Genetic polymorphism of merozoite surface protein-3 in Myanmar Plasmodium falciparum field isolates



Plasmodium falciparum merozoite surface protein-3 (PfMSP-3) is a target of naturally acquired immunity against P. falciparum infection and is a promising vaccine candidate because of its critical role in the erythrocyte invasion of the parasite. Understanding the genetic diversity of pfmsp-3 is important for recognizing genetic nature and evolutionary aspect of the gene in the natural P. falciparum population and for designing an effective vaccine based on the antigen.


Blood samples collected from P. falciparum-infected patients in Naung Cho and Pyin Oo Lwin, Myanmar, in 2015 were used in this study. The pfmsp-3 was amplified by polymerase chain reaction, cloned, and sequenced. Genetic polymorphism and natural selection of Myanmar pfmsp-3 were analysed using the programs DNASTAR, MEGA6, and DnaSP 5.10.00. Genetic diversity and natural selection of the global pfmsp-3 were also comparatively analysed.


Myanmar pfmsp-3 displayed 2 different alleles, 3D7 and K1. The 3D7 allelic type was predominant in the population, but genetic polymorphism was less diverse than for the K1 allelic type. Polymorphic characters in both allelic types were caused by amino acid substitutions, insertions, and deletions. Amino acid substitutions were mainly occurred at the alanine heptad repeat domains, whereas most insertions and deletions were found at the glutamate rich domain. Overall patterns of amino acid polymorphisms detected in Myanmar pfmsp-3 were similar in the global pfmsp-3 population, but novel amino acid changes were observed in Myanmar pfmsp-3 with low frequencies. Complicated patterns of natural selection and recombination events were predicted in the global pfmsp-3, which may act as major driving forces to maintain and generate genetic diversity of the global pfmsp-3 population.


Global pfmsp-3 revealed genetic polymorphisms, suggesting that the functional and structural consequences of the polymorphisms should be considered in designing a vaccine based on PfMSP-3. Further examination of genetic diversity of pfmsp-3 in the global P. falciparum population is necessary to gain in-depth insight for the population structure and evolutionary aspect of global pfmsp-3.


44-48 kDa via removal of the N-terminal region in the parasitophorous vacuole [1,2]. PfMSP-3 plays critical roles in erythrocyte binding of the parasite and protecting the parasite against toxic haem that is inevitably produced by haemoglobin hydrolysis [3]. It is also reported that PfMSP-3 is a target of host immune responses. PfMSP-3 induced naturally acquired immunity by producing antibodies that mediate antibody-dependent cellular inhibition of the parasite [3]. Immunization of PfMSP-3 induced protective immunity in animal models [4,5]. Moreover, anti-PfMSP-3 antibodies suppressed the growth of P. falciparum in vitro in the presence of blood monocytes [2,6]. Vaccines formulated with PfMSP-3 alone or in combination with other vaccine candidate antigens have been designed and their effectiveness in inducing production of cytophilic antibodies that could inhibit P. falciparum erythrocytic growth has been evaluated [7,8]. GMZ2, the vaccine formulated with a chimeric antigen constructed with the C-terminal region of PfMSP-3 and the N-terminal region of glutamate rich protein (GLURP), was well tolerated and immunogenic, and elicited high levels of functional antibodies suppressing multiplication of the parasite [9][10][11][12]. The essential biological functions of PfMSP-3 during parasite survival and the accumulated evidence that specific antibodies for PfMSP-3 are effective in parasite killing and protection against clinical malaria suggest that this protein as a promising vaccine candidate antigen.
Genetic polymorphisms in vaccine candidate antigens of P. falciparum are great hurdles to develop effective vaccines since they could generate a variant-specific immune response that is less effective against parasites with other genetic variants. Like other vaccine candidate antigens, the genetic diversity of pfmsp-3 was also reported in natural parasite populations [13][14][15][16]. The pfmsp-3 consists of 3 separated regions: the N-terminal region containing four alanine heptad repeats (AHR) having the AXXX-AXXX motif, the glutamate rich domain, and the C-terminal region containing a leucine zipper motif [17][18][19][20]. The gene is classified into 2 different alleles, 3D7 and K1, based on the difference in size of the AHR at the N-terminal region [17]. It has been proposed that the N-terminal region was polymorphic, whereas the C-terminal region was relatively well conserved in P. falciparum populations in several countries including Iran [14], Thailand [13,15], Cameroon, Republic of Congo, Burkina Faso, Ghana, and Senegal [16]. In-depth understanding of the population genetic structure of pfmsp-3 in the natural P. falciparum population is important not only for evaluating the genetic nature of the vaccine candidate antigen, but also for advancing effective vaccine development.
However, compared to other vaccine candidate antigens, fewer studies have been done to understand the genetic polymorphisms of pfmsp-3 in the natural P. falciparum population. In this study, the genetic diversity and natural selection of pfmsp-3 in Myanmar P. falciparum isolates were analysed. Comparative analysis of global pfmsp-3 was also performed to gain in-depth understanding of the genetic make-up of the gene in the global P. falciparum population.

Blood samples and extraction of genomic DNA
Seventy-two blood samples were obtained from P. falciparum infected symptomatic patients who live in villages located in Naung Cho and Pyin Oo Lwin, Myanmar in 2015 (Additional file 1: Fig. S1). P. falciparum infection was confirmed by Giemsa-stained thick and thin blood smear examination. All P. falciparum positive samples were further confirmed by polymerase chain reaction (PCR) targeting 18S ribosomal RNA (rRNA) gene [21]. Finger-prick blood samples were collected from the patients prior to drug treatment, air-dried, and stored in individual sealed plastic bag at ambient temperature until use. Genomic DNA was extracted from the blood filters using the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Written informed consent was obtained from all patients before blood collection. The study protocol was approved by either the Ethics committee of the Ministry of Health, Myanmar (97/Ethics 2015) and the Biomedical Research Ethics Review Board of Inha University School of Medicine, Republic of Korea (INHA 15-013).

Amplification and sequencing analysis of pfmsp-3
The full-length pfmsp-3 was amplified by a nested PCR method. The primers used in this study were designed as described previously [13]. The thermal cycling parameters for primary and nested PCRs were as follows: one cycle of initial denaturation at 95 °C for 5 min, 25 cycles of 94 °C for 1 min, annealing at 57 °C for 2 min and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 5 min. Ex Taq DNA polymerase (Takara, Otsu, Japan) with proof-reading activity was used in all PCR amplification steps to minimize the nucleotide misincorporation. PCR products were resolved on a 1.2% agarose gel, and were visualized under ultraviolet (UV). Each PCR product was purified from the gel, and cloned into the T&A vector (Real Biotech Corporation, Banqiao City, Taiwan). Ligation mixture was transformed into Escherichia coli DH5α competent cells, and positive clones with appropriate insert were selected by colony PCR. The nucleotide sequence of cloned insert was analysed by automatic DNA sequencing with M13 forward and M13 reverse primers. Plasmids from at least two independent clones from each transformation mixture were sequenced in both directions to verify the sequence accuracy. The nucleotide sequences reported in this study have been deposited in the GenBank database under the accession numbers MN787600-MN787671.

Analyses of sequence polymorphism and genetic diversity of Myanmar pfmsp-3
The nucleotide and deduced amino acid sequences of pfmsp-3 were analysed using EditSeq and SeqMan in the DNASTAR package (DNASTAR, Madison, WI, USA). The pfmsp-3 sequences of 3D7 (XM_001347593) and K1 (U08851) were used as reference sequences. The value of singleton variable sites, parsimony informative sites, total number of mutations, segregating sites (S), average number pair-wise nucleotide difference (K), haplotype diversity (Hd), and nucleotide diversity (π) were calculated using DnaSP version 5.10.00 [22]. The π was also measured on sliding window plot of 10 bases with a step size of 5 bp to estimate the stepwise diversity across the sequences. The rate of synonymous (dS) and non-synonymous (dN) substitutions were estimated and compared by the Z-test (P < 0.05) in order to test the null hypothesis of strict neutrality of pfmsp-3 in MEGA6 program [23] using the Nei and Gojobori's method [24] with the Juke and Cantor correction (1000 bootstrap replications). Tajima's D test [25], and Fu and Li's D and F statistics [26] were performed to evaluate the neutral theory of natural selection using DnaSP version 5.10.00 [22]. The Tajima's D values were also calculated on sliding window plot of 10 bases with a step size 5 bp to estimate the natural selection parameter across the gene.

Genetic diversity of pfmsp-3 among the global P. falciparum isolates
The genetic diversity of global pfmsp-3 from different P. falciparum populations was analysed. The pfmsp-3 sequences included in this study were from Thailand (AM161544-AM161593), India (HM568669-HM568724), Kenya (KU527019-KU527058), and Nigeria (AM161594-AM161644). These sequences covered full-length or partial of pfmsp-3. Genetic polymorphism and tests of neutrality of each population were analysed or calculated using DNASTAR package, DnaSP version 5.10.00 [22] and MEGA6 [23] as describe above. A logo plot was constructed for each pfmsp-3 population to analyse the polymorphic patterns in the B cell epitopes using the WebLogo program (https ://weblo go.berke ley.edu/ logo.cgi).

Sequence polymorphism and genetic diversity of global pfmsp-3
Comparative analysis of genetic polymorphism of pfmsp-3 from Myanmar and other different geographical areas was performed. Given the partial sequence information of pfmsp-3 from other global populations, analysis focused on the 3 regions corresponding to AHR, the glutamate rich domain and the leucine zipper domain. For 3D7 allelic types, most amino acid polymorphisms were accumulated at glutamate rich domains, but the frequencies were different by country (Fig. 3). The deletion of E272 (E272-) and an amino acid change (D280E) were found in all analysed populations. Insertions of 1 to 3 glutamate residues at the position of 272 (E272 + , E272 ++, and E272 +++) were more prevalent in African pfmsp-3 than in Asian pfmsp-3. The AHR and leucine zipper domains were relatively well conserved in 3D7 allelic types of global pfmsp-3. Amino acid changes at 9 positions were identified in AHR domains of global pfmsp-3, most of them only in Myanmar pfmsp-3. Only 2 amino acid changes (A103S and K164E) were found in Thailand and Kenya pfmsp-3. The five amino acid substitutions (N311D, K331E, G332E, Q335R, and V344I) in the leucine zipper domain were detected only in Myanmar pfmsp-3. Polymorphic patterns of amino acid changes were more diverse and greater in global K1 allelic types (Fig. 4). Most amino acid changes detected at AHR domains were shared by the global pfmsp-3 population with high frequencies. Only five amino acid substitutions, G75I, T81C, S98F, T165A, and K188R, were uniquely identified in Myanmar pfmsp-3. Although the  The amino acid residues constructing the B-cell epitopes were tightly conserved in global pfmsp-3 allelic types (Fig. 5). However, di-morphic and tri-morphic amino acid changes at the corresponding residues were detected in Myanmar pfmsp-3.

Genetic diversity and natural selection of global pfmsp-3
Influence of natural selection on the genetic diversity of the global pfmsp-3 populations was analysed. Nucleotide diversity and patterns of natural selection predicted in 3D7 and K1 allelic types were different by country (Table 1). In the 3D7 allelic types, the values of nucleotide diversity (π) were similar for the pfmsp-3 of each country, ranging from 0.0030 to 0.0050. The dN-dS value was negative for all countries analysed, indicating that negative natural selection may occur in global pfmsp-3. However, the value of Tajima's D was different by country: negative for Myanmar and Kenya, but positive for Nigeria and Thailand. The values of Fu and Li's D and F for each country also showed a similar trend of negative or positive values. Meanwhile, more complex patterns of natural selection were predicted in K1 allelic types ( Table 1). The values of nucleotide diversity (π) of K1 allelic types were greater than those of 3D7 allelic types in all countries analysed. The values ranged from 0.0230 to 0.0420. The value of dN-dS was positive in Kenya, but in other countries including Myanmar, Nigeria, and Thailand was negative. Unlike the 3D7 allelic types, the values of Tajima's D of all countries were positive, indicating they were under balancing selection. The values of Fu and Li's D and F were also positive in all three countries except for Myanmar. These collectively suggested that the overall genetic diversity in the K1 allelic types was greater than in the 3D7 allelic types in global pfmsp-3. Both allelic types of global pfmsp-3 may be affected by natural selection, but the direction of natural selection differed by country.

Discussion
PfMSP-3 is one of the leading vaccine candidates for malaria vaccine development, but its genetic makeup and evolutionary aspect in the global P. falciparum population are less understood that are other major vaccine candidate antigens, such as MSP-1, MSP-2, apical membrane antigen-1 (AMA-1), and circumsporozoite surface protein (CSP) of the parasite. In this study, genetic diversity and natural selection of Myanmar pfmsp-3 were analysed. Two allelic types of pfmsp-3, 3D7 and K1, were identified in Myanmar pfmsp-3 with the predominance of the 3D7   0   50   100   S96T  E98G  A103T  A103S  T117A  D142E  D142G  A163V  K164E  Q179H  E186K  K236E  E245G  E249G  E253K  T256P  S261T  D263E  E267V  E271-E272-E272K  272E+  272E++  272E+++  E274R  E275K  E26K  N277K  D280E  D280M  K281E  K285T  N311D  K331E  G332E  Q335R  allelic type (59.7%). This is consistent with other reports that 3D7 allelic types are more prevalent in Peru [27], East Republic of Congo and Burkina Faso [16], and the Thailand-Laos border [15]. Meanwhile, the recombinant allelic type, which was reported in pfmsp-3 from Iran and Africa countries with a very low frequency [14,16], was not identified in Myanmar pfmsp-3. The lack of a recombinant allelic type may result from the large sequence difference between the two dimorphic alleles, which impedes recombination during meiosis. Otherwise, the sequence dimorphism among pfmsp-3 may be strictly maintained in the population by balancing selection [28,29]. However, recombination between and among the same allelic type of Myanmar pfmsp-3 is likely to occur, considering the Rm value in each allelic type. Particularly, the K1 allelic type of Myanmar pfmsp-3 revealed many recombination events, indicating that active intragenic recombination is occurring in the population.  T81S  T81C  E85Q  M88I  E96Q  S98F  A112S  E113K  Q114E  Q114D  E116G  Q117E  S119A  K120Q  D121E  E123V  K124N  E130K  V145A  S161T  T165A  N166S  E167A  T168D  G169P  G169-R171K  N172P  N175I  F177L  Y178F  T179S  T180R  G187E  K188Q  K188R  V189A  E190K  E235K  K248E  E271G  280ETEEEELEEKNNE+  D289G  E297K  E297-298E+  298E++  298E+++  K310R  N335S  N336S  T350A  A352T  were shared by global pfmsp-3, but their frequencies differed by geographical origin. Interestingly, novel amino acid changes that were not reported previously for either allelic type of Myanmar pfmsp-3, which renders genetic diversity of Myanmar pfmsp-3 more complex; 31 and 25 haplotypes for the 3D7 and K1 allelic types, respectively. Although incidence of malaria has been remarkably decreased in Myanmar in recent years [30], it seems likely that much genetic diversity is still maintained in Myanmar P. falciparum population. Regardless of the declining population in recent years, substantial levels of genetic heterogeneities in pfmsp-1, pfmsp-2, pfama-1, and pfcsp have been reported in the Myanmar P. falciparum population [31][32][33][34]. The reason for this is not clearly demonstrated yet and therefore further study to understand this phenomenon would be necessary. Despite different rates of amino acid changes and insertions/deletions in pfmsp-3 from each country, the overall patterns of amino acid polymorphisms were similar in the global pfmsp-3 population. Major amino acid changes and insertions/deletions found in both 3D7 and K1 allelic types were observed at the AHR and glutamate rich domains. The repeats of the AXXAXXX motif, which plays a role in maintaining and forming an α-helical secondary structure of PfMSP-3 [19], were highly conserved in global pfmsp-3 regardless of diversity within and flanking the ARH. The B-cell epitopes of 3D7 allelic types were also well conserved in the global pfmsp-3 population. The N-terminal region of pfmsp-3 including the B-cell epitopes is a target of naturally acquired immunity. In particular, the regions corresponding to amino acids 21 to 238 not only is an important immunogen to produce a protective immune response but also is involved in the interaction with PfMSP-1 [35]. The prevalence of opsonizing antibodies against the N-terminal region rather than the C-terminal region of pfmsp-3 also suggests a critical role of the N-terminal region as a target of opsonizing antibodies. Therefore, the N-terminal region of pfmsp-3 may represent an attractive candidate for formulation of a PfMSP-3-based vaccine. Meanwhile, the leucine zipper domain was relatively well conserved in global pfmsp-3. The limited polymorphic pattern of the C-terminal region, especially in the leucine zipper domain, may result from its crucial function in the formation of protein oligomerization [3,36]. Moreover, the C-terminal region of pfmsp-3 plays an essential function in the invasion process of merozoite by mediating specific binding with erythrocytes [37].
The dN-dS values for 3D7 allelic types of global pfmsp-3 were negative, implying that negative selection might act in the gene. Meanwhile, the values of Tajima's D and Fu and Li's D and F revealed complicated patterns that were different by country. These suggest that a complicated natural selection may act on 3D7 allelic types of the global pfmsp-3, in which either positive selection or purifying selection might have occurred in the population, depending on the geographical origin. As expected, overall nucleotide diversity in K1 allelic types was greater than that in 3D7 allelic types in the global isolates. The positive values of Tajima's D indicated that K1 allelic types of global pfmsp-3 evolved under balancing selection. Amino acid substitutions, insertion or deletions may be the main pressure to generate genetic diversity of the allelic types. High recombination parameters also support the notion that recombination may be one of a force contributing to generate genetic diversity of K1 allelic types of global pfmsp-3.

Conclusion
Myanmar pfmsp-3 showed genetic polymorphisms. Two different alleles, 3D7 and K1, were detected in Myanmar pfmsp-3, with a higher prevalence of 3D7 allelic types. Overall patterns of nucleotide diversity and distributions of amino acid changes and insertions/deletions found in Myanmar pfmsp-3 were similar to those from global pfmsp-3, even though minute regional differences were also identified between and among each population. Complicated patterns of natural selection and recombination events were predicted in the global pfmsp-3, which may act as major driving forces to maintain and generate genetic diversity of the global pfmsp-3 population. These results suggest that the functional consequences of the polymorphism should be considered for development of a vaccine based on PfMSP-3. This study provides in-depth insight into the genetic nature and population structure of global pfmsp-3. Considering that rather few global pfmsp-3 sequences were currently available, further examination of the genetic diversity and evolutionary aspect of pfmsp-3 with more global pfmsp-3 sequences would be necessary to better understand the genetic make-up of pfmsp-3 in the natural P. falciparum population.