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

Diversifying Selection on the Thrombospondin-Related Adhesive Protein (TRAP) Gene of Plasmodium falciparum in Thailand

  • Jun Ohashi ,

    juno-tky@umin.ac.jp

    Affiliation Molecular and Genetic Epidemiology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

  • Yuji Suzuki,

    Affiliation Molecular and Genetic Epidemiology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

  • Izumi Naka,

    Affiliation Molecular and Genetic Epidemiology, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

  • Hathairad Hananantachai,

    Affiliation Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

  • Jintana Patarapotikul

    Affiliation Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

Diversifying Selection on the Thrombospondin-Related Adhesive Protein (TRAP) Gene of Plasmodium falciparum in Thailand

  • Jun Ohashi, 
  • Yuji Suzuki, 
  • Izumi Naka, 
  • Hathairad Hananantachai, 
  • Jintana Patarapotikul
PLOS
x

Abstract

Sporozoites of Plasmodium falciparum are transmitted to human hosts by Anopheles mosquitoes. Thrombospondin-related adhesive protein (TRAP) is expressed in sporozoites and plays a crucial role in sporozoite gliding and invasion of human hepatocytes. A previous study showed that the TRAP gene has been subjected to balancing selection in the Gambian P. falciparum population. To further study the molecular evolution of the TRAP gene in Plasmodium falciparum, we investigated TRAP polymorphisms in P. falciparum isolates from Suan Phueng District in Ratchaburi Province, Thailand. The analysis of the entire TRAP coding sequences in 32 isolates identified a total of 39 single nucleotide polymorphisms (SNPs), which comprised 37 nonsynonymous and two synonymous SNPs. McDonald–Kreitman test showed that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. falciparum was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. falciparum and P. reichenowi. Furthermore, the rate of nonsynonymous substitution was significantly higher than that of synonymous substitution within Thai P. falciparum. These results indicate that the TRAP gene has been subject to diversifying selection in the Thai P. falciparum population as well as the Gambian P. falciparum population. Comparison of our P. falciparum isolates with those from another region of Thailand (Tak province, Thailand) revealed that TRAP was highly differentiated between geographically close regions. This rapid diversification seems to reflect strong recent positive selection on TRAP. Our results suggest that the TRAP molecule is a major target of the human immune response to pre-erythrocytic stages of P. falciparum.

Introduction

Malaria infection is initiated during the sporozoite stage of the Plasmodium when the parasite is injected into the bloodstream by an infected female Anopheles mosquito. Sporozoites quickly pass into the liver, where they infect hepatocytes. Thrombospondin-related adhesive protein (TRAP) [1] is stored in the micronemes of sporozoites [2] and is released onto the cell surface at the anterior tip on contact of sporozoites with host cells [3]. TRAP has been shown to play a crucial role in sporozoite gliding, motility, and invasion of hepatocytes [4][6].

P. falciparum-infected hepatocytes can be recognized and eliminated by cytotoxic T lymphocytes (CTLs). The CTL-based adaptive immune response appears to be important for protection against the pre-erythrocytic stages of malaria. Presentation of pathogen-derived peptides by human leukocyte antigen (HLA) class I molecules on infected cells to T cell receptors (TCRs) on CTLs is a central event in CTL-mediated elimination of target cells. TRAP is one of the potential proteins that has epitopes presented by HLA class I molecules on infected hepatocytes to TCRs on CTLs. This has been supported by the identification of a CTL response to certain TRAP epitopes in humans [7][11]. These observations raise the question of whether the TRAP gene of P. falciparum has been under strong selective pressure due to human immune response.

A previous study [12] revealed a higher ratio of the number of nonsynonymous to synonymous polymorphisms of TRAP within the Gambian P. falciparum population compared with that of nonsynonymous to synonymous substitutions between the Gambian P. falciparum population and P. reichenowi. A significant difference was observed in this ratio (i.e., the former was higher than the latter) by the McDonald–Kreitman test, suggesting that the TRAP gene is under diversifying selection in the Gambian P. falciparum population. Furthermore, the observed Tajima’s D and Fu and Li’s F values for TRAP in the Gambian P. falciparum population were significantly higher than those from a coalescent simulation under neutrality, suggesting that the TRAP gene is subject to balancing selection, which maintains diversity in the Gambian P. falciparum population [12]. However, no evidence for balancing selection was reported for samples obtained from Tak province, Thailand [12]. Thus, it remains unclear whether positive selection acts on the TRAP gene of P. falciparum in other geographic regions apart from Africa. The aims of the present study were to investigate whether the TRAP gene is under diversifying selection in Thai P. falciparum and to elucidate how TRAP gene is differentiated among P. falciparum populations. Our results would be helpful not only for understanding the molecular evolution of the TRAP gene in P. falciparum but also for designing peptide vaccines based on the TRAP antigen.

Results

TRAP Polymorphisms of P. falciparum in Thailand

To detect TRAP polymorphisms within P. falciparum isolates from Suan Phueng District in Ratchaburi Province, Thailand, we performed direct PCR sequencing of DNA samples from 32 patients with P. falciparum malaria. The sequence reads obtained from direct sequencing of both DNA strands did not show ambiguous electropherogram peaks at any site, indicating that the single or most abundant allele in each blood sample was successfully determined. These 32 single allele sequences of the entire coding region of the TRAP gene (GenBank Accession No. AB807828–AB807859) were statistically analyzed as below, and among them, 23 different allelic haplotypes were identified (Figure 1).

thumbnail
Figure 1. Alignment of amino acid sequences of the TRAP gene of 32 P. falciparum isolates.

Only polymorphic residues in 32 isolates are shown. Residues identical with those of YS001 are indicated by dots, and deletions in the region of the P-N-P repeat are indicated by dashes.

https://doi.org/10.1371/journal.pone.0090522.g001

A total of 39 single nucleotide polymorphisms (SNPs), comprising 37 nonsynonymous and two synonymous SNPs, were found in the coding region of the TRAP gene of 32 Thai P. falciparum isolates. Amino acid alterations caused by single point mutations included 33 diallelic and two triallelic amino acid polymorphisms (Figure 1). Therefore, 35 amino acid residues were polymorphic among a total of 559 residues of the TRAP gene (Figures 1 and 2B). The density of polymorphic residues was relatively high in von Willebrand factor A domains and in the proline-rich repeat regions (Figure 2A), with 13 and 17 amino acid polymorphisms in A domains and proline-rich repeat regions, respectively. In contrast, the N-terminal signal sequence and transmembrane domain had no amino acid polymorphisms, apparently reflecting the difference in the functional constraints among domains.

thumbnail
Figure 2. Polymorphic amino acid residues of the TRAP gene in 32 P. falciparum isolates.

A, Density of polymorphic amino acid residues in the TRAP gene. A sliding window analysis of amino acid polymorphisms (substitutions) was conducted using a sliding window size of 20 amino acids and a step size of one amino acid. B, Schematic representation of the TRAP domain composition, signal sequence (SS), von Willebrand factor A domain (A domain), thrombospondin type 1 domain (TSP-1), and transmembrane domain (TM). The proline-rich repeat region lies between TSP-1 and TM. Positions of polymorphic amino acid residues are indicated by the thin red line above the box.

https://doi.org/10.1371/journal.pone.0090522.g002

In addition to nonsynonymous SNPs, the TRAP gene had a repeat region with a nonanucleotide repeat unit encoding proline-asparagine-proline (P-N-P; Figure 1). The number of repeats varied from 0 to 5. As found in a previous study of TRAP polymorphisms in P. falciparum field isolates from Tak Province, Thailand [13], only simple P-N-P repeats were detected in the present samples from Ratchaburi Province, Thailand. Unlike Thai P. falciparum, glutamate-asparagine-proline-serine-asparagine-proline (E-N-P-S-N-P) repeats were observed in the Gambian P. falciparum population [12]. The allelic haplotypes containing P-N-P and E-N-P-S-N-P repeats exclusively have P and S, respectively, at position 353 in the Gambian P. falciparum population, whereas no haplotypes contain both P-N-P and E-N-P-S-N-P repeats in any P. falciparum population. Because the S allele at position 353 is found in the Thai P. falciparum population (e.g., YS021 in Figure 1), allelic haplotypes containing E-N-P-S-N-P repeats may also exist at a low frequency in Thailand or may appear in the future. P-N-P repeat regions were excluded from the analyses below unless otherwise stated.

Polymorphic Sites within Species and Fixed Sites between Species

The McDonald–Kreitman test [14] for the aligned sequence data of TRAP sequences of 32 Thai P. falciparum isolates and P. reichenowi (GenBank Accession No. AY880881) revealed that the ratio of the number of nonsynonymous (N) to synonymous (S) polymorphic sites within P. falciparum (PN/PS = 36/2) was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between Thai P. falciparum and P. reichenowi (DN/DS = 36/12; P-value = 0.018 by Fisher’s exact test).

Nonsynonymous and Synonymous Substitution Rates within the Thai P. falciparum Population

The significantly higher ratio of the number of nonsynonymous to synonymous polymorphisms within P. falciparum compared with that of nonsynonymous to synonymous substitutions between P. falciparum and P. reichenowi (i.e., PN/PS>DN/DS) in the McDonald–Kreitman test implies that either purifying selection (negative selection) or diversifying selection (positive selection) has been acting on the TRAP gene in P. falciparum. To evaluate this, Z-test statistic, (dNdS)/sqrt{var(dNdS)}, was calculated, where rates of nonsynonymous substitution (dN) and synonymous substitution (dS) were calculated for each pair of sequences within Thai P. falciparum and the variance of (dNdS) was computed using the bootstrap resampling method. Most pairs gave positive Z-test statistics (Figure 3), and binomial tests revealed that dN was significantly higher than dS (P-value = 8.4×10−101). The present results suggest that the TRAP gene in Thai P. falciparum has been subject to diversifying selection.

thumbnail
Figure 3. Distribution of the Z-test statistic for pairwise comparison of the rate of nonsynonymous substitution (dN) with that of synonymous substitution (dS) among 32 P. falciparum isolates.

https://doi.org/10.1371/journal.pone.0090522.g003

Neutrality Test based on Frequency Distribution of Segregating Sites

Tajima’s D statistic [15] was calculated to test the departure from neutral evolution under constant population size. This test compares population genetic parameters estimated from mean pairwise nucleotide differences with those estimated from the number of segregating sites. Tajima’s D value was −0.13 for TRAP sequences of 32 Thai P. falciparum isolates. A coalescence-based computer simulation revealed no significant differences in Tajima’s D value between observed and expected values under neutrality (P-value = 0.86). A sliding window analysis (window size of 250 and step size of 10 bp) of Tajima’s D across the entire TRAP gene for 32 Thai P. falciparum isolates also showed no remarkable deviation of Tajima’s D from 0 (Figure S1). Unlike the Gambian P. falciparum population [12], balancing selection, which maintains polymorphisms with intermediate-frequency alleles, may not apparently operate in the Thai P. falciparum population.

Distribution of Allele Frequencies at the P-N-P Repeat

We detected six P-N-P repeat alleles of different lengths (Figure 1). Each P-N-P repeat was encoded by the nonanucleotide sequence motif CCAAATCCA. To determine whether the observed distribution of allele frequencies at the P-N-P repeat deviated from the expected value under neutrality, we performed the Ewens–Watterson homozygosity test [16]. A two-sided test showed neither significant excess nor deficiency of F in the observed data (two-sided P-value = 0.22), suggesting that the P-N-P repeat is not subject to strong positive selection.

Genetic Differentiation between P. falciparum Populations

To elucidate geographic differences in the nucleotide sequence of TRAP between Thailand and Gambia, a phylogenetic tree was constructed using the neighbor-joining method for individual sequences (Figure 4A). A phylogenetic analysis indicated that P. falciparum malaria parasites are broadly classified into the following three geographical groups: Ratchaburi, Tak, and Gambia. The STRUCTURE analysis for K = 3 also showed three distinct clusters that correspond to the geographic regions where samples were obtained (Figure 4B).

thumbnail
Figure 4. Phylogenetic tree (A) and population structure (B) of the TRAP gene of 110 P. falciparum isolates from three geographic regions.

Samples included 32 isolates from Ratchaburi Province, Thailand (investigated in this study), 29 from Tak Province, Thailand, and 49 from Gambia. A, The phylogenetic tree of the TRAP sequences was constructed using the neighbor-joining method. B, Membership coefficients from STRUCTURE analysis (K = 3) were plotted for 110 isolates. Each isolate is represented by a vertical bar displaying the proportion of membership in each of the three hypothetical clusters.

https://doi.org/10.1371/journal.pone.0090522.g004

To examine the genetic differentiation at each SNP, Fst values were calculated for three pairs of P. falciparum populations (Figure 5). As expected, P. falciparum population in Ratchaburi showed the genetic similarity with that in Tak. Most of SNPs with high Fst values between these Thai P. falciparum populations were located in A-domain of TRAP molecule. Although neutrality test using the Tajima’s D statistic did not detect signature of positive selection in any region of the TRAP gene (Figure S1), A-domain of TRAP molecule may be evolving rapidly to escape the human immune response.

thumbnail
Figure 5. Fst value at each SNP.

A total of 94 SNPs were detected in three P. falciparum populations. Fst values were calculated for three population pairs (Ratchaburi and Tak, Tak and Gambia, and Gambia and Ratchaburi). The locations of four TRAP domains, signal sequence (SS), von Willebrand factor A domain (A domain), thrombospondin type 1 domain (TSP-1), and transmembrane domain (TM) were indicated by thick horizontal lines.

https://doi.org/10.1371/journal.pone.0090522.g005

Discussion

In the present study, we examined whether the TRAP gene has been subject to positive selection in Thai P. falciparum. The ratio of the number of nonsynonymous to synonymous polymorphic sites within P. falciparum was significantly higher than that of nonsynonymous to synonymous fixed sites between P. falciparum and P. reichenowi. This observation excludes the possibility of directional selection after divergence of P. falciparum from P. reichenowi. The rate of nonsynonymous substitutions was found to be significantly higher than that of synonymous substitutions within Thai P. falciparum. Thus, we conclude that diversifying selection has acted on the TRAP gene of P. falciparum in Southeast Asia as previously reported in Africa [12].

If diversifying selection has acted on the TRAP gene for a long evolutionary time, the rate of nonsynonymous substitution (dN) should be higher than that of synonymous substitution (dS) between P. falciparum and P. reichenowi, because fixation eventually occurs even under diversifying selection. However, the dN/dS ratio between a Thai P. falciparum isolate and P. reichenowi was not significantly higher than 1 (data not shown), suggesting that diversifying selection on the TRAP gene may have started to act recently in the Thai P. falciparum population.

Of 94 SNPs across the three P. falciparum populations, five, eight, and 40 population-specific SNPs were found in Ratchaburi, Tak, and Gambia, respectively (data not shown). In addition, four SNPs were completely differentiated, with only a single allele observed in each population, leading to three distinct clusters corresponding to three geographic regions in both cluster analyses (Figures 4 and 5). This high level of genetic differentiation of TRAP implies the following four possibilities: the first is that the TRAP gene has diversified in a short period of time due to “strong” diversifying selection. The second is that long-term isolation due to the lack of migration of P. falciparum along with humans has caused the genetic differentiation. However, with only 300 km between provinces of Ratchaburi and Tak, it seems unlikely that migration has not occurred between P. falciparum populations in Ratchaburi province and in Tak province for a long time. The third is that high mutation rate has accelerated the genetic differentiation. However, the mutation rate at TRAP seems not high, since only 48 fixed sites were observed between P. reichenowi and P. falciparum (DN and Ds in McDonald–Kreitman test). These two parasites are suggested to have diverged at the time of the divergence between humans and chimpanzees [17]. The forth is that the samples in three regions were collected at different times. As shown in a phylogenetic tree (Figure 4A), the genetic differentiation does not come from random genetic drift but from mutation. Considering the low mutation rate at TRAP, the difference in time of sampling would not have largely affected the degree of genetic differentiation. Thus, the high level of genetic differentiation is considered to be one of evidences for strong diversifying selection that has acted on the TRAP gene. To examine how TRAP is markedly differentiated, we would need to compare the degree of differentiation in TRAP with that in other genes.

At the liver stage of malaria infection by P. falciparum, P. falciparum-derived antigens, including TRAP, are processed through the endogenous pathway, and the resulting antigenic peptide fragments are presented by HLA class I molecules to TCRs on CTLs. Previous studies have indicated that a wide range of pre-erythrocytic TRAP antigens are targeted by naturally acquired CTLs [7][11]. Together with the fact that TRAP has CTL epitopes, we conclude that the TRAP molecule is a major target of the human immune response to pre-erythrocytic stages of malaria due to P. falciparum.

Amino acid changes in a CTL epitope can affect processing. Previously detected HLA-A2.1-restricted CTL epitopes [7], HLGNVKYLV (residues 3–11) and GIAGGLALL (residues 500–508), and HLA-B8-restricted CTL epitopes [7], [8], ASKNKEKAL (residues 107–115) and KNKEKALII (residues 109–117), were not found to be variable in P. falciparum isolates in Ratchaburi province, Thailand as well as Tak province [13], suggesting that malaria vaccines based on these TRAP epitopes would be effective in Thailand. To develop optimal malaria vaccine, more CTL epitopes restricted by various HLA molecules need to be detected in the conserved region of TRAP. Our findings on TRAP polymorphisms will be useful not only for investigating the molecular evolution but also for developing malaria vaccine.

Materials and Methods

Ethics Statement

This study was approved by the Institutional Review Board of the Faculty of Tropical Medicine, Mahidol University, and the Research Ethics Committee of the Faculty of Medicine, University of Tsukuba. Written informed consent was obtained from all patients.

Subjects

A total of 32 adult patients with malaria infection due to P. falciparum living in Suan Pung, Ratchaburi, Thailand, participated in this study. All patients underwent treatment at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Thailand. Malaria infection by P. falciparum was confirmed by positive blood smear results for the asexual form of P. falciparum.

Sequencing Analysis of the Entire TRAP Gene from Genomic DNA Samples

Genomic DNA was extracted from pretreatment peripheral blood samples from patients with malaria due to P. falciparum using a QIAamp Blood Kit (Qiagen, Hilden, Germany). Direct PCR sequencing was performed to detect TRAP polymorphisms in Thai P. falciparum populations using the following four sets of primers: TRAP1, 5′-GTTGTTGTGTATTTCACTATAT-3′ (forward) and 5′-GAAACAAAGTGACCCCCAAA-3′ (reverse); TRAP2, 5′-TTCAGGATGTTTTGGAGTATCG-3′ (forward) and 5′-GTTCCACAAGAACCCGAAGA-3′ (reverse); TRAP3, 5′-GGATTTGGTGGATTTTCTGG-3′ (forward) and 5′-GAACAAACTTAAGTGATGCACTGTT-3′ (reverse); and TRAP4, 5′-GATCATTTAATTTTCTTGATTCT-3′ (forward) and 5′-TGTGCATGCGTACAAGAAAA-3′ (reverse). These primers were designed based on the reference sequence (GenBank Accession No. U67764.1) and included the entire coding sequence of the TRAP gene of P. falciparum. PCR amplification was performed using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). PCR amplification using TRAP1 or TRAP3 primer pair was performed in a 10 µL reaction mixture containing 0.2 µL (0.2 µM) each of forward and reverse primers, 5 µL 2×AmpliTaq Gold 360 Master Mix, 0.5 µL (5 ng) of genomic DNA, and 4.1 µL dH2O. PCR amplification using TRAP2 or TRAP4 primer pair was performed in a 10 µL reaction mixture containing 0.2 µL (0.2 µM) each of forward and reverse primers, 0.08 µL (2 units) of Fast start Taq DNA polymerase, 0.2 µL (200 µM) of dNTP, 1 µL of 10×PCR buffer with 20 mM MgCl2, 0.5 µL (5 ng) of genomic DNA, and 7.82 µL dH2O. The PCR cycling condition for TRAP1 or TRAP3 primer pair was: 10 minutes initial denaturation at 95°C, followed by 40 cycles with 30 seconds denaturation at 95°C, 30 seconds annealing at 56°C, 45 seconds extension at 72°C and a final 7 minutes extension at 72°C. The PCR cycling condition for TRAP2 or TRAP4 primer pair was: 4 minutes initial denaturation at 95°C, followed by 40 cycles with 30 seconds denaturation at 95°C, 30 seconds annealing at 56°C, 45 seconds extension at 72°C and a final 7 minutes extension at 72°C. The PCR products were sequenced using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). All isolates gave a clear single allele sequence (no superimposed electropherogram peaks at identified polymorphic sites), corresponding to the single or most abundant allele in each blood sample.

Statistical Analysis

To calculate the proportion of amino acid polymorphisms (substitutions) to amino acid sites (i.e., density of polymorphic amino acid residues), a sliding window analysis of amino acid polymorphisms (substitutions) was conducted using a sliding window size of 20 amino acids and a step size of one amino acid.

The TRAP sequence of P. reichenowi, which is a malaria strain found in primates such as chimpanzees and gorillas and is the closest known relative of P. falciparum, was retrieved from the GenBank database (GenBank Accession No. AY880881) and then aligned with 32 TRAP sequences of Thai P. falciparum. Based on the aligned sequence data, the numbers of nonsynonymous (N) and synonymous (S) polymorphic sites within P. falciparum and the numbers of nonsynonymous and synonymous fixed sites between Thai P. falciparum and P. reichenowi were counted. To test the neutral hypothesis that nonsynonymous and synonymous nucleotide differences occur in the same ratio for fixed (between species) and polymorphic (within species) differences, the McDonald–Kreitman test [14] was performed using DnaSP v5 software [18]. In the present study, two-sided P-values were calculated for all statistical tests, and the significance level was set at 0.05.

For each pair of 32 TRAP sequences of Thai P. falciparum, the number of nonsynonymous substitutions per nonsynonymous site (dN) and the number of synonymous substitutions per synonymous site (dS) were estimated using the Nei–Gojobori method with the Jukes–Cantor model [19]. The variance of (dNdS) was estimated using the bootstrap resampling method (1,000 resamplings), and Z-test statistics were calculated for each pair using MEGA 5.0 software [20]. Binomial tests were used to test the neutral hypothesis that the number of pairs with dN higher than dS is not different from the number of pairs with dS higher than dN.

Tajima’s D test [15] was performed for 32 TRAP nucleotide sequences of Thai P. falciparum using Arlequin version 3.5 software [21], where the probability distribution of Tajima’s D under neutrality was generated by coalescent simulation. A sliding window analysis of Tajima’s D statistic across the TRAP gene for the nucleotide sequences was also performed (a window size of 250 bp and a step size of 10 bp) using a web tool, SLIDER (http://genapps.uchicago.edu/slider/index.html).

Using Arlequin version 3.5 software [22], the Ewens–Watterson test [16], which is based on Ewens sampling theory of neutral alleles [23], was performed to assess whether the observed distribution of allele frequencies at the P-N-P repeat was different from the expected value that was based on neutrality.

After eliminating P-N-P and E-N-P-S-N-P repeats from the TRAP sequences of 110 P. falciparum isolates comprising 32 isolates from Ratchaburi Province, Thailand (investigated in this study); 29 from Tak Province, Thailand; and 49 from Gambia, West Africa, the number of pairwise nucleotide substitutions per site was estimated using the two-parameter method of Kimura [24]. The calculation was performed using MEGA 5.0 software [20] with the “Complete Deletion” option. Based on the estimated number of substitutions, a phylogenetic tree was constructed using the neighbor-joining method [25].

Based on SNPs of the TRAP gene, the population structure of 110 P. falciparum isolates was inferred using the Bayesian clustering algorithm implemented in STRUCTURE software v. 2.3.3 [26]. STRUCTURE was run for K (a user-defined number of clusters) = 3 for 10,000 Monte Carlo Markov Chain (MCMC) iterations after a burn-in period of 10,000 using an admixture model and correlated allele frequencies.

Fst values [27] for three population pairs (Ratchaburi and Tak, Tak and Gambia, and Gambia and Ratchaburi) were calculated using the population genetics software package Genepop 4.2 [28]. The negative Fst values were regarded as 0 in this study.

Supporting Information

Figure S1.

A sliding window analysis of Tajima’s D. A sliding window analysis of Tajima’s D statistic across the entire TRAP gene for 32 Thai P. falciparum isolates was conducted using a sliding window size of 250 bp and a step size of 10 bp. The locations of four TRAP domains, signal sequence (SS), von Willebrand factor A domain (A domain), thrombospondin type 1 domain (TSP-1), and transmembrane domain (TM) were indicated by thick horizontal lines.

https://doi.org/10.1371/journal.pone.0090522.s001

(TIF)

Acknowledgments

We sincerely thank all the subjects for their participation in the study. We thank two anonymous reviewers for their valuable comments and suggestions.

Author Contributions

Conceived and designed the experiments: JO JP. Performed the experiments: YS IN HH JP. Analyzed the data: JO. Contributed reagents/materials/analysis tools: JO JP. Wrote the paper: JO. Assembled the data: JO YS. Statistically analyzed the data: JO.

References

  1. 1. Robson KJ, Hall JR, Jennings MW, Harris TJ, Marsh K, et al. (1988) A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature 335: 79–82.
  2. 2. Rogers WO, Malik A, Mellouk S, Nakamura K, Rogers MD, et al. (1992) Characterization of Plasmodium falciparum sporozoite surface protein 2. Proc Natl Acad Sci U S A 89: 9176–9180.
  3. 3. Gantt S, Persson C, Rose K, Birkett AJ, Abagyan R, et al. (2000) Antibodies against thrombospondin-related anonymous protein do not inhibit Plasmodium sporozoite infectivity in vivo. Infect Immun 68: 3667–3673.
  4. 4. Muller HM, Reckmann I, Hollingdale MR, Bujard H, Robson KJ, et al. (1993) Thrombospondin related anonymous protein (TRAP) of Plasmodium falciparum binds specifically to sulfated glycoconjugates and to HepG2 hepatoma cells suggesting a role for this molecule in sporozoite invasion of hepatocytes. EMBO J 12: 2881–2889.
  5. 5. Robson KJ, Frevert U, Reckmann I, Cowan G, Beier J, et al. (1995) Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes. EMBO J 14: 3883–3894.
  6. 6. Sultan AA, Thathy V, Frevert U, Robson KJ, Crisanti A, et al. (1997) TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites. Cell 90: 511–522.
  7. 7. Aidoo M, Lalvani A, Allsopp CE, Plebanski M, Meisner SJ, et al. (1995) Identification of conserved antigenic components for a cytotoxic T lymphocyte-inducing vaccine against malaria. Lancet 345: 1003–1007.
  8. 8. Wizel B, Houghten RA, Parker KC, Coligan JE, Church P, et al. (1995) Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum sporozoite surface protein 2. J Exp Med 182: 1435–1445.
  9. 9. Wizel B, Houghten R, Church P, Tine JA, Lanar DE, et al. (1995) HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. J Immunol 155: 766–775.
  10. 10. Plebanski M, Aidoo M, Whittle HC, Hill AV (1997) Precursor frequency analysis of cytotoxic T lymphocytes to pre-erythrocytic antigens of Plasmodium falciparum in West Africa. J Immunol 158: 2849–2855.
  11. 11. Doolan DL, Hoffman SL, Southwood S, Wentworth PA, Sidney J, et al. (1997) Degenerate cytotoxic T cell epitopes from P. falciparum restricted by multiple HLA-A and HLA-B supertype alleles. Immunity 7: 97–112.
  12. 12. Weedall GD, Preston BM, Thomas AW, Sutherland CJ, Conway DJ (2007) Differential evidence of natural selection on two leading sporozoite stage malaria vaccine candidate antigens. Int J Parasitol 37: 77–85.
  13. 13. Jongwutiwes S, Putaporntip C, Kanbara H, Tanabe K (1998) Variation in the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum from Thai field isolates. Mol Biochem Parasitol 92: 349–353.
  14. 14. McDonald JH, Kreitman M (1991) Adaptive protein evolution at the Adh locus in Drosophila. Nature 351: 652–654.
  15. 15. Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123: 585–595.
  16. 16. Watterson GA (1978) The homozygosity test of neutrality. Genetics 88: 405–417.
  17. 17. Escalante AA, Ayala FJ (1994) Phylogeny of the malarial genus Plasmodium, derived from rRNA gene sequences. Proc Natl Acad Sci U S A 91: 11373–11377.
  18. 18. Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451–1452.
  19. 19. Nei M, Gojobori T (1986) Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 3: 418–426.
  20. 20. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.
  21. 21. Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online 1: 47–50.
  22. 22. Excoffier L, Lischer HE (2010) Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10: 564–567.
  23. 23. Ewens WJ (1972) The sampling theory of selectively neutral alleles. Theor Popul Biol 3: 87–112.
  24. 24. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16: 111–120.
  25. 25. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425.
  26. 26. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155: 945–959.
  27. 27. Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38: 1358–1370.
  28. 28. Raymond M, Rousset F (1995) GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J Heredity 86: 248–249.