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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Genetic diversity; Merozoite surface protein-3; Myanmar; Natural selection; Plasmodium falciparum.

Fig. 1

Sequence polymorphisms of 3D7 allelic types of Myanmar pfmsp-3. A total of 31 haplotypes of 3D7 allelic types were detected in Myanmar pfmsp-3. The dots present amino acid residues identical to the reference sequence of 3D7 (XM_001347593). Dashes are gaps introduced to maximize the alignment. Numbers above the alignment are amino acid positions with reference to the 3D7 sequence. The di-morphic and tri-morphic amino acid changes at particular positions are shaded by blue and red, respectively. The insertion found in Myanmar pfmsp-3 is marked by green. The number of each haplotype found in Myanmar pfmsp-3 is indicated at right

Fig. 2

Amino acid polymorphisms of K1 allelic types of Myanmar pfmsp-3. A total of 25 haplotypes of K1 allelic types were detected in Myanmar pfmsp-3. The dots present residues identical to the reference sequence of K1 (U08851). Dashes are gaps introduced to maximize the alignment. Numbers above the alignment are amino acid positions with reference to the K1 sequence. The di-morphic and tri-morphic amino acid changes at particular positions are shaded by blue and red, respectively. The insertions found in Myanmar pfmsp-3 are marked by green. The amino acid positions with insertion are numbered with red bold. The number of each haplotype found in Myanmar pfmsp-3 is indicated at right

Fig. 3

Comparison of amino acid polymorphisms in 3D7 allelic types of pfmsp-3 from different geographical areas. Positions and frequencies of amino acid changes found in pfmsp-3 from different countries were compared. Each domain is presented by a different color bar: alanine heptad repeat domains (black), glutamate rich domain (orange), and leucine zipper domain (green). The dotted red bar in India pfmsp-3 means a missed sequence. The E271- and E272-represent deletion of an amino acid in the corresponding site. The 272E + , 272E ++, and 272E +++ indicate the numbers of inserted glutamate residues at the positions with 1, 2, and 3, respectively

Fig. 4

Comparison of amino acid polymorphism of K1 allelic types of pfmsp-3 between Myanmar and different geographical areas. Positions and frequencies of amino acid changes found in pfmsp-3 of Myanmar and other countries were compared. Each domain is presented by different color bar; alanine heptad repeat domains (black), glutamate rich domain (orange), and leucine zipper domain (green). The dotted red bar in India pfmsp-3 means missed sequence. The G169- and E297- represent deletion of an amino acid in the corresponding site. E272ETEEEELEEKNNE + means insertion in the position. The 298E + , 298E ++, and 298E +++ mean the numbers of inserted glutamate residues at the positions with 1, 2, and 3, respectively

Fig. 5

Polymorphic patterns of B-cell epitope in 3D7 allelic types of pfmsp-3 from Myanmar and different P. falciparum isolates. A logo plot was constructed for each pfmsp-3 population using the WebLogo program

Fig. 6

Nucleotide diversity and test of natural selection in Myanmar pfmsp-3. a Sliding window plot presented nucleotide diversity (π) values in Myanmar pfmsp-3. A window size of 10 bp and step size of 5 bp were used. b Sliding window calculation of Tajima’s D statistic was performed in Myanmar pfmsp-3. A window length of 10 bp and step size of 5 bp were used. Each domain is presented by different color bar on the graphs: alanine heptad repeat domains (black), glutamate rich domain (orange), and leucine zipper domain (green)

Fig. 7

The linkage disequilibrium (LD) of Myanmar pfmsp-3. The LD plots show non-random associations between nucleotide variations in Myanmar pfmsp-3 at different polymorphic sites. The R² values are plotted against the nucleotide distance with two-tailed Fisher’s exact test of significance