Positive selection on the Plasmodium falciparum clag2 gene encoding a component of the erythrocyte-binding rhoptry protein complex

Abstract

A protein complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Clarification of the level of polymorphism and the evolutionary processes is important both for vaccine design and for a better understanding of the evolution of cell invasion in this parasite. In a previous study on 5 genes encoding RhopH1/Clag proteins, positive diversifying selection was detected in clag8 and clag9 but not in the paralogous clag2, clag3.1 and clag3.2. In this study, to extend the analysis of clag polymorphism, we obtained sequences surrounding the most polymorphic regions of clag2, clag8, and clag9 from parasites collected in Thailand. Using sequence data obtained newly in this study and reported previously, we classified clag2 sequences into 5 groups based on the similarity of the deduced amino acid sequences and number of insertions/deletions. By the sliding window method, an excess of nonsynonymous substitutions over synonymous substitutions was detected in the group 1 and group 2 clag2 and clag8 sequences. Population-based analyses also detected a significant departure from the neutral expectation for group 1 clag2 and clag8. Thus, two independent approaches suggest that clag2 is subject to a positive diversifying selection. The previously suggested positive selection on clag8 was also supported by population-based analyses. However, the positive selection on clag9, which was detected by comparing the 5 sequences, was not detected using the additional 34 sequences obtained in this study.

Keywords: malaria; polymorphism; rhoptry.

Fig. 1.

Regions used for the analysis. Bars with arrows under each schematic for Clag protein indicate the regions used for the analysis in this study. Vertical bars and a gap with asterisk above each schematic indicate polymorphic sites among 5 P. falciparum laboratory isolates and an indel reported previously [14]. Amino acid position is according to the 3D7 line sequence.

Fig. 2.

Clag2 is classified into 5 groups based on the similarity in the variable region. Dots and bars indicate identical amino acid residues with the 3D7 line sequence and gaps. Asterisks indicate sequences reported previously [14]. PrCL2 indicate Plasmodium reichenowi Clag2. Amino acid positions shown above the sequences are according to the 3D7 line sequence.

Fig. 3.

Sliding window plot of d_N/d_S ratio for Plasmodium falciparum clag2 (group 1 and 2) and clag8 in Thai isolates and in a set combined with laboratory isolates. Nucleotide positions are according to the 3D7 line sequence. Window length is 90 bp, and step size is 3 bp. n indicates the number of samples analyzed. Asterisks indicate the region where a significant excess of nonsynonymous substitutions (d_N) over synonymous substitutions (d_S) was observed (single for p<0.05, and double for p<0.02). The statistical difference between d_S and d_N was tested using a one-tailed Z-test with 500 bootstrap pseudosamples implemented in MEGA4.0. Others indicate sequence reported previously and shown in Fig. 2 [14].

Fig. 4.

Sliding window plot of Tajima’s D, Fu & Li’s D* and F* tests for Plasmodium falciparum clag2 (group 1) and clag8 genes in Thai isolates. Nucleotide positions are according to the 3D7 line sequence. Window length is 90 bp, and step size is 3 bp. n indicates the number of samples analyzed. Asterisks indicate the region where a significant departure from the neutrality was observed (single for p<0.05, and double for p<0.02).