Background
Malaria is the parasitic disease having the greatest impact on public health [
1]. It is caused by different species from the
Plasmodium genus, these being widely distributed throughout the world’s tropical and sub-tropical regions [
2]. These parasites cause 140–300 million clinical cases and more than half a million deaths annually [
3,
4].
Plasmodium falciparum is considered the most lethal species, mainly affecting vulnerable populations in sub-Saharan Africa [
4]. Even though efforts were initially concentrated on controlling this species, reports of ever-increasingly severe cases caused by
Plasmodium vivax [
5] and the appearance of drug-resistant strains during the last few years [
6,
7] has made this species a growing public health problem, affecting more than a third of the world’s population, having high prevalence in Asia and South and Central America [
3,
7,
8].
Designing an anti-malarial vaccine against
P. vivax (as for
P. falciparum) has been focused on blocking parasite-host interactions during different parasitic stages, especially during the blood phase responsible for the disease’s clinical manifestations [
9,
10]. A large amount of
P. vivax antigens have been characterized to date [
10,
11], however, their genetic diversity should be assessed for selecting the best antigens for vaccine development [
10,
12]. Highly polymorphic antigens can provoke allele-specific immune responses leading to protection having low efficacy after vaccination. On the contrary, those having limited diversity are attractive targets for being evaluated as candidates as they avoid an allele-specific immune response [
13].
Most antigens characterized to date have been merozoite proteins [
10,
11], including the microneme AMA1 protein and rhoptry neck (RONs) proteins. The interaction between these proteins (specifically AMA1–RON2) has been well described in
Toxoplasma gondii and
P. falciparum, these being the structural basis for the tight junction (TJ), a connective ring through which a parasite enters a host cell [
14‐
18].
The RON protein complex (characterized in
P. falciparum) consists of RON2, RON4 and RON5 proteins [
14,
17,
19]. Even though the mechanisms regarding function and interaction between the complex’s proteins are not clear, they are considered important targets for blocking invasion. Various studies have highlighted the potential of AMA1 and RON2 as vaccine candidates, however, current knowledge concerning the other RONs is deficient. Co-localization studies and invasion models described for
Plasmodium spp and
T. gondii have led to establishing RON4’s convincing participation in the TJ [
15,
17,
20,
21]. Likewise, its expression in the parasite’s invasive forms [
21‐
23], the ability to provoke an immune response in natural malarial infections [
23] and the protein’s conserved nature, specifically towards the C-terminal (inferred by comparative analysis between
PfRON4 and
TgRON4 amino acid sequences) [
24] suggest that this protein plays an important role for the parasite and could thus be evaluated as vaccine candidate.
The
P. falciparum RON4 orthologue has recently been characterized in the
P. vivax VCG-I strain (Vivax Colombia Guaviare-I) [
22]. PvRON4 (
P. vivax RON4) is encoded by a gene having around 2657 bp in this species, expressed during the last hours of the intra-erythrocyte cycle and secreted from the rhoptry neck. This consists of signal peptide sequence, a low complexity domain formed by two types of tandem repeats, a double spiral alpha helix domain and five conserved cysteines towards the C-terminal [
22]; the latter region seems to be highly conserved among
P. vivax and parasite species infecting monkeys [
25].
Bearing RON4’s potential participation in invasion in mind and given that parasite antigen genetic diversity is an obstacle for designing a completely effective vaccine against P. vivax, this study was thus aimed at using Colombian clinical isolates for evaluating pvron4 locus genetic diversity and the evolutionary mechanisms determining its variation pattern.
Discussion
Various proteins contained within the parasite’s apical organelles seem to be crucial for host cell invasion and thus represent promising vaccine targets. RON complex proteins are among the proteins localized in the apical organelles, forming part of the TJ [
17,
21,
63,
64]. This TJ plays a decisive role in parasite entry to a host cell and closure of the parasitophorous vacuole [
17]. The RON4 protein located in the invasion complex is present in Phylum Apicomplexa members [
15,
23,
24], suggesting that it forms part of a conserved invasion pathway. This protein has thus been described as a potential vaccine candidate.
A vaccine candidate must have several characteristics [
10,
65]; one of them is to have low genetic diversity to avoid allele-specific immune responses, which could reduce vaccine efficacy. The analysis of
P. falciparum laboratory strains from different geographically origins showed the
pfron4 locus as being a highly conserved locus, having just one amino acid substitution [
23].
Plasmodium vivax ron4 seems to have the same pattern, as analysing five reference sequences revealed low genetic diversity [
25]. This study thus analysed 73 clinical isolates from the Colombian population for confirming
pvron4 as a highly conserved gene in
P. vivax. In spite of increasing the number of sequences analysed,
pvron4 diversity remained low, the present study’s results showing that
pvron4 had lower genetic diversity than in a previous report [
25]. Only eight SNPs were identified in the 80 available sequences compared to 14 previously identified ones [
25]. Such high number of previously reported SNPs (i.e., 14) was due to erroneous repeat region alignment.
pvron4 had a similar pattern to that of other apical organelle proteins [
66,
67].
pvrap1 and
pvrap2 had 0.0009 to 0.001 π [
67] while
pvron4 had a much lower value than
pvrap1, suggesting it had low genetic diversity, the locus being more conserved to date for
P. vivax. As it has been suggested for
pvrap1 and
pvrap2 [
67] the low diversity in
pvron4 could be the consequence of functional/structural constraint (see below) due to the key role of this protein in parasite invasion.
Even though
pvron4 was a highly conserved sequence regarding SNP occurrence, it had high polymorphism regarding size. Previous studies have identified two types of repeats towards the N-terminal of the encoded protein [
22]. These repeats were reported as being imperfect copies of amino acids GGEH/SGEH/S and G/AEH. However, the analysis here performed showed that the
pvron4 repeat region consisted of two types of repeats having 100 % identity; the first encoded three GES amino acids (one to three copies) and the second one GEHGEHAEHGE amino acids (one to seven copies). These repeats gave a high number of different haplotypes (alleles) in
P. vivax.
Previous studies have suggested that tandem repeats could play an important role as host immune response evasion mechanism [
68‐
71]. In this study, 21 haplotypes were identified in PvRON4 when the InDels were analysed. PvRON4 N-terminal region seems to be the most exposed protein according to solvent availability and hydrophobicity results. This region (between signal peptide and repeat region) seems to be a potential antigenic target due to this being where the largest amount of potential B-cell linear epitopes was predicted. The repeat sequences identified broadened the solvent-exposed region and the protein’s antigenic potential. The PvRON4 N-terminal region could thus be the region exposed to a host’s immune system and repeats could be acting as an immunological smokescreen. Further antigenic and immunogenic studies are needed to confirm such hypothesis. As the repeat region was highly conserved regarding sequence, it could play an important structural or functional role, as has been suggested recently for the CSP [
72,
73].
While the N-terminal region might to be exposed to the immune system, the central and C-terminal regions seem to be under functional constraint. Neutrality tests (e.g., Tajima, Fu and Li) gave no statistically significant values and neutrality was thus not ruled out. If
pvron4 is under neutrality then it should show high polymorphism unless there is a functional or structural constraint [
74]. Given that this locus was highly conserved regarding sequence, functional/structural constraint is probable. However, selection at translational level could also be responsible for high conservation in the
pvron4 sequence. Analysing regarding preferential codon use did not reveal bias regarding codon use (ENC = 53–55). In fact this value was similar to that reported for the complete genome (ENC = 52.18) [
75], suggesting that high
pvron4 locus conservation was not due to selection at translational level and could have been a result of strong purifying selection.
The d
S rate was significantly greater than the d
N rate according to Fisher’s exact test, suggesting that this locus has evolved under purifying selection. However, it is not easy to evaluate how natural selection acts in highly conserved antigens [
66,
76,
77]. Previous studies have compared
P. vivax sequences to phylogenetically related species to evaluate the effect of selection on parasite antigens [
66,
76‐
78]. Sliding window analysis of ω rate gave values less than 1 towards the protein’s central region as well as towards the C-terminal. The K
S was statistically greater than K
N and various sites under purifying selection between species were detected in these regions, suggesting that purifying selection plays an important role during the locus’ evolution in the genus. Bearing in mind that functional regions tend to have slower evolution and are usually conserved between species [
79], these results suggest that the PvRON4 C-terminal and central regions could be functionally important. The presence of conserved cysteines in the C-terminal portion (usually associated with protein–protein interaction) could be mediating the interaction between RON4 and AMA-1 and/or other RONs [
16,
80], while the presence of a putative esterase/lipase domain in the protein’s central region could be involved in RON4 entry to the host cell.
Plasmodium falciparum and
T. gondii studies have shown that the RON4 C-terminal region seems to play an important role in invasion [
16,
24]. RON4 is located inside red blood cells (RBC), anchoring the AMA1/RON2 complex [
17,
18,
24]; RON4 must thus be secreted and enter RBC during initial invasion stages by a yet-unknown mechanism. The presence of an esterase/lipase domain in the PvRON4 central region could provide a clue regarding the action mechanism. This is one of the protein’s most structured regions, being highly conserved among species and containing several sites under purifying selection, suggesting a functional/structural role. Therefore, while the PvRON4 N-terminal region seems to be associated with evasion of the immune response, the central region (containing the esterase/lipase domain) could be associated with the rupture of ester bonds in the phospholipids constituting host cell membrane. Such rupture would enable RON4 entry to RBC or hepatocyte cytoplasm. Once inside, the RON4 C-terminal region anchors RON proteins, which, in turn, enable AMA1-mediated interaction between the parasite and host cells. It can thus be hypothesized that such putative esterase/lipase domain could play a role regarding RON4 entry to a host cell, however, further functional assays are needed to confirm this.
In spite of
ron4 being highly conserved between species and that purifying selection seems to be important during this locus’ evolution in
Plasmodium, some sites under positive selection were identified, coinciding with the regions where ω > 1 was observed. Previous studies have shown that some antigens (regardless of their genetic diversity) have regions/codons under episodic positive selection, which could have enabled adaptation to different hosts [
76,
81,
82]. The topology obtained for
ron4 was similar to that obtained when analysing mitochondrial DNA [
83]. The phylogenetic relationships of species infecting rodents and hominids can be seen in
ron4 phylogeny, however, such relationships have not been seen for species infecting monkeys. These species have a complex evolutionary history, which includes biogeographic aspects, adaptation to new macaque hosts and even a change from monkeys to humans [
81,
84]. The episodic selection observed here might thus have been a consequence of this group of parasites’ rapid diversification (in the N-terminal region and a small portion of the C-terminal region) thereby enabling RON4 to adapt from an ancestral population to new available hosts, as previously suggested [
76,
81,
82,
84].
A relatively high number of haplotypes has been found in the
pvron4 locus in Colombia, resulting from a combination of SNPs and tandem repeats. AMOVA analysis and median joining showed that Colombian regions shared most haplotypes and seemed to be genetically similar. However, it was observed that a 6 % of estimated variation between these regions was due to differences between the subpopulations constituting them. The F
ST value showed that some subpopulations might not be genetically similar; this could be associated with the presence of unique haplotypes. This agreed with studies in Colombia involving other parasite antigens [
77], as well as mitochondrial DNA studies in America [
85], suggesting that the parasite population in America is structured and has limited gene flow. However, since some subpopulations analysed here had limited sample size, the number of sequences must be increased for such results to be confirmed.