrimethamine is used for treatment of falciparum malaria, because of the high frequency of mixed infections. The previously reported S114N mutation within the dhfr gene of P. malariae supports this [5], [6]. The P. malariae gene for dhps, the target for sulfadoxine, has not been studied to date.

Materials and Methods

In this study, the dhps gene of 44 P. malariae strains from Asian clinical samples (34 from Thailand, 6 from Laos, 3 from Viet Nam, and 1 from Bangladesh) were isolated and analysed. Genomic DNA of P. brasilianum, a New World monkey malaria parasite considered to be genetically indistinguishable from P. malariae, purified from a cloned line maintained in Saimiri monkeys was obtained from MR4 (MR4-349). Samples were obtained from patients enrolled in a variety of previous treatment studies. All patients provided written informed consent. The protocol for this study was reviewed and approved from the Faculty of Tropical Medicine, Mahidol University, Thailand (reference no. MUTM2011-049-03). To design primers for isolation of the dhps gene from P. malariae and P. brasilianum, the nucleotide sequences of dhps gene from other human Plasmodium spp. were aligned, including P. falciparum (accession number XM_001349382), P. vivax (accession number XM_001617159), and P. knowlesi (accession number XM_002262146). A nested and seminested PCR approach was used to increase sensitivity of the amplification product. The sequences of primers, Mg2+concentrations, annealing temperatures, numbers of cycles, and sizes of products were individually determined for the different primer pairs shown in Table 1. Amplified PCR products were then cloned into the pCR 2.1 vector (Invitrogen, USA), and the plasmids purified from the bacterial clones were submitted for DNA sequencing.

thumbnail
Table 1. Primer sequences and PCR condition for isolation of pppk-dhps gene from P. malariae and P. brasilianum.

https://doi.org/10.1371/journal.pone.0093942.t001

Results and Discussion

The partial pppk-dhps gene was isolated from both P. malariae (1,953 bp) and P. brasilianum (1,914 bp). The partial 1,953 bp Pmpppk-dhps included one intron (167 bp) at the carboxyl terminus coding region, which position and size was established by reverse-transcriptase PCR amplification and sequencing from corresponding mRNA. DNA sequences of pppk-dhps are available from 3 other species infecting humans: P. falciparum, P. vivax and P. knowlesi. In all species the pppk-dhps gene has two introns, one of which is located near the N-terminus coding region of the pppk gene and one near the C-terminal. This study revealed an 167 bp intron within dhps gene of P. malariae. AT content of the pppk-dhps gene from P. malariae, P. brasilianum and P. falciparum was high (72.2–76.5%), whereas this is low in P. vivax and P. knowlesi (56.8% and 62.2% respectively). The pppk-dhps gene of P. falciparum appeared to be located on chromosome 8, whereas in P. vivax and P. knowlesi this is on chromosome 14.

Specific primers were designed to isolate the Pmdhps gene from all 44 P. malariae isolates. In the amplified product 1,140 bp (corresponding to 379 amino acids) of Pmdhps were analyzed, which revealed 2 and 3 positions of synonymous and nonsynonymous mutations which were predicted to change amino acids located outside the binding pocket of the enzyme for sulfadoxine. This was assessed through amino acid alignment with DHPS of P. falciparum and P. vivax (Figure 1), showing that equivalent variation in these other species are not associated with changes in the binding pocket for sulfadoxine, nor with sulfadoxine resistance. The 44 Pmdhps sequences could be categorized into 4 haplotypes; the wild type haplotype 1 was most prevalent (41/44 isolates; Table 2).

thumbnail
Figure 1. Deduced partial PPPK-DHPS amino acid sequences alignment.

https://doi.org/10.1371/journal.pone.0093942.g001

thumbnail
Table 2. Nonsynonymous mutations observed in P. malariae dhps.

https://doi.org/10.1371/journal.pone.0093942.t002

The current study investigated 27 isolates from which we have previously reported details on dhfr gene polymorphisms [6]. Two of these isolates contained the S114N-mutation within dhfr, which are predicted to confer antifolate resistant, and thus suggest antifolate drug pressure on the parasite populations. However, these dhfr mutations were not accompanied by mutations in the dhps gene thought to confer sulfadoxine resistance: one of these two samples contained wild type dhps gene, while the other strain showed 2 SNPs (one synonymous and one nonsynonymous). The nonsysnonymous mutation was located at the equivalent position 516 in P. falciparum and 488 in P. vivax, which does not code for the enzyme binding pocket. The dhfr gene sequences were then assessed in the 17 P. malariae isolates from Thailand, none of which showed mutations, and all were classified as haplotype 4 [6]. Thus far all available published and unpublished Pmdhfr sequences [5], [6] show a high prevalence of the wild type gene. Homology with other Plasmodium species suggest that the initial mutation associated with drug pressure may be at position S114N. In order to assess this point mutation more efficiently (analogous to S108N and S117N in P. falciparum and P. vivax), a PCR-RFLP method was developed and investigated. Patterns of wild type S114 contain two bands of 401 bp and 172 bp+168 bp product, and mutant type 114N showed two bands of 569 bp and 172 bp product. Forty-four samples of P. malariae were tested and PCR-RFLP results were in accordance with the sequence data. Using PCR-RFLP, we analysed 17 isolates recently collected in Thailand which revealed no mutations, with all strains classified as the common haplotype 4 [6].

Comparing homology of dhps genes isolated from P. falciparum, P. vivax, P. knowlesi and P. malariae has some limitations. Amino acid alignment of DHPS in each Plasmodium spp. determined equivalent position of nonsynonnymous mutations in association to sulfadoxine resistance in P. falciparum and P. vivax (Table 2). All 44 isolates of P. malariae showed wild type amino acids at these equivalent positions, whereas the three nonsynonymous mutations present in three P. malariae isolates have not been described in these other species. They are equivalent to T537, L516 and E339 in P. falciparum (Table 2). Amino acids found in P. vivax and P. knowlesi at these three residues are the same as in P. falciparum, suggesting that these three residues are conserved among Plasmodium spp. It is therefore interesting to construct an in silico comparative structure model to further explore whether the nonsynonymous mutations found in P. malariae at these positions have an effect on molecular docking of sulfadoxine.

Our findings suggest that drug pressure of sulphadoxine-pyrimethamine on P. malariae in the region has been lower than on P. falciparum or P. vivax. Mutations in the PmDHPS and PmDHFR genes are rare in Thailand, suggesting absence of selection of SP resistant parasite populations. We plan to extend our observations to P. malariae isolates collected from African countries where SP drug pressure is more prominent and where prevalence of P. malariae is higher compared to Southeast Asia. The nonsynonymous mutations identified in PmDHPS and PmDHFR require further in vivo and in vitro studies to elucidate their significance in SP resistance in P. malariae.

Acknowledgments

We would like to thank MR4 for the provision of Plasmodium brasilianum.

Author Contributions

Conceived and designed the experiments: NT NJW NPJD AMD MI. Performed the experiments: NT. Analyzed the data: NT NJW NPJD AMD MI. Contributed reagents/materials/analysis tools: MM PNN FN CD TTH. Wrote the paper: NT MM PNN AMD MI.

References

  1. 1. Imwong M, Pukrittakayamee S, Looareesuwan S, Pasvol G, Poirreiz J, et al. (2001) Association of genetic mutations in Plasmodium vivax dhfr with resistance to sulfadoxine-pyrimethamine: geographical and clinical correlates. Antimicrob Agents Chemother 45: 3122–3127.
  2. 2. Imwong M, Pukrittayakamee S, Renia L, Letourneur F, Charlieu JP, et al. (2003) Novel point mutations in the dihydrofolate reductase gene of Plasmodium vivax: evidence for sequential selection by drug pressure. Antimicrob Agents Chemother 47: 1514–1521.
  3. 3. Plowe CV, Cortese JF, Djimde A, Nwanyanwu OC, Watkins WM, et al. (1997) Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance. J Infect Dis 176: 1590–1596.
  4. 4. Zolg JW, Plitt JR, Chen GX, Palmer S (1989) Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum. Mol Biochem Parasitol 36: 253–262.
  5. 5. Khim N, Kim S, Bouchier C, Tichit M, Ariey F, et al. (2012) Reduced impact of pyrimethamine drug pressure on Plasmodium malariae dihydrofolate reductase gene. Antimicrob Agents Chemother 56: 863–868.
  6. 6. Tanomsing N, Imwong M, Pukrittayakamee S, Chotivanich K, Looareesuwan S, et al. (2007) Genetic analysis of the dihydrofolate reductase-thymidylate synthase gene from geographically diverse isolates of Plasmodium malariae. Antimicrob Agents Chemother 51: 3523–3530.