Global sequence diversity of the lactate dehydrogenase gene in Plasmodium falciparum

A total of 50 isolates of the human malaria parasite P. falciparum used in the present study were maintained from the Malaria Research Laboratory, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. These parasites were isolated from Thai patients who were admitted to malaria clinical centres at the Thailand–Myanmar border in Tak (n = 1), Mae Hong Son (n = 10), Kanchanaburi (n = 14), and Ranong (n = 11), the Thailand–Laos border in Ubon Ratchathani (n = 4) and the Thailand–Cambodia border in Trat (n = 10) [14] (see Additional file 1). After microscopic examination, the parasites were culture-adapted using a standard candle jar method [15]. The parasite lines were genotyped with microsatellites and merozoite surface protein-3 gene for species identification [16, 17].

Blood-stage malaria parasites were expanded in laboratories and harvested for genomic DNA preparation. The phenol/chloroform extraction method was employed, as previously described [14]. Genomic DNA was dissolved in TE buffer (pH 8.0) and stored at − 20 °C until use. PCR amplification of the ldh gene utilized two PCR primers with the sequences LDH-F1 5′-ATG GCA CCA AAA GCA AAA ATC GTT T-3′ and LDH-R1 5′-TTG CAT TTG TTT CTC TCT TTG TTG CA-3′, corresponding to nucleotide positions 1041824–1041850 and 1041358–1041382 of chromosome 13 from P. falciparum strain 3D7 (Accession Number: NC_004331), respectively. Total PCR volumes of 50 μl contained 200 μM of dNTPs, 2 mM of MgCl₂, 0.2 μM of primers, 200–300 ng of parasite DNA and 1 unit of Taq polymerase enzyme in 1× Taq PCR buffer (Biotechrabbit, Germany). PCR amplification started with 95 °C for 1 min, followed by 35 cycles of 95 °C for 40 s, 60 °C for 40 s and 72 °C for 1.20 min, with a final extension at 72 °C for 5 min. PCR products were analysed by standard agarose gel electrophoresis. All reactions produced a single band with an estimated size of ~ 1000 bp. Subsequently, DNA sequencing was performed using BigDye™ Terminator Cycle Sequencing kit (Applied Biosystems) on an ABI3730XL DNA analyser. Sequencing primers were the PCR primers as well as two additional sequencing primers (LDH-F2 5′-CAT CAA GAT TGA AGT ATT ACA TAT CTC-3′ and LDH-R2 5′-TCT TGT AAA GGG ATA CCA CCT ACA GTA-3′) corresponding to nucleotide positions 1041936–1041962 and 1042384–1042409 of chromosome 13 from P. falciparum strain 3D7. Sequences of the ldh gene were visualized using BioEdit (version 7.2.6).

Nucleotide sequences of P. falciparum ldh genes from India, Iran and Madagascar were available from the published literature [11‐13] (see Additional file 2). ldh sequences of P. vivax in South Korea, China, India and Iran were previously described [11, 12, 18], while the ldh sequence of P. ovale strain Harding was retrieved from a previous report by Brown et al. [19] (see Additional file 3). ldh sequences of malaria parasites in gorillas and chimpanzees from Cameroon and Democratic Republic of Congo were retrieved from two reports by Liu [20, 21].

Furthermore, to retrieve deposited sequences of Plasmodium ldh gene from the NCBI database, BLAST searches were performed using the nucleotide sequence 5′ ATG GCA CCA AAA GCA AAA ATC GTT TTA GTT 3′ as a query sequence against the nucleotide collection (nr/nt) database of all plasmodia (Taxid, 5820). Then, complete or partial sequences with an annotation “lactate dehydrogenase” from Plasmodium species of humans (P. falciparum), gorillas (Plasmodium gaboni), chimpanzees (Plasmodium reichenowi) and macaques (Plasmodium knowlesi) were manually selected (see Additional file 3). Homology of the putative ldh sequences was determined by sequence alignment.

Additional unpublished ldh nucleotide sequences from Plasmodium parasites deposited in the PlasmoDB database [22] were obtained using a key word search for “lactate dehydrogenase”. A total of 202 P. falciparum ldh sequences were deposited by Christopher V Plowe (Howard Hughes Medical Institute/Center for Vaccine Development, University of Maryland), Dan Neafsey (The Broad Institute), Elizabeth Winzeler (The Scripps Research Institute), Alfred Amambua-Ngwa (Medical Research Council, Fajara Banjul, The Gambia), and Dominic Kwiatkowski (Oxford/Wellcome Trust Sanger Institute) (unpublished data). The P. knowlesi ldh sequence was deposited by Arnab Pain [23]. The Plasmodium cynomolgi ldh sequence was deposited by Tachibana et al. [24]. The P. gaboni and P. reichenowi ldh sequences were deposited by Sundararaman et al. [25] and Otto et al. [26], respectively.

ldh sequences from Plasmodium parasites were aligned and edited using the MUSCLE alignment algorithm [27] and the Alignment Editor tool in MEGA 7.0 [28]. The numbers of ldh alleles, non-synonymous and synonymous SNP sites, and LDH isoforms (variants of polypeptides) in each Plasmodium species were analysed using the BioEdit sequence editor (version 7.2.6) and DnaSP (version 5.10.1) [29]. Nucleotide diversity (π) was extrapolated from the number of nucleotide differences between two sequences per nucleotide site as previously described [30]. The haplotype diversity index (H) was calculated from a relative frequency of each haplotype and the numbers of sequences in the dataset [31]. The mean numbers of non-synonymous substitutions per non-synonymous sites (d _N ) and synonymous substitutions per synonymous sites (d _S ) were calculated using the method of Nei and Gojobori [32] with the Hasegawa–Kishino–Yano nucleotide substitution model. d _N ‐d _S values were calculated to investigate evidence of positive selection. Significant positive values at P < 0.05 indicated an overabundance of non-synonymous mutation, suggesting directional selection [33, 34].

Departures from the predictions of the neutral mode of molecular evolution were determined using Tajima’s D, Fu and Li’s D* and Fu and Li’s F* indices, implemented in DnaSP 5.10.1 software [35, 36]. The results of the neutrality tests were deemed to be statistically significant if the P value was less than 0.05 (P < 0.05). In addition, sliding window plots, with a window length of 90 bases and a step size of 3 bp, were generated for d _N ‐d _S and neutrality test analyses to identify regions of ldh where a significant departure from neutrality was observed (P < 0.05). Divergence in the distributions of ldh alleles of P. falciparum populations in Africa, Asia and South America was tested for using Wright’s fixation index (F _st ) with Arlequin version 3.5 [37]. Significant differences of genetic variance for any parasite population pairs at P < 0.05 indicated population differentiation.

Unique ldh haplotypes of the malaria parasites from humans (P. falciparum, P. malariae, P. ovale, P. vivax), gorillas (Plasmodium praefalciparum, Plasmodium alderi, Plasmodium billcollinsi), chimpanzees (P. reichenowi, Plasmodium blacklocki, P. gaboni), and macaques (P. knowlesi, P. cynomolgi) were selected from the putative ldh sequences in the NCBI and PlasmoDB databases. Sixty-one ldh sequences were included in the phylogenetic tree reconstruction (Additional files 2, 3). The best-fit substitution model for the multiple sequence alignment generated by MUSCLE was determined using the Bayesian Information Criteria (BIC) strategy in jModelTest [38]. Evolutionary relationships of the aligned sequences were determined using neighbour-joining (NJ) and maximum likelihood (ML) approaches in MEGA 7.0 [28] based on the p-distance method and general time reversible substitution model with a gamma distributed shape parameter (GTR+G), respectively. The robustness of the tree topology was tested with 1000 bootstrap replicates.

The ldh gene of P. falciparum was amplified from genomic DNA of 50 P. falciparum isolates and sequenced using a Sanger sequencing method as described in “Methods”. An analysis of the 918-bp partial sequences of the ldh gene, corresponding to nucleotide positions 31–948 in P. falciparum strain 3D7, revealed a single allelic type of ldh, named L1a (Table 1 and Additional file 1). The L1a allele was also identical to the sequence of P. falciparum strain 3D7.

Having demonstrated the fixation of the ldh allele in Thai P. falciparum populations, the global diversity of the ldh gene in P. falciparum was further investigated. Nucleotide sequences of the ldh gene in P. falciparum from 23 countries worldwide were retrieved from the PlasmoDB and NCBI databases and the literature (Table 2 and Additional file 2), thereby generating a database of ldh sequences containing 268 worldwide P. falciparum isolates. A comparison of the 918-bp nucleotide sequences revealed 15 single nucleotide polymorphism (SNP) sites, revealing 9 ldh gene alleles with a haplotype diversity index (H) of 0.203, an average pairwise nucleotide difference between sequences (k) of 0.355 and an average nucleotide diversity at each locus (π) of 0.0004.

In Table 1, 7 SNPs detected at positions 36 (TCA/TCG), 399 (GTA/GTC), 450 (TTA/TTG), 513 (CCA/CCC), 783 (TCA/TCG), 858 (GTT/GTC) and 891 (GAG/GAA) resulted in synonymous mutations at amino acid residues 12 (S), 133 (V), 150 (L), 171 (P), 261 (S), 286 (V) and 297 (E), respectively. The nucleotide substitution frequencies at positions 12, 133, 150, 171, 261, 286, and 297 were A/G (99.8%/0.2%), A/C (99.8%/0.2%), A/G (99.8%/0.2%), A/C (99.8%/0.2%), A/G (99.8%/0.2%), T/C (99.8%/0.2%) and G/A (99.8%/0.2%). The other 8 SNPs were detected at nucleotide positions 73 (CAG/AAG), 85 (GGA/CGA), 259 (GGA/CGA), 407 (TTA/TCA), 451 (GGT/CGT), 560 (GTT/GGT), 563 (CTT/CCT) and 814 (GAT/AAT), resulting in non-synonymous amino acid substitutions at residues 25 (Q/K), 29 (G/R), 87 (G/R), 136 (L/S), 151 (G/R), 187 (V/G), 188 (L/P) and 272 (D/N), respectively. The nucleotide substitution frequencies at positions 73, 85, 259, 407, 451, 560, 563 and 814 were C/A (99.8%/0.2%), G/C (99.8%/0.2%), G/C (99.8%/0.2%), T/C (99.8%/0.2%), G/C (99.8%/0.2%), T/G (99.8%/0.2%), T/C (99.8%/0.2%), and G/A (91.1%/8.9%), respectively.

Of the 7 synonymous SNP sites, 2 SNPs at positions 36 and 891 were unique to L1b, whereas SNPs at positions 513 and 858 were unique to L1c (Table 1). The other 2 SNPs at positions 450 and 783 were unique to L1d and L1e, respectively. L2, L3, L4 and L5 alleles of the ldh gene were characterized by 8 non-synonymous SNPs. Two SNPs at positions 814 and 73 were unique to the L2 and L3 alleles, respectively. Four SNP sites at positions 85, 259, 451 and 560 were specific to the L4 allele, while 2 SNP sites at positions 407 and 563 were specific to the L5 allele.

Because of the observed low levels of polymorphism, the next goal was to determine signatures of purifying selection on this gene. Three neutrality tests revealed significantly negative Tajima’s D, Fu and Li’s D* and Fu and Li’s F* statistics of − 1.967 (P < 0.05), − 7.065 (P < 0.02) and − 6.196 (P < 0.02), respectively. These results indicated that the ldh gene of P. falciparum was under negative purifying selection. To determine whether specific region(s) of the ldh region were under selection, a sliding window plot analysis was performed, with a window of 90 bp and a step size of 3 bp. Significantly negative D* and F* statistics were detected at nucleotide positions 333–558 (Fig. 1). A sliding window plot of D statistics also showed negative values (0 to − 1.802), although the values did not show a significant departure from zero (Fig. 1c). Examination of the patterns of synonymous and non-synonymous substitutions was also indicative of purifying selection, which is not surprising given the low levels of observed polymorphism and the results from the Fu and Li’s D* and F* tests. The average values of the numbers of non-synonymous substitution per non-synonymous site (d _N ) and number of synonymous substitution per synonymous site (d _S ) were 0.02763 ± 0.2040 and 0.0138 ± 0.0854, respectively. The d _N –d _S values of the ldh gene did not show a significant departure from zero (Fig. 2). Taken together, these results supported the view that the genetic conservation in the ldh gene of P. falciparum was mainly attributed to purifying selection.

The 918-nucleotide fragments of the ldh gene were translated to 306 amino acid residues, corresponding to amino acid positions 11–316 in P. falciparum strain 3D7. An analysis of amino acid sequences revealed 5 distinct isoforms of LDH (Table 1). Five alleles, named L1a, L1b, L1c, L1d, and L1e, produced the same amino acid sequence, corresponding to the LDH-1 isoform. The other 4 allelic types, L2, L3, L4 and L5, encoded different LDH amino acid sequences and were designated the LDH-2, LDH-3, LDH-4 and LDH-5 isoforms, respectively. Different alleles of ldh showed different geographical distributions. Table 2 shows that L1a was the most abundant allele, and was distributed across almost all P. falciparum populations. L1b, L1c, and L1d were detected in P. falciparum populations in Iran, India and China, while L1e was present in Mali. The second most abundant allele was L2, which was reported in Iran, The Gambia, Madagascar, and Uganda. In contrast, 3 alleles, L3, L4 and L5, were rare ldh alleles that were detected in single parasite isolates. L3 was reported only in Madagascar, while the L4 and L5 isoforms were found in P. falciparum populations in China.

The distribution of different LDH isoforms also reflected different levels of genetic diversity of ldh in P. falciparum populations. The results showed that genetic diversity of the ldh gene was lowest in South America, where one isoform of LDH was detected in all P. falciparum populations. In contrast, P. falciparum populations in Iran and many African countries, including the Gambia, Ghana, Senegal, and Uganda carried the 2 most abundant isoforms of LDH (LDH-1 and LDH-2). Madagascar was the only endemic area in which 3 isoforms (LDH-1, LDH-2, LDH-3) were detected.

To further determine whether parasite populations in Asia, Africa and South America are genetically isolated, pairwise inter-population comparisons were performed for each parasite population using Wright’s fixation index (F _st ). Table 3 shows that the F _st values from the pairs of P. falciparum populations between South America and Asia and between South America and Africa were low and non-significant. The analysis indicated that the P. falciparum population in South America was genetically similar to P. falciparum populations in Africa and Asia. In contrast, a significant F _st value was detected between parasite populations from Africa and Asia (P < 0.05), suggesting genetic differentiation between these parasite populations.

A final goal of the present study was to compare levels of genetic diversity of ldh sequences in different Plasmodium species infecting humans and non-human primates. Nucleotide sequences of ldh genes of the human malaria parasites P. vivax, P. ovale and P. malariae were retrieved from the NCBI database, as described in “Methods”. The 49 ldh sequences of P. vivax contained 21 non-synonymous SNPs and 10 synonymous SNPs, which could be classified into 11 alleles, while the 3 ldh sequences of P. ovale contained 5 non-synonymous SNPs and 27 synonymous SNPs classified into 3 alleles (Table 4 and Additional file 3). There was only one ldh sequence from P. malariae. The level of ldh nucleotide diversity (π) in P. ovale (π = 0.0208) was greater than those of P. falciparum (π = 0.0004) and P. vivax (π = 0.0021), although the number of available sequences was much lower. The non-synonymous mutations were outnumbered by synonymous mutations in P. ovale ldh sequences, which again suggest that the P. ovale ldh gene is under strong purifying selection. In addition to the ldh sequences of the human malaria parasites, ldh sequences from parasites of non-human primates were obtained from the NCBI database, including 9 ldh sequences from 3 species of malaria parasites in the gorilla: P. praefalciparum (n = 4), P. alderi (n = 4) and P. billcollinsi (n = 1), 29 ldh sequences from 3 species of malaria parasites in chimpanzees: P. reichenowi (n = 11), P. gaboni (n = 15) and P. blacklocki (n = 3), and 2 ldh sequences from 2 species of malaria parasites in macaques: P. cynomolgi and P. knowlesi (Additional file 3). An analysis of non-human primate malaria ldh sequences showed that the highest numbers of ldh alleles were detected in malaria parasites isolated from chimpanzees, P. gaboni and P. reichenowi. There were, however, no differences in the levels of the nucleotide diversity of ldh in P. gaboni and P. reichenowi compared to other non-human primate malaria parasites, including P. praefalciparum, P. alderi and P. blacklocki. It should also be noted that the levels of nucleotide diversity of ldh genes in the non-human malaria parasites were much higher than that of P. falciparum but were similar to that of P. vivax. These data demonstrate, therefore, the different levels of genetic diversity in ldh genes across malaria parasite species of humans and non-human primates.

Phylogenetic trees of 61 ldh gene alleles from the 12 species of Plasmodium species in human and non-human primates were constructed using the neighbour joining and maximum likelihood methods. As shown in Fig. 3, the phylogenetic tree from the maximum likelihood method yielded 2 branches of ldh genes. The same tree topology was generated using neighbour-joining method (Additional file 4). The major branch is represented by the sequences of the 3 human malaria parasites, P. falciparum, P. malariae and P. ovale, as well as the gorilla and chimpanzee Plasmodium sequences. The tree also inferred that the P. falciparum ldh gene clustered closely together with the gorilla and chimpanzee malaria parasites. It should be noted that P. ovale and P. malariae formed a sister branch to the P. falciparum lineage, but the sequences were more diverse and distantly related to P. falciparum. Interesting, the minor branch of the ldh gene tree was represented by the sequences of P. vivax ldh and the malaria parasites in macaques, P. knowlesi and P. cynomolgi. These results imply that the malaria parasites in humans may originate from different sister species of non-human primate malaria.