Sequence variation in Plasmodium falciparum merozoite surface protein-2 is associated with virulence causing severe and cerebral malaria

Abstract

Parasite virulence, an important factor contributing to the severity of Plasmodium falciparum infection, varies among P. falciparum strains. Relatively little is known regarding markers of virulence capable of identifying strains responsible for severe malaria. We investigated the effects of genetic variations in the P.f. merozoite surface protein 2 gene (msp2) on virulence, as it was previously postulated as a factor. We analyzed 300 msp2 sequences of single P. falciparum clone infection from patients with uncomplicated disease as well as those admitted for severe malaria with and without cerebral disease. The association of msp2 variations with disease severity was examined. We found that the N allele at codon 8 of Block 2 in the FC27-like msp2 gene was significantly associated with severe disease without cerebral complications (odds ratio = 2.73, P = 0.039), while the K allele at codon 17 of Block 4 in the 3D7-like msp2 gene was associated with cerebral malaria (odds ratio = 3.52, P = 0.024). The data suggests possible roles for the associated alleles on parasite invasion processes and immune-mediated pathogenicity. Multiplicity of infection was found to associate with severe disease without cerebral complications, but not cerebral malaria. Variations in the msp2-FC27-block 2-8N and 3D7-block 4-17K allele appear to be parasite virulence markers, and may be useful in determining the likelihood for severe and cerebral malaria. Their interactions with potential host factors for severe diseases should also be explored.

Fig 1. Structure of the variable region of the Plasmodium falciparum msp2 gene showing block 2, 3, and 4.

FC27 and 3D7-like structures were presented in the upper and lower panels, respectively. Amino acid (aa.) lengths of each block are indicated. Block 3 consist of 2 different repeat units (R1 and R2), which were separated by a non repeat region (NR). For 3D7-like sequences, R1 had Glycine (G), Serine (S), and Alanine (A) enriched sequences, while R2 featured Threonine (T) repeats.

Fig 2. Sequence alignment of Msp2 FC27 family of Plasmodium falciparum showing the polymorphisms in each block.

Changes of nt. / aa. are shaded with gray / yellow. The Msp2 sequence of K1 (FC27-liked) (GenBank accession number: M59766.1) was used as a reference for Block 2 and 4 alignment. (A) Block 2: the first 6 nt. residues of 57 (19 aa.) were not analyzed. An indel and 7 non-synonymous SNPs are shown. (B) Block 4: only the first 90 nt. residues of 144 (48 aa.) are presented. Two non-synonymous SNPs are shown. (C) Block 3, repeat region1 (R1): only the first 90 nt. residues of the 96 nt. repeat are presented, showing 4 repeat variants (R1-A, R1-B, R1-C, R1-D) with different positions of single non-synonymous SNPs. (D) Block 3, repeat region2 (R2): 36 nt. repeat showing 3 repeat variants (R2-1, R2-2, R2-3) with different non-synonymous SNPs.

Fig 3. Sequence alignment of Msp2 3D7 family of Plasmodium falciparum showing polymorphisms in each block.

Changes in nt. / aa. are shaded gray / yellow. The Msp2 sequence for 3D7 (GenBank accession number: PFB0300c) was used as a reference for alignment of Block 2, 4, and the non-repetitive region (NR) of block 3. (A) Block 2: first 6 nt. residues of 21 (7 aa.) are not analyzed. Four non-synonymous SNPs are shown. (B) Block 4: only residues from nt. 31 to 100 of 270 (90 aa.) are presented. Eight non-synonymous SNPs and 2 indel are shown. (C) Block 3, non-repeat region (NR): all 54 nt. (18 aa.) are presented, showing 6 non-synonymous SNPs and 3 indel. (D) Block 3, repeat region 2 (R2): 2, 3, and 4 copies of nanomer (ACT ACC ACA) followed by ACT ACT producing Threonine 8, 11, and 14 residues, respectively.

Fig 4. Linkage disequilibrium (LD) structures of 3D7-liked msp2 of P. falciparum in Thailand.

Pairwise LD plots based on r² between the 10 bi-allelic polymorphisms with minor allele frequency ≥ 10% were calculated for P. falciparum from mild (A), severe (B), and cerebral malaria patients (C). White, shades of grey, and black squares indicate no LD (r² = 0), intermediate LD (0 < r² < 1), and strong LD (r² = 1), respectively. LD structures were plotted using haploview software and amino acid changes are shown. The polymorphisms associated with cerebral malaria are labeled with a red star.