Evolution of tandem repeats in putative CSP to enhance its function: A recent and exclusive event in Plasmodium vivax in India

Background

Plasmodium vivax is the second most common underlying parasite responsible for causing malaria in humans. Malaria control and eradication programs need to redirect their efforts to target this species.Implementing targeted strategies requires understanding the parasites’ genetic features and the evolutionary processes that have molded this species. DNA repeats are common in the protein-coding genes of Plasmodium species. This underlying genetic feature can result in changed or novel protein functions over time, thus influencing parasite dynamics (Davies, et al., 2017). Despite harboring high variation within these repeat sites , few show signs of being under selection and most are following the expected neutral evolution.
Plasmodium proteins’ functions are poorly characterized, which is also true for repeat proteins. The circumsporozoite (CSP) gene is one example in Plasmodium containing variable repeats of clinical importance as some of these are the target of malaria vaccines. The repeat sequence motif in CSP is known to be variable both between and within parasite species, showing differences between strains of P. falciparum (Neafsey et al., 2015). Thus, CSP is an excellent protein model to understand the effects of repeat protein’ variation and its evolutionary significance. P. vivax possesses a locus, PVX_086150, which is a putative CSP gene in this species. The protein encoded by this gene structurally resembles the P. falciparum CSP protein, containing a hypervariable repeat region. In this preprint, the authors analyze the evolution of the putative P. vivax CSP loci and speculate on the selection forces that have allowed the expansion of the repeat region.

Key findings of the preprint.

Qualitative characterization of repeats in PvpuCSP (PVX_086150)
After confirming infection by P. vivax in samples and obtaining sequences of the target loci CSP,the authors screened for repeats using Tandem Repeats Finder (TRF) in PvpuCSP (PVX_086150) sequences. The repeat units were grouped into RATs, repeat allotypes, and PRMs, protein repeat motifs. The consensus of the repeat was found to be AGG[GA/TG]TAA[C/T]GC[C/T], and the variations produced four RATs encoding two PRMs (R-D-N-A; R-C-N-A). The order of the repeats 1-4 was also characterized showing that in most samples the repeats started with R1 and ended with R3, except in 3 isolates (G3948-GUJ, K13-ODI, D2115-DEL). The authors also found variation in the number of repeats within their samples ranging from 2 to 30.

Haplotype diversity and neutrality tests
After characterizing the repeats variation and structure, the authors created haplotypes from the 71 isolates. The haplotypes were found to vary across populations and were “mostly population specific”; furthermore, 5 haplotypes were shared among all analyzed populations. Overall diversity in the PvpuCSP (π= 0.00047, θW=0.00084) was smaller compared to the repeats’ region (π= 0.00513, θW=0.01762), thus leading the authors to conclude that gene diversity is mostly driven by the repeats’ region. Describing diversity among populations in a putative vaccine candidate locus is necessary for efficient vaccine development.

Selection tests and recombination
Tests of neutrality suggested some deviations from neutral evolution, but these were statistically nonsignificant. The authors conducted positive selection tests suggesting there might be some evidence to support directional selection, and results from site tests showed that most sites under positive selection fall within the repeat region. In contrast, branch tests suggested diversifying selection in two branches. Complementary recombination tests showed breaks at the start and end of the repeat region and no pattern was found to be related to any population repeat number of rearrangements.

The authors speculate that the selection of repeats could enhance the surface area for binding proteins targeted for degradation. Finally, the lack of repeat units in PvpuCSP orthologs species could be interpreted as a sign that repeat expansion in P. vivax is a recent evolutionary event, specific to this species populations, which could be targeted by new malaria elimination strategies.

What we like about the preprint

Malaria elimination requires targeting other Plasmodium species besides P. falciparum and this includes P. vivax. Recognizing and identifying evolutionary features that separates P. vivax from other malaria-causing species is necessary to implement targeted elimination.

Questions for the authors

-Do you think that statistically controlling for the number of sites would change any of your interpretations? In other words, do you think your results could be partially explained because repeats increase the power of detecting positive selection using site tests?

-Could you functionally validate that the repeats expansion on PvpuCSP are selected due to their role in protein degradation?

-If you were able to follow up and do a longitudinal collection of samples, do you expect consistency in branch tests where evidence of positive selection is found in the “newest” samples”?

-Given what you found, would you recommend this locus for a vaccine? What other applications would you suggest given your results?

References

Davies HM, Nofal SD, McLaughlin EJ, Osborne AR. Repetitive sequences in malaria parasite proteins. FEMS Microbiol Rev. 2017 Nov 1;41(6):923-940. doi: 10.1093/femsre/fux046. PMID: 29077880; PMCID: PMC5812512.
Neafsey DE, Juraska M, Bedford T, et al. Genetic diversity and protective efficacy of the RTS, S/AS01 malaria vaccine. N Engl J Med 2015;373:2025–37.

 

doi: https://doi.org/10.1242/prelights.36592

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