Low genetic diversity and functional constraint in loci encoding Plasmodium vivax P12 and P38 proteins in the Colombian population
© Forero-Rodríguez et al.; licensee BioMed Central Ltd. 2014
Received: 14 September 2013
Accepted: 13 February 2014
Published: 18 February 2014
Plasmodium vivax is one of the five species causing malaria in human beings, affecting around 391 million people annually. The development of an anti-malarial vaccine has been proposed as an alternative for controlling this disease. However, its development has been hampered by allele-specific responses produced by the high genetic diversity shown by some parasite antigens. Evaluating these antigens’ genetic diversity is thus essential when designing a completely effective vaccine.
The gene sequences of Plasmodium vivax p12 (pv12) and p38 (pv38), obtained from field isolates in Colombia, were used for evaluating haplotype polymorphism and distribution by population genetics analysis. The evolutionary forces generating the variation pattern so observed were also determined.
Both pv12 and pv38 were shown to have low genetic diversity. The neutral model for pv12 could not be discarded, whilst polymorphism in pv38 was maintained by balanced selection restricted to the gene’s 5′ region. Both encoded proteins seemed to have functional/structural constraints due to the presence of s48/45 domains, which were seen to be highly conserved.
Due to the role that malaria parasite P12 and P38 proteins seem to play during invasion in Plasmodium species, added to the Pv12 and Pv38 antigenic characteristics and the low genetic diversity observed, these proteins might be good candidates to be evaluated in the design of a multistage/multi-antigen vaccine.
Malaria is a disease caused by protozoan parasites from the Plasmodium genus, five of which cause the disease in human beings (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi) [1, 2]. This parasite is transmitted by the bite of an infected Anopheles female mosquito. Around 3.3 billon people are at risk of malaria annually, mainly in tropical and subtropical areas of the world, children aged less than five years and pregnant women being the most vulnerable . Plasmodium falciparum is responsible for the disease’s most lethal form, being predominantly found on the African continent whilst P. vivax is widely distributed around the world. Even though it has been thought that infection caused by the latter species was benign, recent studies have shown that P. vivax can cause clinical complications . It has been found that 2,488 million people are at risk of becoming infected by P. vivax on the continents of Asia and America, 132 to 391 million cases occurring annually .
In spite of control strategies having been introduced in different countries, malaria continues to be a public health problem due to the parasite’s resistance to anti-malarial treatments  and the vector’s resistance to insecticides , among other causes. More effective measures have thus to be implemented for controlling such disease, including the development of an anti-malarial vaccine.
Several antigens have been characterized as promising candidates for inclusion in a vaccine [8, 9], however, the genetic diversity of some of them [10–18] has hampered the development of such vaccine [19, 20] as these genetic variations provoke allele-specific responses [21, 22] making them become a mechanism for evading the immune system . It has been necessary to focus vaccine development on conserved domains or antigens to avoid such responses , since these regions could have functional constraint and have had slower evolution .
Developing a multi-antigen vaccine against the parasite’s blood stage has been focused on blocking all host-pathogen interactions to stop merozoite entry to red blood cells (RBC) . A group of proteins anchored to the membrane via glycosylphosphatidylinositol (GPI) has been identified in P. falciparum, predominantly located in detergent-resistant membrane (DRM) domains [27, 28]; they have been implicated in the parasite’s initial interaction with RBC [29–33] and some have been considered as being candidates for being included in a vaccine [34, 35]. One group of proteins belonging to the 6-cystein (6-Cys) family is particularly noteworthy among these DRMs (i.e., Pf12, Pf38, Pf41 and Pf92) as they have been characterized by having s48/45 domains (ID in PFAM: PF07422). Members of this family are expressed during different parasite stages [28, 36] and some of them (e.g., Pf48/45, Pf230) have been considered as vaccine candidates for the sexual stage [36, 37].
Pf12 and Pf38 are expressed during late stages of the intra-erythrocyte cycle, each having two high binding peptides, suggesting an active role during invasion of RBC . Orthologous genes encoding these proteins have been characterized recently in P. vivax[38, 39]. Both proteins have a signal peptide, a GPI anchor sequence and have been associated with DRMs [38, 39]. Pv12 has two s48/45 domains  whilst Pv38 has a single domain located towards the C-terminal end . These proteins have been shown to be antigenic [38–40], suggesting that they are exposed to the immune system, probably during P. vivax invasion of RBC.
The present study involved a population genetics analysis for evaluating the genetic diversity of pv12 and pv38 loci and the evolutionary processes generating this variation pattern; the results revealed these antigens’ low genetic diversity in the Colombian population, possibly due to functional/structural constraints in s48/45 domains. Since the proteins encoded by these genes share structural characteristics with other vaccine candidates, added to the fact that Pv12 and Pv38 are targets for the immune response [38–40] and have conserved domains, they should be considered when designing a multistage/multi-antigen anti-malarial vaccine.
The parasitized DNA used in this study was extracted from total blood collected from different Colombian areas (Antioquia, Atlántico, Bogotá, Caquetá, Cordoba, Chocó, Guainía, Guaviare, Magdalena, Meta, Nariño, and Tolima) from 2007 to 2010. All P. vivax-infected patients who provided blood samples were notified about the object of the study and signed an informed consent form if they agreed to participate. All procedures involved in taking blood samples were approved by Fundación Instituto de Inmunología de Colombia (FIDIC) ethics committee.
Parasitized DNA presence and integrity
Parasitized DNA presence and integrity in 100 samples stored at -20°C (2007-2010) at FIDIC (from different areas of Colombia) were evaluated by 18S ribosomal RNA gene amplification using specific primers for P. vivax (SSU-F 5′-ATGAACGAGATCTTAACCTGC-3′ and SSU-R 5′-CATCACGATATGTA5TGATAAAGATTACC-3′) in a touchdown PCR . The reaction contained: 1x Mango Taq reaction buffer (Bioline), 2.5 mM MgCl2, 0.25 mM dNTPs, 0.5 mM of each primer, 0.1 U Mango Taq DNA polymerase (Bioline) and 10-40 ng gDNA in 10 mL final volume. The PCR thermal profile was: one initial denaturing cycle at 95°C (5 min), followed by ten cycles at 95°C (20 sec), annealing at 65°C (30 sec) and an extension step at 72°C (45 sec). Annealing temperature was reduced by 1°C in each cycle until reaching 55°C; 35 additional cycles were run at this temperature followed by a final extension cycle at 72°C (10 min). PCR products were visualized by electrophoresis on 1.5% agarose gel in 1× TAE, using 1 μL SYBR-Safe (Invitrogen).
Identifying infection caused by single Plasmodium vivax strain
Infection by the single P. vivax strain was identified by PCR-RFLP of the pvmsp-1 polymorphic marker. The pvmsp-1 gene fragment 2 (blocks 6, 7 and 8) was amplified using direct 5′-AAAATCGAGAGCATGATCGCCACTGAGAAG-3′ and reverse 5′-AGCTTGTACTTTCCATAGTGGTCCAG-3′ primers . The amplified fragments were digested with Alu I and Mnl I restriction enzymes, as described elsewhere . The products were visualized by electrophoresis on 3% agarose gel in 1× TAE, using 1 μL SYBR-Safe (Invitrogen).
PCR amplification of pv12 and pv38 genes
A set of primers was designed for amplifying each of the genes based on Sal-I reference strain sequences (accession numbers in PlasmoDB: PVX_113775 for pv12 and PVX_097960 for pv38). The following primers were used: for pv12, pv12- direct 5′-GTACCGCTTAACACCGC-3′ and pv12- reverse 5′-GCACTACATTATAAAGAAAAGGACC-3′ and for pv38, pv38- direct 5′-CGCTTCTTTCACCGCTTC-3′ and pv38- reverse 5′-CACACATTAACGCTGCTTCG-3′. The PCR reaction mixture contained 10 mM Tris HCL, 50 mM KCl (GeneAmp 10× PCR Buffer II [Applied Biosystems]), 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 μM of each primer, 0.76 U Amplitaq Gold DNA polymerase (Applied Biosystems) and 10-40 ng gDNA in a 50 μL final volume. The PCR thermal profile was as follows: one cycle at 95°C (7 min), 40 cycles at 95°C (20 sec), 56°C (30 sec), 72°C (1 min) and a final extension cycle at 72°C (10 min). PCR products were purified using a commercial UltraClean PCR Clean-up kit (MO BIO). The purified PCR products were sequenced in both directions with the amplification primers using the BigDye method with capillary electrophoresis, using ABI-3730 XL (MACROGEN, Seoul, South Korea). Two independent PCR products were sequenced to ensure that errors were ruled out.
Analysing genetic diversity
The electropherograms obtained by sequencing were analysed and forward and reverse sequences were assembled using CLC Main workbench software v.5 (CLC bio, Cambridge, MA, USA). The pv12 and pv38 genes were analysed and compared to reference sequences obtained from several sequencing projects [43, 44] (accession numbers, pv12: XM_001616094.1, AFBK01001496.1, AFNI01000939.1, AFMK01001167.1 and AFNJ01001458.1; pv38: XM_001613202.1, AFNI01000834.1, AFNJ01000090.1, AFMK01001057.1 and AFBK01001340.1) or those reported in the GenBank database (accession numbers for pv12: GU476521.1; and for pv38: JF427569.1 and JF427570.1). Gene Runner software was used for translating the sequences for deducing the amino acid sequences. These sequences were then aligned using the MUSCLE algorithm , and manually edited. Amino acid alignment was then used for inferring DNA using PAL2NAL software .
DnaSP software (v.5)  was used for evaluating intrapopulation genetic polymorphism by calculating: the number of polymorphic segregating sites (Ss), the number of singleton sites (s), the number of parsimony-informative sites (Ps), the number of haplotypes (H), haplotype diversity (Hd, which was multiplied by (n-1)/n according to Depaulis and Veuille [47, 48]), the Watterson estimator (θw) and nucleotide diversity per site (π). DNA sequence variation was calculated using the sequences obtained from the aforementioned databases, plus the Colombian ones (worldwide isolates, global diversity) and just those obtained for the Colombian population (local diversity). The frequency for each Colombian haplotype was also estimated by count and year.
Two test families were used for evaluating the neutral molecular evolution model for the Colombian population: (1) frequency spectrum test, and (2) haplotype test. The former involved calculating Tajima’s D statistics , Fu and Li’s D* and F*  and Fay and Wu’s H statistic . Tajima’s D statistic compares the difference between segregating sites and the average of nucleotide differences between two randomly taken sequences. Fu and Li’s D* statistic takes the difference between the number of singleton sites and the total of mutations, whilst F* takes the difference between the number of singleton sites and the average of nucleotide differences between two randomly taken sequences. Fay and Wu’s H statistic is based on the difference of the average number of nucleotide differences between pairs of sequences and the frequency of the derived variants. Fu’s Fs statistic , K-test and H-test  are tests for calculating haplotype distribution. The Fs statistic compares the number of haplotypes observed to the expected number of haplotypes in a random sample. K-test and H-test  are based on haplotype number and haplotype diversity, respectively; these statistics are conditioned by sample size (n) and the number of segregating sites (Ss). Test significance was determined by coalescence simulations using DnaSP (v.5)  and ALLELIX software (kindly supplied by Dr Sylvain Mousset). Sites having gaps were not taken into account in any of the tests performed.
The effect of natural selection was evaluated regarding intra and interspecies; the average number of non-synonymous substitutions per non-synonymous site (dN) and the average number of synonymous substitutions per synonymous site (dS) were calculated for the former by using the modified Nei-Gojobori method . The significant differences between the above were determined by using Fisher’s exact test (suitable for dN and dS < 10) and codon-based Z-test incorporated in MEGA software (v.5) . Differences between dN and dS per site were calculated by using SLAC, FEL, REL , IFEL , MEME , and FUBAR  methods. The average number of non-synonymous divergence substitutions per non-synonymous site (KN) and the average number of synonymous divergence substitutions per synonymous site (KS) were calculated using the modified Nei-Gojobori method , with Jukes-Cantor correction , to infer natural selection signals which may have prevailed during malarial parasite evolutionary history (interspecies; using Plasmodium cynomolgi (accession number BAEJ01001076.1) and P. knowlesi (accession number NC_011912.1) orthologous sequences). The significant differences between KN and KS were determined by using a codon-based Z-test incorporated in MEGA software (v.5) . The McDonald-Kreitman test  was also calculated; this is based on a comparison of intraspecific polymorphism to interspecific divergence (using Plasmodium cynomolgi (accession number BAEJ01001076.1) and P. knowlesi (accession number NC_011912.1) orthologous sequences). This test involved using a web server , which takes Jukes-Cantor divergence correction into account . All the above tests were calculated using the sequences obtained from the databases plus the Colombian ones and just those obtained for the Colombian population.
ZnS and ZZ  statistics were calculated for evaluating the influence of linkage disequilibrium (LD) and intragenic recombination, respectively. The minimum number of recombination (Rm) events was also calculated; this included calculating effective population size and the probability of recombination between adjacent nucleotides per generation . Additionally, the GARD method  available at the Datamonkey web server  was performed. These tests were performed using the sequences obtained from the Colombian population.
Results and discussion
The presence of genomic DNA (gDNA) and identification of single Plasmodium vivax strain infection
An 18S subunit rRNA gene fragment was amplified from 100 samples of P. vivax collected from different areas of Colombia and stored from 2007 to 2010. Seventy-seven samples revealed an amplicon at the expected size, indicating the presence of P. vivax gDNA. A region of the pvmsp-1 gene was then amplified and digested with restriction enzymes, showing that seven of the 77 samples proving positive for P. vivax had multiple infections. Only 70 samples were thus considered for later analysis. Due to the low number of samples collected from some areas, they were grouped according to geographical localisation and epidemiological conditions (South-west: Chocó, Nariño; South-east: Caquetá, Guainía, Guaviare, Meta; Midwest: Bogota, Tolima; North-west: Atlántico, Antioquia Cordoba, Magdalena).
Genetic diversity in pv12
Seventy samples amplified a 1,200 base pair (bp) fragment corresponding to the pv12 gene (South-west n = 6; South-east: n = 20; Midwest: n = 8; North-west: n =36). These amplicons were purified and sequenced; the sequences were then analysed, compared to different reference sequences obtained from various sequencing projects [43, 44] and those having a different haplotype were deposited in the GenBank database (accession numbers KF667328 and KF667329).
Estimators for pv12 and pv38 global and local genetic diversity
This gene had 0.0004 ± 0.0001 global nucleotide diversity (π) and 0.0003 ± 0.0001 for the Colombian population (Table 1). This value was about 2.5 times less than that reported for its orthologue in P. falciparum (π = 0.001) ; however, both values were low when compared to other membrane proteins [10–14, 17], suggesting that this gene is highly conserved in different Plasmodium species. This value places pv12 among the most conserved antigen-encoding genes characterized to date in P. vivax.
Mutations in pv12 appear to be selectively neutral
pv12 and pv38 neutrality, linkage disequilibrium and recombination tests for the Colombian population
Fu and Li
Fay and Wu’s H
Natural selection in pv12
Synonymous substitution per synonymous site rate (d S ) and non-synonymous substitution per non-synonymous site rate (d N ) for pv12 and pv38 genes
Full length gene
Synonymous divergence substitution per synonymous site (K S ) rate and non-synonymous divergence substitution per non-synonymous site (K N ) rate
P. vivax/P. Cynomolgi
s48/45 domain in region A
s48/45 domain in region B
P. vivax/P. knowlesi
s48/45 domain in region A
s48/45 domain in region B
K S (se)
K N (se)
K S (se)
K N (se)
K S (se)
K N (se)
McDonald-Kreitman test for evaluating the action of natural selection
P. vivax/P. cynomolgi
P. vivax/P. knowlesi
Genetic diversity in pv38
Only 46 out of 70 samples could be amplified for the pv38 gene, giving a 1,121 bp fragment (South-west n = 6; South-east: n = 13; Midwest: n = 4; North-west: n = 23). The 46 sequences obtained from Colombian isolates were compared to and analysed regarding reference sequences obtained from different regions of the world [43, 44]. Colombian sequences that have a different haplotype to that of previously reported ones can be found in GenBank (accession numbers KF667330-KF667340).
Nine SNPs were observed in the pv38 gene (Figure 1B), most of which were no-synonymous (nucleotides: 88 (R30S), 206/207 (A69V), 209 (R70L), 524/525 (T175N), 880 (M294L), and 998 (S333N)), similar to that found in Pf38. Positions 525 and 969 produced synonymous substitutions (a change in protein sequence was generated when the substitution in position 525 was accompanied with another one in position 524). The parasite population in Colombia has eight of these nine SNPs, all being informative-parsimonious sites. Similar to that reported for its orthologue in P. falciparum, most substitutions were found in the gene’s 5′ region.
Seventeen haplotypes were identified from alignment (including sequences from different regions of the world) (Figure 1B), 14 of which were found in Colombia’s parasite population at different frequencies: 11% haplotype 1, 4% haplotype 5, 2% haplotype 6, 20% haplotype 7, 15% haplotype 8, 7% haplotype 9, 2% haplotype 10, 4% haplotype 11, 13% haplotype 12, 4% haplotype 13, 2% haplotype 14, 4% haplotype 15, 7% haplotype 16, and 4% haplotype 17. Most haplotypes were found in intermediate frequencies per year (2007 n = 6; 2008 n = 6; 2009 n = 8; 2010 n = 26) and none exceeded 50% (Figure 2B). The absence of some haplotypes in determined years, or in some locations, could not just have been due to the low frequency which they might have had but also to the difference in the number of samples for each year (n = 6 in 2007, n = 6 in 2008, n = 8 in 2009 and n = 26 in 2010) or because American P. vivax populations appear to be structured and therefore several privative haplotypes might be found .
π in this gene was 0.0026 ± 0.0002 worldwide and 0.0024 ± 0.0002 in the Colombian population (Table 1), this being 1.3 times lower than that for its orthologue in P. falciparum (π = 0.0034) [68, 73] showing that the pv38 gene had low diversity, at least in the two main species affecting human beings.
Deviation from the neutral model of molecular evolution in pv38
Tajima’s D, Fu and Li’s D* and F*, Fay and Wu’s H and Fu’s Fs neutrality tests did not reveal statistically significant values (Table 2), suggesting that the gene might follow the neutral evolution model. However, the presence of 14 haplotypes and 0.890 ± 0.02 haplotype diversity in the Colombian population was greater than that expected under neutrality according to K-test and H-test results (Table 2). This suggested balanced ancestral polymorphism , being similar to that reported for the P. falciparum p38 gene which showed evidence of balanced selection in 5’ region .
Natural selection in pv38
A modified Nei Gojobori method was used for calculating dN and dS rates for showing some type of selection in the pv38 gene. Similar to that used regarding pv12, the pv38 gene was divided into two regions: region A, covering position 1-459 (amino acids 1-153) and region B, nucleotides 460-1,065 (amino acids 154-355 including the s48/45 domain). There were more dN substitutions in region A than dS substitutions, whilst there were more dS substitutions in region B than dN ones, even though no significant values were observed (Table 4 and Additional file 2). Selection tests by codon revealed positive selection in codon 70 and negative selection in codons 175 and 323, suggesting that the gene was influenced by selection. When the long-term effect of natural selection was explored by comparing divergence rates (KS and KN), pv38 had a higher statistically significant KS rate than KN (Table 4), revealing ω values below 1 throughout the gene (Figure 3), suggesting divergence by negative selection.
The McDonald-Kreitman test revealed statistically significant values (Table 4), when intraspecific polymorphism and interspecific divergence was compared, showing PN/PS > DN/DS (p < 0.02). This result could have been the result of either a negative selection or a balanced selection [61, 74]. K-test and H-test results (Table 2) and the presence of different haplotypes at intermediate frequencies (Figure 2B) suggested that it is most probable that pv38 was influenced by balanced selection, similar to that reported for P. falciparum. Such selection seemed to be domain specific. Significant values were observed for region A (p = 0.014) when intraspecific polymorphism and interspecific divergence was calculated in each region (Additional file 3), this being where most of the substitutions found became accumulated, whilst neutrality could not be ruled out for region B (p = 0.1). Functional/structural constraint due to the presence of an s48/45 domain was also probable for pv38, given this region’s low diversity, two negatively selected sites and a statistically significant KS > KN.
Linkage disequilibrium (LD) and recombination
Several statistics were calculated for determining possible associations between polymorphisms and/or the presence of recombination in pv38. ZnS did not reveal statistically significant values, indicating that pv38 polymorphisms were not associated. Lineal regression between linkage disequilibrium (LD) and nucleotide distance revealed a reduction in LD as nucleotide distance increased, indicating that intragenic recombination might have led to new variations being produced.
The ZZ statistic was calculated to confirm whether recombination affected pv38 evolution, showing no significant values (Table 2); however, 2 RM (minimum recombination events) were found. The GARD method (in Datamonkey web server) gave a recombination breakpoint in position 524. Prior studies have suggested that new haplotypes could be produced through recombination in spite of functional constraints . Intragenic recombination could thus be one of the factors promoting diversity in the pv38 gene. Crosslinking during recombination could produce new combinations between the gene’s 5′ (region A) and 3′ region (region B) as the breakpoint found in this gene was located upstream of the region encoding the s48/45 domain (region B). As only one polymorphic site was found in pv12, the aforementioned tests were not carried for this gene.
pv12 and pv38 should be considered for an antimalarial vaccine
The lack of a totally effective vaccine against human malarial parasites is at least partly due to high genetic diversity found in proteins involved in red blood cell invasion. These molecules’ constant exposure to the host’s immune system allows the fixation of mutations generating an adaptive advantage preventing their recognition. Antigens such as pvmsp-1, pvdbp, pvmsp-3 α, pvmsp-5, pvmsp-7C, pvmsp-7H, pvmsp-7I and pvama-1 have shown high genetic diversity which appears to be maintained by positive-balancing selection [10–15, 75–78]; however, other antigens are highly conserved despite being exposed to the host’s immune system. Surface antigens such as pvmsp-4, pvmsp-7A, pvmsp-7 K, pvmsp-8, pvmsp-10, pv230 or others in the rhoptries (pvrap-1 and pvrap-2) appear to evolve more slowly due to a possible functional constraint in their encoded proteins [70, 71, 79–82]. Thus, most mutations have become eliminated from the population, maintaining a conserved protein structure, even throughout these parasites’ evolutionary history [70, 71]. The latter behaviour seems to have been directing pv12 and pv38 evolution, highlighting high conservation at both intra- and inter-species level due to the influence of negative selection exerted on s48/45 domains which are important for red blood cell recognition . Although antigens having low genetic diversity are usually not immunogenic  nor do they induce protection-inducing responses , some limited polymorphism antigens have been shown to be able to induce immunogenicity and protection . Therefore, pv12 and pv38 (or their s48/45 domains) should be evaluated regarding vaccine development because immune responses against 6-Cys family antigens appear to be directed against structural epitopes in s48/45 domains [86–88], blocking such domains should prevent invasion [30, 88] and being highly conserved and having a functional constraint, allele-specific immune responses are thus avoided.
The p12 and p38 genes in P. vivax were seen to have low genetic diversity; the regions encoding the s48/45 domains seemed to be functionally or structurally constrained. Several members of the 6-Cys family are found on the surface of malaria parasites in every stage [28, 36–39, 69] and some of them (e g, P48/45, P230) are considered to be promising (transmission-blocking) vaccine candidates [36, 37, 87]. Epitopes identified by monoclonal antibodies against this type of protein are structural and have been localized within s48/45 domains [86, 87] which seem to be involved in host-pathogen interaction [30, 72]. Since pv12 and pv38 share structural characteristics with members of the 6-Cys family, added to their antigenic characteristics [38–40] and the low genetic diversity found in this study, the proteins encoded by these genes or their functionally/structurally constrained (conserved) regions could be born in mind when designing a multistage, multi-antigen subunit-based anti-malarial vaccine.
We would especially like to thank Dr Sylvain Mousset who provided the ALLELIX software for our analysis. We would also like to thank Jason Garry for translating the manuscript and Professor Manuel E. Patarroyo for his comments and suggestions. This work was financed by the “Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS)” through contract RC # 0309-2013. JF-R received financing through COLCIENCIAS cooperation agreement # 0757-2012.
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