Changing pattern of the genetic diversities of Plasmodium falciparum merozoite surface protein-1 and merozoite surface protein-2 in Myanmar isolates
Plasmodium falciparum merozoite surface protein-1 (PfMSP-1) and -2 (PfMSP-2) are major blood-stage vaccine candidate antigens. Understanding the genetic diversity of the genes, pfmsp-1 and pfmsp-2, is important for recognizing the genetic structure of P. falciparum, and the development of an effective vaccine based on the antigens. In this study, the genetic diversities of pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum were analysed.
The pfmsp-1 block 2 and pfmsp-2 block 3 regions were amplified by polymerase chain reaction from blood samples collected from Myanmar patients who were infected with P. falciparum in 2013–2015. The amplified gene fragments were cloned into a T&A vector, and sequenced. Sequence analysis of Myanmar pfmsp-1 block 2 and pfmsp-2 block 3 was performed to identify the genetic diversity of the regions. The temporal genetic changes of both pfmsp-1 and pfmsp-2 in the Myanmar P. falciparum population, as well as the polymorphic diversity in the publicly available global pfmsp-1 and pfmsp-2, were also comparatively analysed.
High levels of genetic diversity of pfmsp-1 and pfmsp-2 were observed in the Myanmar P. falciparum isolates. Twenty-eight different alleles of pfmsp-1 (8 for K1 type, 14 for MAD20 type, and 6 for RO33 type) and 59 distinct alleles of pfmsp-2 (18 for FC27, and 41 for 3D7 type) were identified in the Myanmar P. falciparum population in amino acid level. Comparative analyses of the genetic diversity of the Myanmar pfmsp-1 and pfmsp-2 alleles in the recent (2013–2015) and past (2004–2006) Myanmar P. falciparum populations indicated the dynamic genetic expansion of the pfmsp-1 and pfmsp-2 in recent years, suggesting that a high level of genetic differentiation and recombination of the two genes may be maintained. Population genetic structure analysis of the global pfmsp-1 and pfmsp-2 also suggested that a high level of genetic diversity of the two genes was found in the global P. falciparum population.
Despite the recent remarkable decline of malaria cases, the Myanmar P. falciparum population still remains of sufficient size to allow the generation and maintenance of genetic diversity. The high level of genetic diversity of pfmsp-1 and pfmsp-2 in the global P. falciparum population emphasizes the necessity for continuous monitoring of the genetic diversity of the genes for better understanding of the genetic make-up and evolutionary aspect of the genes in the global P. falciparum population.
KeywordsPlasmodium falciparum Merozoite surface protein-1 Merozoite surface protein-2 Genetic diversity Myanmar
artemisinin-based combination therapy
Greater Mekong Subregion
multiplicity of infection
polymerase chain reaction (PCR)
merozoite surface protein-1 of Plasmodium falciparum
merozoite surface protein-2 of Plasmodium falciparum
Papua New Guinea
recombination parameter between adjacent sites
recombination parameter for entire gene
minimum number of recombination events between adjacent polymorphic sites
18S ribosomal RNA
Although global malaria cases have remarkably decreased in recent years, malaria is still one of the most important public health concerns worldwide, with an estimated 219 million cases and 435,000 related deaths in 2016 . Many efforts have been undertaken to develop an effective malaria vaccine, but to date, there is no available licensed malaria vaccine. The Greater Mekong Subregion (GMS) has long been one of the most malarious regions in the world . Among the countries in GMS, Myanmar has the highest malaria burden, accounting for an estimated 77% of malaria cases, and approximately 79% of malaria deaths in the GMS . Several interventions have been made in the country to reduce the malaria burden, including the training and deployment of community health workers, the distribution of insecticide-treated bed nets, strategies to improve access to rapid diagnostic tests, and the provision of artemisinin-based combination therapy (ACT) . Due to these nationwide efforts, the annual incidence of malaria in Myanmar has been greatly reduced, with an 81.1% decline between the years 2005 and 2014. However, the recent rise and spread of artemisinin resistance parasite is the greatest threat to the effective control and elimination of malaria in the country .
Understanding the population genetic structure of malaria parasites is necessary to determine the epidemiology, diversity, distribution, and dynamics of the natural population of malaria parasites. Plasmodium falciparum merozoite surface protein-1 (PfMSP-1) and -2 (PfMSP-2) are major blood-stage vaccine candidate antigens, which play important roles in erythrocyte invasion [6, 7], and are targeted by host immune responses [8, 9, 10, 11, 12]. They show high polymorphic patterns in different geographical settings, and have been considered as suitable polymorphic markers for genotyping genetically distinct P. falciparum sub-populations [13, 14, 15, 16, 17]. PfMSP-1 is initially synthesized as a large molecular size precursor with an approximate size of 190 kDa, and then undergoes post-translational proteolytic processing into four fragments, 83 kDa, 30 kDa, 38 kDa and 42 kDa . These fragments persist as a non-covalent linked complex on the surface of mature merozoites [19, 20]. The pfmsp-1 is divided into 17 distinct blocks that are conserved, semi-conserved, and variable [19, 21, 22], among which block 2 is the most polymorphic part of the gene, and is grouped into three allelic types, namely K1, MAD20, and RO33, based on their sequence polymorphic patterns [21, 23]. PfMSP-2 is a glycoprotein that consists of highly polymorphic central repeats (block 3), flanked by unique variable domains (block 2 and block 4), and conserved N- and C-terminal regions. The pfmsp-2 alleles are in general grouped into two dimorphic families, FC27 and 3D7, which are based on block 2 and block 4, while repeat regions in block 3 also differ in the number and sequence of repeat units [24, 25, 26].
Similar to the P. falciparum population in other malaria endemic areas, the Myanmar P. falciparum population has also shown high levels of genetic diversity [26, 27, 28, 29]. Extensive genetic diversity with diverse allele types was also previously identified in pfmsp-1 and pfmsp-2 in P. falciparum Myanmar isolates that were collected in 2004–2006 . This study analysed the genetic polymorphisms of pfmsp-1 block 2 and pfmsp-2 block 3 in Myanmar P. falciparum isolates that were collected in 2013–2015. They were also compared with the sequences from previous years 2004–2006, to understand the temporal changes of genetic heterogeneity of the two genes in the Myanmar P. falciparum population. Comparative analysis of the global pfmsp-1 and pfmsp-2 was also performed, in order to gain in-depth understanding of the genetic make-up of the two genes in the global P. falciparum population.
Blood samples and genomic DNA extraction
Amplification and sequence analysis of pfmsp-1 and pfmsp-2
Genomic DNA was purified from the blood filters by using a QIAamp DNA Blood Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The primers specific for the block 2 of pfmsp-1 and block 3 of pfmsp-2 were designed as described previously [26, 31]. The two genes were amplified by a nested PCR method. Each amplification was done with thermal cycling conditions of 94 °C for 5 min; and 30 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min 30 s, followed by a final extension at 72 °C for 10 min. Ex Taq DNA polymerase (Takara, Otsu, Japan) with proof-reading activity was used in all PCR amplification steps to minimize the nucleotide mis-incorporation. Each PCR product was resolved on a 1.5% agarose gel, and was visualized under ultraviolet (UV). The multiplicity of infection (MOI) was estimated by the average number of PCR fragments for the corresponding locus per infected individual . The PCR products were purified from the gel, and cloned into the T&A cloning vector (Real Biotech Corporation, Banqiao City, Taiwan). Each ligation mixture was transformed into Escherichia coli DH5α competent cells, and colonies were screened for the presence of appropriate insert by colony PCR. The nucleotide sequences of the positive clones of pfmsp-1 and pfmsp-2 were analysed through automatic DNA sequencing with M13 forward and M13 reverse primers by the Sanger methods. Plasmids from at least two independent clones from each transformation mixture were sequenced in both directions, in order to verify the sequence accuracy. The nucleotide and deduced amino acid sequences of pfmsp-1 and pfmsp-2 were analysed using EditSeq and SeqMan in the DNASTAR package (DNASTAR, Madison, WI, USA). The nucleotide sequences reported in this study have been deposited in the GenBank database under the accession numbers MH981972–MH982070 for pfmsp-1, and MH982071–MH982183 for pfmsp-2.
Sequence analysis of pfmsp-1 and pfmsp-2 among the global P. falciparum population
The genetic diversity of pfmsp-1 and pfmsp-2 among global isolates was analysed. Accession numbers of sequences analysed in this study were presented in Additional file 1: Table S1. These sequences cover block 2 of pfmsp-1, and block 3 of pfmsp-2. The genetic polymorphism of each population was analysed by the methods as describe above.
Recombination parameters and linkage disequilibrium
The recombination and linkage disequilibrium of Myanmar pfmsp-1 and pfmsp-2 were analysed. The recombination parameter (R), which contained the effective population size and probability of recombination between adjacent nucleotides per generation, and the minimum number of recombination events (Rm) were measured, using DnaSP ver. 5.10.00 . Linkage disequilibrium (LD) between different polymorphic sites was computed in term of the R2 index, using DnaSP ver. 5.10.00. The R2 values were plotted against the nucleotide diversity distances with the two-tailed Fisher’s exact test of significance .
Sequence polymorphism of Myanmar pfmsp-1 block 2
Dynamic changes of Myanmar pfmsp-1 alleles between 2004–2006 and 2013–2015
Population structure of pfmsp-1 in global P. falciparum isolates
Sequence polymorphism of pfmsp-2 block 3 in Myanmar P. falciparum isolates
Comparison of genetic diversity of R1 and R2 regions in the global pfmsp-2
Linkage disequilibrium of Myanmar pfmsp-1 and pfmsp-2 between 2004–2006 and 2013–2015
Recombinant events in Myanmar pfmsp-1 and pfmsp-2 between 2004–2006 and 2013–2015
The genetic structure of P. falciparum population plays a very important role in the natural acquisition of immunity against malaria infections [33, 34]. Therefore, knowledge of the genetic nature and structure of the P. falciparum population is important to developing strategies to control the disease, including the design of effective vaccines against P. falciparum. Due to their polymorphic features, pfmsp-1 and pfmsp-2 have been employed as polymorphic markers in studies for malaria transmission dynamics in the natural P. falciparum population [21, 24, 35, 36, 37, 38, 39]. Plasmodium falciparum cases in Myanmar have decreased in recent years , but this species is still the most critical priority for malaria control and prevention in the country. In this study, the polymorphic characteristics of pfmsp-1 and pfmsp-2 were analysed in Myanmar P. falciparum isolates, to gain in-depth insight into the genetic nature of the Myanmar P. falciparum population.
High levels of genetic polymorphisms were identified in the Myanmar pfmsp-1 and pfmsp-2. Sequence analysis of 99 pfmsp-1 block 2 sequences revealed that a total of 28 distinct alleles (8 for K1 type, 14 for MAD20 type, and 6 for RO33 type) were identified in the Myanmar P. falciparum population that was analysed in this study. Similar to previous studies, different numbers and arrangements of unique tripeptide repeats were the main factors to contribute to the allelic diversity in the K1 and MAD20 types of Myanmar pfmsp-1 [15, 26]. In the case of pfmsp-2, 113 Myanmar sequences were classified into FC27 and 3D7 types, which showed highly different dimorphic structures in the variable central regions. The alleles of Myanmar pfmsp-2 belonging to the FC27 family were polymorphic, which was characterized by various numbers of FC27 family specific repeats in the R1 and R2 regions, in accord with previous reports [24, 40]. The 3D7 type showed more extensive sequence diversity. Besides the major polymorphic characters in the R1 and R2 variable regions, non-synonymous amino acid changes were also found in family specific regions (E1, E2, and E3) of 3D7 type alleles, which make the genetic diversity of 3D7 type alleles much greater than that of FC27 type alleles. Interestingly, the overall genetic diversity of Myanmar pfmsp-1 and pfmsp-2 increased in recent years (2013–2015) compared with the previous years (2004–2006), even though the incidence of malaria in the studied areas has reduced in recent years . A total of 14 distinct alleles (5 alleles for K1 type, and 9 alleles for MAD20 type) of pfmsp-1 were previously identified from 63 Myanmar P. falciparum isolates collected in 2004–2006 . Meanwhile, 28 alleles for all 3 types of pfmsp-1, K1, MAD20, and RO33, were identified in 99 sequences in recent years (2013–2015). Moreover, comparative analysis of alleles belonging to K1 and MAD20 types in the previous and recent years revealed that they have changed remarkably in recent years, with the appearance of new alleles, and disappearance of pre-existing alleles. The pfmsp-2 also showed a similar pattern of genetic diversity to pfmsp-1 in the Myanmar P. falciparum population. Twenty-two different alleles (7 for FC27 type, and 15 for 3D7 type) in 148 sequences were identified in previous years, but 59 alleles (18 for FC27, and 41 for 3D7) were identified in 113 sequences in recent years. As the numbers of P. falciparum isolates analysed both in previous and recent years were limited, it may not be easy to insist that the overall genetic diversity of Myanmar pfmsp-1 and pfmsp-2 has increased in recent years, compared to previous years. However, considering that the number of alleles for Myanmar pfmsp-1 and pfmsp-2 over the total number of sequences analysed has increased in recent years compared with those of previous years, it would be plausible to propose that the genetic diversity of the Myanmar pfmsp-1 and pfmsp-2 in the P. falciparum population in the studied areas has increased recently, and high levels of genetic complexity are still maintained in the population. High levels of the genetic diversity of pfmsp-1 and pfmsp-2 in the recent P. falciparum population in Southeast and Western Myanmar have also been reported .
It is not clear why the Myanmar pfmsp-1 and pfmsp-2 population structure in the studied areas has diversified so drastically in recent years, compared to the previous years, 2004–2006, despite the remarkable reduction of transmission in the last decade. In a declining population, rare alleles usually disappear, and the probability of multiplicity of infection (MOI) is reduced, so inbreeding is likely to increase. Overall decline of MOI was also identified in the Myanmar P. falciparum population analysed in this study. These declining patterns have been identified in both the P. falciparum and P. vivax populations, even in low transmission areas and pre-elimination settings [41, 42, 43, 44, 45]. This could be explained by asymptomatic carriers acting as fundamental reservoirs contributing to malaria transmission. Indeed, a substantial level of asymptomatic infections in the studied areas has been reported . Asymptomatic patients can facilitate superinfection, and the genotypes infected in asymptomatic patients may contribute to the maintenance or generation of genetic complexity of the Myanmar P. falciparum population. Another plausible explanation for the increased genetic diversity of Myanmar pfmsp-1 and pfmsp-2 in recent years can be speculated as the higher values of Rm for both genes, compared to those of previous years. The high values of recombination parameters identified in the recent pfmsp-1 and pfmsp-2 suggest that high levels of meiotic recombination events may occur in the genes, which render the genetic make-up of the genes more complex. The finding that high levels of MOI comparable to previous years (2.03 for pfmsp-1 and 2.35 for pfmsp-2) were still maintained in recent years (1.98 for pfmsp-1 and 2.41 for pfmsp-2) also supported this notion. Further combined analyses of other polymorphic markers, such as circumsporozoite protein and apical membrane antigen-1, with larger number of isolates, may be necessary for in-depth understanding of the genetic nature and genetic flow of the Myanmar P. falciparum population.
The comparative population structural analysis of pfmsp-1 and pfmsp-2 in the global P. falciparum population also suggests that high levels of genetic diversity of the two genes are maintained in the global P. falciparum population. Interestingly, the overall distribution of allele types of pfmsp-1 differed by geographical origin. Although K1 and MAD20 types were largely predominant in the global pfmsp-1, the overall prevalence and distribution of pfmsp-1 allelic types differed by country or continent. In Southeast Asia countries, MAD20 was the most prevalent type, followed by K1 type. The MAD20 type was also dominant in Pacific countries, but RO33 allele type was also identified as of high proportion in these countries. Meanwhile, K1 type was the most prevalent allele type in the South American and African pfmsp-1. Further analysis of the global pfmsp-1 sequences showed more complicated patterns of genetic diversity of the global pfmsp-1. Most pfmsp-1 alleles among the total 267 global pfmsp-1 alleles were identified in a country-specific manner. The genetic diversity of K1 and MAD20 types was much greater than that of RO33 type. The pfmsp-2 also showed high level of genetic diversity in the global pfmsp-2 population. The R1 and R2 regions were highly polymorphic, with different numbers and arrangements of repeat units. The geographic patterns of genetic differentiation suggest that the functional consequences of the polymorphism should be considered for designing a vaccine based on pfmsp-1 or pfmsp-2. The results of this study contribute insight into the genetic nature of Myanmar pfmsp-1 and pfmsp-2, as well as aid understanding of the genetic diversity of the global pfmsp-1 and pfmsp-2, and may provide valuable information for the development of an effective vaccine based on pfmsp-1 and pfmsp-2.
The Myanmar pfmsp-1 and pfmsp-2 populations showed high levels of genetic diversity, and have remarkably diversified in recent years, despite the rapid decline of malaria cases in the country for the last decade. These suggest that the Myanmar P. falciparum population still remains of sufficient size to allow the generation and maintenance of genetic diversity. Therefore, continuous molecular epidemiological surveillance to supervise the genetic variation of the parasite in Myanmar would be necessary. Additional studies to examine the dynamics of the genetic diversity and gene flow of the P. falciparum population combined with factors including the malaria transmission intensity based on entomological inoculation rates, and the immune status of P. falciparum-infected individuals, may contribute to the guidance of malaria interventions. The extreme genetic diversity patterns of pfmsp-1 and pfmsp-2 found in the global P. falciparum population also warrant continuous monitoring of the genetic diversity of the two genes in the global population, to better understand the polymorphic nature and evolutionary aspect of the vaccine candidate antigens in the global P. falciparum population.
We thank the staffs in Department of Medical Research Pyin Oo Lwin Branch and the health professionals in Naung Cho, Pyin Oo Lwin and Tha Beik Kyin townships for their contribution and technical support in field study.
HGL and JMK carried out genetic analysis of pfmsp-1 and pfmsp-2. HJ, JL, TLT, MKM and KSA contributed the blood sample collection. HGL, JMK, WMS, HJS, TSK and BKN analysed and interpreted the data. MKM, TSK and BKN designed and supervised the experiments. HGL and BKN wrote the draft of the manuscript. All authors read and approved the final manuscript.
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2018M3A9H5055614).
Ethics approval and consent to participate
This study was approved by the Ethics Review Committee, Department of Medical Research, Myanmar (97/Ethics 2015), and by the Ethical Review Committee of Inha University School of Medicine, Korea (INHA 15-013). Informed written consent and permission were obtained from each individual.
Consent for publication
The authors declare that they have no competing interests.
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