Size polymorphism and low sequence diversity in the locus encoding the Plasmodium vivax rhoptry neck protein 4 (PvRON4) in Colombian isolates

Abstract

Background: Designing a vaccine against Plasmodium vivax has focused on selecting antigens involved in invasion mechanisms that must have domains with low polymorphism for avoiding allele-specific immune responses. The rhoptry neck protein 4 (RON4) forms part of the tight junction, which is essential in the invasion of hepatocytes and/or erythrocytes; however, little is known about this locus' genetic diversity.

Methods: DNA sequences from 73 Colombian clinical isolates from pvron4 gene were analysed for characterizing their genetic diversity; pvron4 haplotype number and distribution, as well as the evolutionary forces determining diversity pattern, were assessed by population genetics and molecular evolutionary approaches.

Results: ron4 has low genetic diversity in P. vivax at sequence level; however, a variable amount of tandem repeats at the N-terminal region leads to extensive size polymorphism. This region seems to be exposed to the immune system. The central region has a putative esterase/lipase domain which, like the protein's C-terminal fragment, is highly conserved at intra- and inter-species level. Both regions are under purifying selection.

Conclusions: pvron4 is the locus having the lowest genetic diversity described to date for P. vivax. The repeat regions in the N-terminal region could be associated with immune evasion mechanisms while the central region and the C-terminal region seem to be under functional or structural constraint. Bearing such results in mind, the PvRON4 central and/or C-terminal portions represent promising candidates when designing a subunit-based vaccine as they are aimed at avoiding an allele-specific immune response, which might limit vaccine efficacy.

Keywords: Functional restriction; Genetic diversity; Natural selection; Plasmodium vivax; Rhoptry; Tandem repeat; pvron4.

Fig. 1

Representation of pvron4 encoding DNA sequence and sliding window for ω rate. ron4 ω values (d_N/d_S) within P. vivax are shown in green throughout. Likewise, ω divergence rates (K_N/K_S) between the species infecting primates and P. vivax are shown in blue (for the species evolutionarily closest to P. vivax) and red (most distant). A representation of the ron4 gene is given below the sliding window indicating signal peptide (blue), the repeat region (black) and the putative esterase/lipase domain (purple). The P. vivax SNP positions are shown as discontinue lines, their numbering is based on Additional file 2 alignment. The sites under purifying selection within species are represented by light green lines and dark green between species while positively selected sites between species are shown by red lines

Fig. 2

Lineages subject to episodic diversifying selection in ron4. The branches (lineages) under episodic positive selection were identified by the Branch-site REL method. The figure shows the ω⁺ values, the percentage of sites (Pr [ω = ω+]) and p values. The shade of each colour on branches indicates strength of selection (red shows ω⁺ > 15, blue ω⁻ ≤ 1 and grey ω = 1). The size of each colour represents the percentage of sites in the corresponding class. Branches have been classified as undergoing episodic diversifying selection by the p value corrected for multiple testing using the Holm-Bonferroni method at p < 0.05

Fig. 3

Median-joining network for Colombian regions. The Figure shows the pvron4 haplotypes identified from the isolates from the three regions of Colombia. Haplotypes 22 and 28 were included within haplotype 15 using the contraction star algorithm [86] for simplifying interpretation of the network. Each node is a haplotype and its size indicates its frequency. The lines connecting the haplotypes represent the different mutational paths and the median vectors are the ancestral sequences explaining the relationship and evolutionary origin