Sequence diversity and positive selection at the Duffy-binding protein genes of Plasmodium knowlesi and P. cynomolgi: Analysis of the complete coding sequences of Thai isolates

Infection, Genetics and Evolution 44 (2016) 367–375

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Research paper

Sequence diversity and positive selection at the Duffy-binding protein genes of Plasmodium knowlesi and P. cynomolgi: Analysis of the complete coding sequences of Thai isolates Chaturong Putaporntip ⁎, Napaporn Kuamsab, Somchai Jongwutiwes Molecular Biology of Malaria and Opportunistic Parasites Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

a r t i c l e

i n f o

Article history: Received 7 June 2016 Received in revised form 25 July 2016 Accepted 28 July 2016 Available online 30 July 2016 Keywords: Plasmodium knowlesi Plasmodium cynomolgi Duffy binding protein Sequence diversity Natural selection Macaque

a b s t r a c t Plasmodium knowlesi and P. cynomolgi are simian malaria parasites capable of causing symptomatic human infections. The interaction between the Duffy binding protein alpha on P. knowlesi merozoite and the Duffy-antigen receptor for chemokine (DARC) on human and macaque erythrocyte membrane is prerequisite for establishment of blood stage infection whereas DARC is not required for erythrocyte invasion by P. cynomolgi. To gain insights into the evolution of the PkDBP gene family comprising PkDBPα, PkDBPβ and PkDBPγ, and a member of the DBP gene family of P. cynomolgi (PcyDBP1), the complete coding sequences of these genes were analyzed from Thai field isolates and compared with the publicly available DBP sequences of P. vivax (PvDBP). The complete coding sequences of PkDBPα (n = 11), PkDBPβ (n = 11), PkDBPγ (n = 10) and PcyDBP1 (n = 11) were obtained from direct sequencing of the PCR products. Nucleotide diversity of DBP is highly variable across malaria species. PcyDBP1 displayed the greatest level of nucleotide diversity while all PkDBP gene members exhibited comparable levels of diversity. Positive selection occurred in domains I, II and IV of PvDBP and in domain V of PcyDBP1. Although deviation from neutrality was not detected in domain II of PkDBPα, a signature of positive selection was identified in the putative DARC binding site in this domain. The DBP gene families seem to have arisen following the model of concerted evolution because paralogs rather than orthologs are clustered in the phylogenetic tree. The presence of identical or closely related repeats exclusive for the PkDBP gene family suggests that duplication of gene members postdated their divergence from the ancestral PcyDBP and PvDBP lineages. Intragenic recombination was detected in all DBP genes of these malaria species. Despite the limited number of isolates, P. knowlesi from Thailand shared phylogenetically related domain II sequences of both PkDBPα and PkDBPγ with those from Peninsular Malaysia, consistent with their geographic proximity. © 2016 Published by Elsevier B.V.

1. Introduction Despite a complex life cycle of malaria parasites in vertebrate and invertebrate hosts, only their intraerythrocytic asexual development is responsible for clinical symptoms. A crucial event prior to establishment of asexual blood stage development of malaria parasites is specific interaction between ligands on the merozoite surface and corresponding receptors on the host erythrocytes. Both Plasmodium vivax and P. knowlesi deploy the Duffy-antigen receptor for chemokines (DARC) as a common path for invasion into host erythrocytes although reticulocyte receptor is also mandatory for invasion of P. vivax (Miller et al., 1975; Miller et al., 1976). Recognition of DARC receptor on human erythrocytes by the merozoites principally depends on protein-protein interaction mediated by parasite-encoded Duffy binding proteins (DBP) (Adams ⁎ Corresponding author at: Molecular Biology of Malaria and Opportunistic Parasites Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. E-mail address: [email protected] (C. Putaporntip).

http://dx.doi.org/10.1016/j.meegid.2016.07.040 1567-1348/© 2016 Published by Elsevier B.V.

et al., 1992; Chitnis et al., 1996). Interruption of P. vivax DBP (PvDBP) binding to DARC receptor by specific antibodies can inhibit erythrocyte invasion; thereby, halting intraerythrocytic cycle of malaria parasites and abrogating subsequent pathogenesis (Grimberg et al., 2007; King et al., 2008). Therefore, PvDBP has been considered to be an attractive target for vaccine development. The gene encoding Duffy binding protein of P. vivax contains 5 exons located on chromosome 6 (Adams et al., 1992; Carlton et al., 2008). Although the complete genome of P. vivax derived from the Salvador I strain reveals a single copy of the PvDBP gene, a recent analysis has shown a high prevalence of tandem duplication at this locus among clinical samples in Madagascar (Menard et al., 2013). The DBP genes of P. cynomolgi and P. knowlesi have been identified on the orthologous chromosomes, referred to as PcyDBP1 and PkDBPα, respectively. However, a paralogous gene of PcyDBP1, designated PcyDBP2, has been mapped on chromosome 3 and two paralogs of PkDBPα, i.e. PkDBPβ and PkDBPγ are found in the genome of P. knowlesi, one of which is known to reside on chromosome 13 (Pain et al., 2008; Tachibana et al., 2012). Importantly, all these genes of both simian malaria

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parasites possess similar coding and noncoding regions akin to that of PvDBP except the lack of intron 4 in PkDBPβ (Adams et al., 1992). Meanwhile, it appears that PkDBPα per se serves as a binding ligand for the DARC receptors of human and rhesus erythrocytes whereas the functional roles for PkDBPβ and PkDBPγ remain unknown (Chitnis et al., 1996). Sequence comparison of the DBP genes of P. vivax and its related simian malaria species and the erythrocyte binding protein 175 (EBP175) of P. falciparum that mediates binding of the merozoites to sialic acid on human erythrocytes has led to partition the DBP-EBP gene family into 7 domains in which domains II and VI are relatively conserved across species (Adams et al., 1992). Specific DARC binding site of PvDBP has been mapped within the α-helix central cysteinerich region in domain II of PvDBP (PvDBPII) spanning conserved cysteine residues 4–8 (C4–C8) (VanBuskirk et al., 2004) or residues 5–8 (C5–C8) (Singh et al., 2003, 2006). Sequence analysis of PvDBP from field isolates has shown evidence of positive selection in domain II, particularly in the DARC binding domain spanning C4–C10 in which immunodominant epitopes have been identified (Cole-Tobian and King, 2003). Structural and functional analyses have revealed that binding of PvDBPII to DARC receptor on host erythrocyte triggers homodimerization of PvDBPII, creating the putative sulfotyrosinebinding pocket and heterotrimer formation consisting of two PvDBPII and one DARC molecules. This in turn causes additional DARC molecule coming into contact with the preformed heterotrimer complex resulting in heterotetramer formation consisting of two molecules of DARC and two molecules of PvDBPII (Batchelor et al., 2011). A number of critical binding residues involving in this multi-step binding process have been identified within subdomain II of PvDBPII (Batchelor et al., 2011) and the N-terminal extracellular domain of DARC receptor spanning residues 19–30 (Batchelor et al., 2014). Importantly, naturally acquired inhibitory antibodies to PvDBPII among semi-immune individuals in endemic areas were variant-specific (Cole-Tobian et al., 2002; Xainli et al., 2003; Ceravolo et al., 2009), an issue that could complicate an effective vaccine design. The emergence of naturally acquired P. knowlesi malaria in humans in Southeast Asia has highlighted the importance of zoonotic and possibly anthroponotic transmission of this simian malaria parasites whose main natural hosts comprising Macaca fascicularis (long-tailed macaques) and M. nemestrina (pig-tailed macaques) co-exist and share overlapping ecological niche with humans (Putaporntip et al., 2010; Jongwutiwes et al., 2011). Furthermore, a human malaria case caused by natural P. cynomolgi infection has been recently reported (Ta et al., 2014) although further extensive epidemiological surveillance is required. Therefore, analysis of parasite ligands relevant to host cell invasion would provide a better understanding of the evolutionary aspects of these P. vivax-related simian malaria parasites. Recent studies on sequence diversity in domain II of PkDBPα among isolates from humans and long-tailed macaques (Macaca fascicularis) in Peninsular Malaysia and North Borneo have shown substantial nucleotide substitutions (Fong et al., 2014, 2015). However, evidence of purifying selection reported among P. knowlesi isolates in these studies was in stark contrast to positive selection observed in PvDBPII. To date, the complete coding sequences of the 3 members of PkDBP have been available from the H strain and its clones (Moon et al., 2013) while PcyDBP sequences were obtained from 3 monkey strains (Tachibana et al., 2012). To gain further insight into the extent of sequence diversity and evolution of these genes, we determined the complete coding sequences of PkDBP gene family from naturally acquired human and macaque infections and those of PcyDBP1 from wild macaque isolates in Thailand. 2. Materials and methods 2.1. DNA samples Blood samples (~ 1 ml) were obtained from P. knowlesi-infected patients during our previous studies during 2006–2010 by screening

N5000 individual blood samples across major endemic areas of Thailand (Jongwutiwes et al., 2004, 2011; Putaporntip et al., 2009). Between 2007 and 2009, 743 macaques (449 M. nemestrina and 294 M. fascicularis) inhabiting Prachuab Khirikhan, Ranong, Pattalung, Pattani, Yala and Narathiwat Provinces were screened for simian malaria parasites in their blood. Definite species identification of primate malaria were done by PCR targeting the 18S rRNA genes and confirmed by sequencing (Seethamchai et al., 2008; Putaporntip et al., 2010). Of these, 18 blood samples harboring P. knowlesi from humans (n = 8) and macaques (n = 10) were available in this study. Likewise, 17 isolates of P. cynomolgi were included in this analysis. These studies were approved by the Institutional Review Board of Faculty of Medicine, Chulalongkorn University (No. 018/2006 and No. 338/51). The monkey studies were conducted in compliance with standard of animal care and use established under ethical principles and guidelines for the use of animals for scientific purposes and policies of Chulalongkorn University (No. 02/55). 2.2. PCR amplification and sequencing of the PkDBPs and PcyDBP1 genes The complete nucleotide sequences of all three members in the PkDBP gene family and the PcyDBP1 locus were amplified by nested PCR using forward and reverse primers derived from the upstream and downstream sequences encompassing the complete coding regions whose sequences were designed from the PkDBP3α, PkDBP3β and PkDBP3γ gene sequences of P. knowlesi Malayan H strain (GenBank accession numbers M90466, M90694 and M90695, respectively) (Adams et al., 1992). Likewise, the complete coding sequence of PcyDBP1 was obtained by nested PCR using primers derived from the sequence of P. cynomolgi strain B (accession number NC020399) (Tachibana et al., 2012). Forward and reverse primers for these genes and the expected size of the PCR products were listed in Supplementary Table 1. Attempt to obtain the complete PcyDBP2 was not done due to insufficient amount of available DNA. Amplification of each gene was carried out in a total volume of 25 μl of the PCR reaction containing corresponding malarial DNA, 2.5 mM MgCl2, 300 mM each deoxynucleoside triphosphate, 3 μl of 10× LATaq PCR buffer, 0.3 μM of each primer and 0.5 units of LATaq DNA polymerase (Takara, Seta, Japan). The PCR cycling profiles included the preamplification denaturation at 94 °C for 1 min, followed by 35 cycles of 96 °C for 20 s, 55 °C for 30 s and 62 °C for 4 min, and a final extension at 72 °C for 10 min. Two microliters of the products from primary PCR were used as template for secondary PCR in a total volume of 30 μl. Amplification conditions for the secondary PCR were identical with those of primary PCR. All amplification reactions were done in an Applied Biosystem GeneAmp® PCR System 9700 thermocycler (PE Biosystems, Foster City, CA). PCR products were analyzed by 1% agarose gel electrophoresis. DNA sequencing was performed directly from the purified PCR product using Qiagen PCR purification kit (Qiagen, Hilden, Germany). Singleton substitutions were verified by sequencing of the independent PCR products generated from the same genomic DNA samples. Intron sequences were excluded from this analysis due to imperfect quality of signals in the electropherograms. 2.3. Data analysis Sequences were aligned by the MUSCLE algorithm implemented in MEGA 6.0 with manual editing. The sequences of P. knowlesi DBP gene family and PcyDBP1 from this study were aligned separately for each gene member. Related sequences available in the GenBank database were included for comparison as follows: PkDBPα (M90466, AM910988, HF564624, HF564625, HF564626, KM926563-KM926611 and KT238344-KT238382); PkDBPβ (M90694); PkDBPγ (M90695, AM910995, KR053974-KR054020 and KU216673-KU216702); PcyDBP1 (JQ422035, AB617788, XM004221494, KT588668, KT596773-

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KT596778 and KT714073) and PcyDBP2: (JQ422036, AB617789, XM004220981, KT625994-KT625999, KT596779 and KT596780). For interspecies family comparison with the DBP gene of P. vivax, 23 complete coding sequences of PvDBP from diverse geographic origins were included whose GenBank accession numbers and origins and their isolate/strain names were DQ156513 (Colombia, isolate Colombia), DQ156514 (India, isolate 03-1), DQ156515 (Korea, isolate 02-13), DQ156516 (India, isolate India I), DQ156517 (Honduras, isolate Hondurus III), DQ156518 (Vietnam, isolate Vietnam IV), DQ156519 (Papua New Guinea, isolate New Guinea), DQ156520 (Brazil, isolate Brazil I), DQ156521 (Indonesia, isolate Indonesia VII), DQ156522 (Korea, isolate Korea 02-9), DQ156523 (Korea, isolate Korea 02-25), EU395587 (Brazil, strain Belem), EU395588 (Brazil, isolate Brazil I), EU395589 (Papua New Guinea, strain Chesson), EU395590 (India, isolate India VII), EU395591 (Indonesia, isolate Indonesia XIX), EU395592 (North Korea, isolate North Korea), EU395593 (Vietnam, strain PaloAlto), KQ234239 (India, isolate India VII), KQ234786 (Brazil, isolate Brazil I), KQ235026 (Mauritania, isolate Mauritania I), KQ235311 (North Korea, isolate North Korea) and M61095 (El Salvador, strain Salvador 1). Domains in malarial DBP were assigned per previous report (Adams et al., 1992). Identification of tandem repeats was performed by using the Tandem Repeats Finder program version 4.0 using the default parameters (Benson, 1999). Nucleotide diversity (π) was determined from the mean value of pairwise sequence differences in the sample and its standard deviation was the square root of the variance (Nei, 1987). Haplotype diversity (h) and its standard deviation were computed using an unbiased estimate of h, designated ĥ (Nei, 1987) as implemented in the DnaSP version 5.10.01 (Librado and Rozas, 2009). Evidence for departure from neutral evolution was tested by comparing the number of synonymous substitutions per synonymous site (dS) with the number of nonsynonymous substitutions per nonsynonymous site (dN) calculated by using Nei and Gojobori's method (Nei and Gojobori, 1986) with Juke and Cantor correction (Jukes and Cantor, 1969) and the regions containing repeats were excluded from analysis. Standard errors of these parameters were

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determined by the bootstrap method implemented in the MEGA 6.0 program (Tamura et al., 2013). Significant differences in dS and dN indicating non-neutral evolution on tested regions were considered when the corresponding p value was b 0.05. The minimum number of recombination events in the history of the sample or recombination parameter (Rm) (Hudson and Kaplan, 1985) was estimated using the DnaSP version 5.10.01 software (Librado and Rozas, 2009). Evidence of intragenic recombination was also determined by using the RDP4 package that include RDP, GENECONV, BOOTSCAN, MAXCHI, CHIMERA, SISCAN, 3SEQ, PHYLPRO and LARD programs using the default parameters (Martin et al., 2010). Parsimony informative sites were used in all analyses pertaining to recombination. Phylogenetic tree was constructed by the maximum likelihood using the Tamura-Nei model implemented in the MEGA program version 6.0 (Tamura et al., 2013). Reliability of tree topology was determined by the bootstrap method with 500 pseudoreplicates. 3. Results 3.1. Parasite isolates Of 18 P. knowlesi isolates, the PkDBPα, PkDBPβ and PkDBPγ genes could be amplified by PCR from 11, 14 and 12 samples, respectively. Three positive samples for PkDBPβ and two for PkDBPγ had multiple clone infections as viewed from superimposed signals at certain positions in the electropherograms. Therefore, the complete nucleotide sequences of PkDBPα and PkDBPβ were obtained from 11 isolates: five from humans (CT273, YL978, NR280, MC128 and BMC151 for PkDBPα; and NR234, NR280, NR600, MC128 and BMC151 for PkDBPβ) and six from macaques (HB63, HB126, HB144, HB323, HB439 and HB488 for PkDBPα; and HB63, HB132, HB321, HB323, HB530 and HBP11 for PkDBPβ) while 10 complete sequences of PkDBPγ were from 5 human cases (BMC151, CT157, CT190, CT273, and MC128) and 5 macaque natural hosts (HB63, HB132, HB323, HB530 and HBP11). Meanwhile, 14 of 17 P. cynomolgi isolates were successfully amplified by PCR for PcyDBP1.

Table 1 Nucleotide and amino acid repeats in domain I of the Duffy-binding proteins of Plasmodium cynomolgi and Plasmodium vivax.

P. cynomolgi

Locus

Haplotype

DBP1

I-I

I-II

I-III

I-IV

DBP2

P. vivax

DBP

I-I

I-I

I-II

Nucleotide/amino acid repeats

Isolates/genbank accession no.

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Table 2 Nucleotide and amino acid repeats in domain III of PkDBPβ.

Haplotype

Nucleotides/amino acids

Three of these positive samples were mixed clone infections; thereby, 11 sequences of this locus (HB7, HB122, HB135, HB147, HB148, HB308, HB414, HB530, M140, MNR8 and MNR33,) were obtained

Isolates

in this study. The geographical origins of isolates whose sequences were successfully obtained in this study are listed in Supplementary Fig. 1.

Table 3 Repeats in domain V of the Duffy binding protein family of Plasmodium knowlesi.

Locus

Haplotype

Amino acid sequences

Isolates/genbank accession no.

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Table 4 Nucleotide diversity (π) and the rates of synonymous (dS) and nonsynonymous (dN) substitutions per 100 sites in the DBP genes of P. knowlesi, P. cynomolgi and P. vivax. Gene

Domain

Sites (repeats excluded)

π ± S.E.

dS ± S.E.

dN ± S.E.

PkDBPα PkDBPβ PkDBPγ PcyDBP1 PvDBP PkDBPα PkDBPβ PkDBPγ PcyDBP1 PvDBP PkDBPα PkDBPβ PkDBPγ PcyDBP1 PvDBP PkDBPα PkDBPβ PkDBPγ PcyDBP1 PvDBP PkDBPα PkDBPβ PkDBPγ PcyDBP1 PvDBP PkDBPα PkDBPβ PkDBPγ PcyDBP1 PvDBP PkDBPα PkDBPβ PkDBPγ PcyDBP1 PvDBP PkDBPα PkDBPβ PkDBPγ PcyDBP1 PvDBP

I

606 606 606 585 (561) 615 (555) 975 1032 1013 975 978 348 516 (465) 348 480 519 357 423 351 309 339 473 (188) 543 (188) 453 (188) 303 303 218 324 324 318 318 189 189 189 186 186 3166 3633 3284 3156 3258

1.98 ± 0.32 1.08 ± 0.27 2.70 ± 0.42 3.00 ± 0.77 0.22 ± 0.07 1.29 ± 0.19 1.85 ± 0.27 1.35 ± 0.20 5.28 ± 0.44 0.93 ± 0.17 2.27 ± 0.41 1.68 ± 0.39 2.27 ± 0.51 4.83 ± 0.61 0.78 ± 0.18 1.69 ± 0.39 3.06 ± 0.55 1.05 ± 0.30 4.41 ± 0.71 0.22 ± 0.10 2.79 ± 0.77 3.30 ± 0.48 3.84 ± 0.54 2.96 ± 0.59 0.22 ± 0.14 1.06 ± 0.33 0.74 ± 0.27 1.22 ± 0.44 2.54 ± 0.53 0.90 ± 0.31 1.58 ± 0.54 0.94 ± 0.33 0.61 ± 0.25 1.18 ± 0.46 0.58 ± 0.27 1.78 ± 0.14 1.98 ± 0.14 1.93 ± 0.15 3.93 ± 0.20 0.60 ± 0.08

3.33 ± 0.87 1.09 ± 0.65 2.02 ± 0.85 6.71 ± 1.79 0.00 ± 0.00 2.05 ± 0.61 2.06 ± 0.60 1.71 ± 0.56 6.49 ± 1.07 0.33 ± 0.23 4.34 ± 1.61 1.23 ± 0.75 5.41 ± 1.76⁎ 6.95 ± 1.81 0.69 ± 0.40 4.83 ± 1.75 1.93 ± 0.88 1.06 ± 0.74 3.18 ± 1.23 0.00 ± 0.00 6.90 ± 2.81 6.97 ± 2.91 9.42 ± 3.58 1.34 ± 1.13 0.75 ± 0.71 1.83 ± 1.01 1.66 ± 1.01 2.28 ± 1.29 2.61 ± 1.39 1.25 ± 0.94 3.63 ± 2.57 2.56 ± 1.55 3.61 ± 2.06 2.84 ± 1.79 2.10 ± 1.57 2.60 ± 0.36⁎⁎⁎

1.71 ± 0.35 1.22 ± 0.33 3.07 ± 0.56 3.19 ± 0.53 0.73 ± 0.23⁎⁎ 1.56 ± 0.29 1.82 ± 0.32 1.68 ± 0.32 4.97 ± 0.52 1.10 ± 0.23⁎ 3.25 ± 0.67 1.79 ± 0.51 1.51 ± 0.50 6.54 ± 0.93 1.53 ± 0.43 2.03 ± 0.56 3.58 ± 0.74 1.29 ± 0.46 4.24 ± 0.74 1.20 ± 0.53⁎ 1.60 ± 0.49 1.91 ± 0.60 2.78 ± 0.73 4.59 ± 1.03⁎ 0.49 ± 0.29 1.11 ± 0.39 0.81 ± 0.34 1.03 ± 0.54 4.24 ± 1.00 1.19 ± 0.41 1.12 ± 0.49 1.69 ± 0.67 0.78 ± 0.60 1.30 ± 0.71 0.94 ± 0.45 1.40 ± 0.13 1.73 ± 0.17 1.65 ± 0.18 3.81 ± 0.25 0.66 ± 0.10

II

III

IV

V

VI

VII

Total

1.80 ± 0.31 2.42 ± 0.39 4.36 ± 0.50 0.39 ± 0.15

PkDBPα (n = 16), PkDBPβ (n = 13), PkDBPγ (n = 12), PcyDBP1 (n = 14) and PvDBP (n = 23). dS and dN are computed excluding repeats. Z-tests of the hypothesis that dS = dN. ⁎ p b 0.05. ⁎⁎ p b 0.01. ⁎⁎⁎ p b 0.005.

3.2. Variation in repeats of the PkDBP gene family and PcyDBP1 Short repeats containing two copies of sequences encoding KTDS(N or I) were found in domain I of PcyDBP1 although a deletion of one repeat unit was previously noted in the Berok strain (JQ422035) (Table 1). Imperfect docapeptide repeats were also observed in domain I of PvDBP that have not been previously recognized (Table 1). All these repeats were located at almost equivalent positions (codons 119–138 and 125–132 of the P. vivax Salvador I and P. cynomolgi strain B sequences, respectively). Meanwhile, the tripeptide repeats encoding D(E)NS occurred in domain III of PkDBPβ, but none were observed in two other paralogs. Size variation in these tripeptide repeats of PkDBPβ, ranging from 3 to 6 units, was observed among isolates (Table 2). On the other hand, domain V of all members in PkDBP family contained highly related pentapeptide repeats encoding SS(F)D(N or G)H(Q or E)T(N, I or R). Sequence and size variation in the repeats of domain V generated 8, 9 and 7 different haplotypes for PkDBPα, PkDBPβ and PkDBPγ, respectively (Table 3). Interestingly, identical repeats in domain V were shared between haplotype V-VI of PkDBPα and haplotype V-III of PkDBPγ. Size differences among isolates for each member were mainly caused by variation in repeat units. In contrast to the PkDBP family, the number of nucleotides in the coding region of PcyDBP1 did not exhibit extensive

size variation. All except one isolate had 3144 nucleotides; the remaining isolate had 3156 nucleotides as a result from 3 inserted codons in domain II and an extra codon in domain IV.

Table 5 Numbers of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per 100 sites in domain II of the Duffy-binding protein genes of Plasmodium knowlesi, P. cynomolgi and P. vivax. Gene (n)a

PkDBPα (16) PkDBPβ (12) PkDBPγ (90) PcyDBP1 (14) PcyDBP2 (11) PvDBP (23)

DARC-binding siteb

Remainder of domain II

dS

dN

dS

dN

0.390 ± 0.295 2.797 ± 0.990 3.365 ± 1.005 6.859 ± 1.768 7.391 ± 2.408 0.446 ± 0.448

1.420 ± 0.430# 2.219 ± 0.529 2.551 ± 0.577 6.136 ± 0.988 3.734 ± 0.726 1.600 ± 0.374#

5.424 ± 1.632 1.400 ± 0.742 3.996 ± 1.141 8.715 ± 2.049 5.792 ± 2.191 0.391 ± 0.276

2.511 ± 0.588 1.504 ± 0.396 1.798 ± 0.356 6.525 ± 0.864 3.255 ± 0.833 0.974 ± 0.342

Tests of the hypothesis that mean dS equals that for dN. a Full sequences used in Table 3 and reported partial sequences (Fong et al., 2016; Sutton et al., 2016) are included. b Codons 256–426 of the Salvador I sequence (GenBank accession number M61095). # p b 0.05.

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3.3. Nucleotide diversity Nucleotide diversity and its standard deviations across the complete coding regions of PkDBPα, PkDBPβ and PkDBPγ of isolates in this study including available sequences from the GenBank database were 0.0178 ± 0.0014, 0.0198 ± 0.0014 and 0.0193 ± 0.0015, respectively, that were not significantly different from one another (Z = 0.238– 0.999; p = 0.31–0.81). However, the PcyDBP1 sequences exhibited significantly greater nucleotide diversity (0.0393 ± 0.0020) than the three members of PkDBP (p b 0.0001). In contrast, 23 complete DBP sequences of P. vivax isolates from worldwide origins displayed significantly lower level of nucleotide diversity (0.0060 ± 0.0008) than those of P. knowlesi and P. cynomolgi (p b 10−5). Domain V of PkDBPα, PkDBPβ and PkDBPγ displayed more pronounced nucleotide diversity than other domains. For PcyDBP1, the greatest nucleotide diversity occurred in domain II, followed by domains III and IV. In contrast, domains II, III and VI of PvDBP displayed higher levels of nucleotide diversity than the remaining domains (Table 4). Therefore, the coding sequences of malarial DBP genes exhibit differential sequence diversity across species. 3.4. Deviation from neutrality Analysis of the complete coding sequences excluding repeatcontaining regions has shown that the rate of synonymous substitutions per synonymous site (dS) was greater than that of nonsynonymous substitutions per nonsynonymous site (dN) for all three members of PkDBP and PcyDBP1 while the reverse was true for PvDBP. However, these differences were not statistically significant except for the PkDBPα locus (Table 4). Meanwhile, dN was significantly greater than dS (p b 0.05) in domains I, II and IV of PvDBP and in domain V of PcyDBP1. Despite no signature of positive selection was detected in domain II of PkDBPα, dN significantly outnumbered dS in the region spanning codons 256 to 426 (p b 0.05) equivalent to a region spanning α1 to α8 domains (Batchelor et al., 2011) while the remaining regions outside these domains did not yield significant difference between these parameters (p N 0.05) (Table 5). Importantly, a similar finding was noted in the equivalent region of the PvDBP sequences. No statistically significant difference between dN and dS was observed in orthologous regions in domain II of PkDBPβ, PkDBPγ, PcyDBP1 and PcyDBP2 (Table 5). 3.5. Intragenic recombination Analysis of recombination events (Rm) implemented in the DnaSP program among Thai isolates has revealed 19, 25, 8 and 11 recombination sites in PkDBPα, PkDBPβ, PkDBPγ and PcyDBP1, respectively. Additional analysis using the RDP4 program package with the default parameters has identified 3, 4, 3 and 1 recombination breakpoints in these genes, respectively. Likewise, a minimum of 14 recombination sites were observed in PvDBP from 23 P. vivax strains using the DnaSP program while RDP4 program detected 3 recombination breakpoints. All recombination breakpoints identified using the RDP4 program were in good agreement with those analyzed by using the parameter Rm.

Fig. 1. Maximum likelihood phylogenetic relationships inferred from the complete coding region of the DBP genes of P. knowlesi (PkDBPα, PkDBPβ and PkDBPγ), P. cynomolgi (PcyDBP1 and PcyDBP2) and P. vivax (PvDBP). Taxons describe names of isolates, strains or countries. Parentheses are GenBank accession numbers of publicly available sequences or host origins. Percentage of bootstraps ≥50% are shown along the branch.

Peninsular Malaysia and North Borneo (Fong et al., 2014; Fong et al., 2015; Fong et al., 2016), it was evident that all Thai isolates form the same cluster with those from Malaysian mainland (Fig. 2). 4. Discussion

3.6. Phylogeny Maximum likelihood tree inferred from the complete DBP sequences of P. knowlesi (3144–3234, 3363–3513 and 3132–3243 bp for PkDBPα, PkDBPβ and PkDBPγ, respectively), P. cynomolgi (3132–3159 and 3147–3150 bp for PcyDBP1 and PcyDBP2, respectively) and P. vivax (3210–3231 bp) has revealed that the three gene members of PkDBP were clustered together characterized by PkDBPα being more closely related with PkDBPγ than with PkDBPβ (Fig. 1). PcyDBP1 and PcyDBP2 were closely related and formed a close neighbor with PvDBP. When comparison was made between Thai isolates of P. knowlesi and partial sequences of PkDBPα and PkDBPγ of human and macaque origins from

Analysis of the complete coding sequences of the PkDBP gene family has shown similar levels of nucleotide diversity in each member gene that were approximately three-fold greater than that of PvDBP. Meanwhile, PcyDBP1 exhibits higher level of nucleotide diversity than all PkDBP member genes. The extent and pattern of nucleotide diversity in each domain of these orthologs seem to be different. For instance, a significantly higher rate of synonymous substitutions per synonymous site than that of nonsynonymous substitutions per nonsynonymous site was observed in domain III of PkDBPγ, a signature of purifying selection in this domain that could be due to some unknown functional constraint of this protein. On the other hand, dN significantly outnumbered

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Fig. 2. Maximum likelihood tree inferred from domains II of PkDBPα (A) and PkDBPγ (B). Taxons include isolates from Thailand (filled circles), North Borneo (filled triangles) and Peninsular Malaysia (open circles). Scale represents nucleotide substitutions per site.

dS in domains I, II, IV of PvDBP and in domain V of PcyDBP1, suggesting positive selection in these regions. Therefore, differential sequence diversity in malarial DBP genes could have arisen from structural or functional divergence of these protein families. Previous studies on sequence diversity in domain II of PkDBPα using P. knowlesi isolates from Peninsular Malaysia and North Borneo have suggested purifying selection in this domain because the rate of synonymous substitutions per synonymous site was significantly greater than that of nonsynonymous substitutions per nonsynonymous site (Fong et al., 2014, 2015). Such discrepancy could stem from differences in parasite populations or sequencing artifacts from the methods used, i.e. sequencing from plasmid clones (Fong et al., 2014, 2015) versus direct sequencing of the PCR products in our study. Nevertheless, it is interesting to note that the DARC receptor binding region in domain II of both PkDBPα and PvDBP, spanning amino acid residues 256–426 (Batchelor et al., 2011), were under positive selection because the rate of nonsynonymous substitutions per nonsynonymous site significantly exceeded the rate of synonymous substitutions per synonymous site. Positive selection in domain II of PvDBP has been suggested to be rendered by host immune pressure (Cole-Tobian and King, 2003) because antibodies against domain II of PvDBP that blocked erythrocyte entry exhibit strain-transcending responses (Cole-Tobian et al., 2002; Xainli et al., 2003; Ceravolo et al., 2009). Importantly, P. knowlesi has been found to cause natural infections in humans; thereby, it is likely that sequence diversity in the DARC-binding region in domain II of PkDBPα could be a result of selective pressure exerted by the immune responses from either humans or macaque natural hosts, or both (Jongwutiwes et al., 2004, 2011; Putaporntip et al., 2009, 2010). On the other hand, the putative DARC recognition sites (Y295, Q296, K297, R304, L369 and I376) and the potential interacting residues with sulfotyosine of DARC (K297, K301, R304 and K378) in subdomains II of PkDBPα and PvDBPII (Singh et al., 2006) are perfectly conserved among field isolates, reaffirming functional importance of these residues. Alteration of each of these residues by site-directed mutagenesis reportedly abrogated the interaction between PkDBPα or PvDBP and DARC (Hans et al., 2005; VanBuskirk et al., 2004). Short repeats have been identified in domain V of the PkDBP family encoding SSDQT and related sequences while the corresponding regions

in PcyDBP1 and PvDBP did not possess apparent repeats. Additional short repeats that have not been previously recognized occurred in domain III of PkDBPβ and in domain I of both PcyDBP1 and PvDBP. It is noteworthy that the amino acid composition in the DBP repeats of malaria parasites analyzed herein contained mostly neutral and hydrophilic residues while hydrophobic amino acids were relatively scarce (Tables 1–3). This is in line with a previous analysis by Verra and Hughes showing that hydrophobic amino acids in repeats of Plasmodium antigens were reduced comparing with non-repeat regions of malaria antigens (Verra and Hughes, 1999). Such biased amino acid composition in repeat regions of malaria antigens could be due to the requirement of antigen-antibody recognition mediated by polar interaction from Van der Waals forces and hydrogen bonds. Although the role of repeats in Plasmodium DBPs remains unknown, short repeats in several malarial proteins have been hypothesized to be targets for a relatively less effective T-cell-independent antibody response while a strong response toward repeats-containing epitopes could lead to an insufficient response against protective epitopes of malarial proteins; thereby, favoring parasite evasion of host immune attack (Kemp et al., 1987). However, the repeat regions in the DBP families of malaria parasites span relatively small segments of the proteins with little variation in PcyDBP and PvDBP. Intriguingly, these repeats could possess some unknown functions yet to be of further investigation. It is noteworthy that all isolates in this study were obtained from infected subjects harboring very low parasite density (Putaporntip et al., 2009; Putaporntip et al., 2010; Jongwutiwes et al., 2011). Therefore, it is likely that PCR amplification failure could stem from very low level of malarial DNA in these samples or poor DNA quality after long-term storage rather than the absence of some member genes in the PkDBP and PcyDBP gene families. Undoubtedly, further investigation using more appropriate methodology such as next generation sequencing could elucidate this point. Nevertheless, the phylogenetic tree of PkDBP and PcyDBP genes seems to support the concerted model for the generation of these gene families because paralogous rather than orthologous genes of these member genes were clustered together (Nei and Rooney, 2005). The PvDBP gene seems to be more closely with the PcyDBP genes than with those of the PkDBP gene family as viewed from the tree topology. Importantly, variation in repeat units

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in the PkDBP family was more pronounced than those of PcyDBP1 and PvDBP while the repeats of PkDBPs were located at different domain from those of PcyDBP and PvDBP. It is therefore likely that the generation of repeats in the PkDBP gene family occurred independently from those of PcyDBP and PvDBP, probably after the divergence of PkDBP from the common ancestral gene of PcyDBP and PvDBP. Subsequently, the expansion or contraction of the repeat region could follow the concerted mode of repeat evolution by means of slippage-strand mispairing, unequal crossing over or intragenic gene conversion while point mutations could shape characteristics of repeats. The presence of identical or nearly identical repeat units of the PkDBP gene families (Table 3) could suggest recent expansion of the repeat array by duplication of repeat units (Jongwutiwes et al., 1994; Hughes, 2004). Like most other Plasmodium genes, evidence for intragenic recombination has been detected in all malarial DBP genes analyzed in this study. Recombination could further increase the number of haplotypes in these genes. However, there was no significant correlation between nucleotide diversity and the minimum number of recombination events or the recombination breakpoints detected by RDP4 program (Pearson's linear r = −0.892 to 0.698; p = 0.107 to 0.301). Therefore, recombination might not be the main mechanism generating sequence diversity at these loci. A recent genome sequence analysis of P. knowlesi population from North Borneo and some laboratory lines originated from Peninsular Malaysia and the Philippines has revealed remarkably higher nucleotide diversity than that of the two major human malaria pathogens, P. falciparum and P. vivax. Importantly, these P knowlesi isolates could be assigned to three genetically distinct clusters (Assefa et al., 2015). Furthermore, recent analysis of the gene encoding the normocyte binding protein of P. knowlesi (Pknbpxa) has shown three clusters with two of these lineages were more closely related (Ahmed et al., 2016). Meanwhile, our analyses of domain II of PkDBPα comparing with those from Peninsular Malaysia have shown three distinct clusters, two of which were closely related and found exclusively either in North Borneo or Peninsular Malaysia. A few samples from North Borneo displayed the third unique cluster. Likewise, the domain II sequences of PkDBPγ of all Thai isolates were closely related with those from Malaysian mainland. Despite a limited sample size of Thai isolates, concordant results obtained from phylogenetic analyses of domains II of both loci were consistent with geographic proximity of this simian malaria pathogen (Fig. 2).

5. Conclusions This study provides a detailed analysis of diversity in the entire coding sequences of the DBP gene families of two simian pathogens capable of causing disease in humans, P. knowlesi and P. cynomolgi. Evidence of positive selection in the DARC receptor binding site in domain II of PkDBPα suggests that similar selective pressure, probably from host immune responses, could shape diversity of the receptor binding site of this protein akin to those observed in PvDBP. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.07.040.

Acknowledgements We are grateful to all patients who donated their blood samples; to Siriporn Thongaree and staff at Hala Bala staff of Hala-Bala Wildlife Research Station, National Park and Wildlife Research Division, Department of National Parks, Wildlife and Plant Conservation for assistance in field work; and to Urassaya Pattanawong for excellent laboratory assistance. This research was supported by grants from the Thai Government Research Budget to S. J. and C.P. (GRB-APS-12593011) and the Thailand Research Fund (RSA5480008) to C.P.

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