Plasmodium falciparum surf4.1 in clinical isolates: From genetic variation and variant diversity to in silico design immunopeptides for vaccine development

Abstract

SURFINs protein family expressed on surface of both infected red blood cell and merozoite surface making them as interesting vaccine candidate for erythrocytic stage of malaria infection. In this study, we analyze genetic variation of Pfsurf4.1 gene, copy number variation, and frequency of SURFIN4.1 variants of P. falciparum in clinical isolates. In addition, secondary structure prediction and immunoinformatic were employed to identify immunogenic epitopes in humoral response as proposed vaccine candidates. Overall, our data demonstrate extensive polymorphism of SURFIN4.1 in both genetic and protein level. The surf4.1 gene showed extensive genetic variation with total of 447 polymorphic sites with maximum of three variants as well as singlet/triplet bases indels and mini/microsatellites in the coding sequence. The exon1 encoding extracellular region exhibited higher variation compared to exon2 which coding for intracellular domain. Interestingly, selective pressure was detected on both extracellular region (Var1 and Var2) as well as intracellular region (WRD2 and WRD3). Importantly, extensive full gene analysis suggests adenosine insertion at three key points nucleotide bases (nt 2409/2410, 3809/3810, and 4439/4440) of exon2 could lead to frameshift mutation resulted in four different SURFIN4.1 variants (TMs, WD1, WD2 and WD3). The SURFIN4.1 variant TMs was the most observed type with 67% frequency (51/76). Along with more than one copy number of surf4.1 gene was observed with frequency of 13% (9/70). Despite substantial polymorphism, analysis of relatedness within P. falciparum population using full coding sequence was able to group SURFIN4.1 protein into five distinct clades and reduced into four clades when using only exon1 coding sequence. Also, predicted secondary structure revealed conserved structure of five helix domains of extracellular region which similar among four SURFIN4.1 variant types. In addition, in silico design eight immunopeptides derived from SURFIN4.1, four of which are highly conserved and four of dimorphic epitopes, as potential vaccine candidates.

Fig 1. The organization of Plasmodium falciparum surf_4.1 gene and nucleotide diversity.

(A) Schematic structure of full-length surf_4.1 gene and coding region including the N-terminal sequence (NTS), cysteine-rich domain (CRD), variable regions (Vars), transmembrane region (TM), tryptophan-rich domain (WRD). The length in nucleotide base pair of the reference P. falciparum strain 3D7 is shown in parenthesis. (B) Distribution of nucleotide diversity and accumulation of mutation, insertion and deletion (InDels) across surf_4.1 gene. Asterisks (*) indicate positions of single base indels. Nucleotide position is after the P. falciparum strain 3D7.

Fig 2. Test of neutrality for Plasmodium falciparum surf_4.1 across 6.7 kb in Thai isolates (n = 30) using a sliding window approach (window size = 90bp, step size = 3bp).

Allele frequency indices (A) Tajima’s D, (B) Fu and Li D* and (C) Fu and Li F* were calculated. Sites above the solid line (P<0.05) and broken line (P<0.01 for Tajima’s D and P<0.02 for Fu and Li’s D* and F*) depart significantly from neutrality (two-tailed), suggesting diversifying selection. Asterisks (*) indicate the region where positive deviation from neutrality were detected in this study. Nucleotide positions are P. falciparum strain 3D7.

Fig 3. The amino acid sequence of SURFIN_4.1 and type of variants.

(A) Schematic structure of SURFIN_4.1 including the N-terminal sequence (NTS), cysteine-rich domain (CRD), variable regions (Vars), transmembrane region (TM), tryptophan-rich domain (WRD). Accumulation of amino acid residue substitution, insertion and deletion (InDels) across the SURFIN_4.1; the length in amino acids of the reference P. falciparum strain 3D7 is presented in parenthesis. (B) Different types of SURFIN_4.1 variants; transmembrane variants (TMs), WD1 variant containing one WRD (WRD1), WD2 variant containing two WRDs (WRD1 and WRD2), and WD3 variant containing three WRDs (WRD1, WRD2 and WRD3). The length of amino acids shown in parenthesis is after the reference P. falciparum strain 3D7.

Fig 4. Phylogenetic tree for P. falciparum isolates.

Constructed using (A) coding nucleotide sequence (exon1 and exon2, n = 46) (B) exon 1(n = 185), and (C) exon 2 (n = 92) of surf_4.1.

Fig 5. The transmembrane location predicted by DeepTMHMM.

(A) Truncated transmembrane types (TM1 and TM2). (B) Tryptophan rich domain types (WD1-3).

Fig 6. Secondary structure of SURFIN_4.1 different variants.

(A) Truncated transmembrane types (TM1 and TM2). (B) Tryptophan rich domain types (WD1-3). Relative surface accessibility showed exposed in red and buried in blue at threshold 25%; secondary structure showed helix, strand and coil; disorder represented by the thickness of line equals probability of disordered residue.

Fig 7. B cell epitope prediction of extracellular domain of SURFIN_4.1.

(A) BepiPred-V3.0 predicting B-cell epitope of extracellular domain (amino acids of exon 1, aa1-790) using BepiPred-v3.0, default is 0.1512. (B) Identify B-cell epitope of four clades was underlined.