Variation in selective constraints along the Plasmodium life cycle

Abstract

Plasmodium parasites, the cause of malaria, have a complex life cycle, infecting alternatively vertebrate hosts and female Anopheles mosquitoes and undergoing intra- and extra-cellular development in several organs of these hosts. Most of the ~5000 protein-coding genes present in Plasmodium genomes are only expressed at specific life stages, and different genes might therefore be subject to different selective pressures depending on the biological activity of the parasite and its microenvironment at this point in development. Here, we estimate the selective constraints on the protein-coding sequences of all annotated genes of rodent and primate Plasmodium parasites and, using data from scRNA-seq experiments spanning many developmental stages, analyze their variation with regard to when these genes are expressed in the parasite life cycle. Our study reveals extensive variation in selective constraints throughout the parasites' development and highlights stages that are evolving more rapidly than others. These findings provide novel insights into the biology of these parasites and could provide important information to develop better treatment strategies or vaccines against these medically-important organisms.

Keywords: Plasmodium; Selection; scRNA-seq.

Figure 1:. Protein-coding sequences of genes expressed in one life stage evolve faster, on average, than genes expressed in multiple life stages.

(A) Range and distribution of dN/dS for genes expressed in several P. berghei life cycle stages (n = 3506) and genes expressed in a single stage (n = 533). (B) Proportion of genes that have a dN/dS value amongst the top 5% of highest values (green) according to their expression in multiple vs. single stage.

Figure 2:. Protein-coding sequences tend to be more conserved as the genes are more broadly expressed.

(A) Estimates of dN/dS for each rodent Plasmodium gene (y-axis) represented according to the number of life stages each gene is expressed in (x-axis) (p = 2.04 × 10⁻²⁰, Spearman’s rho = −0.15). (B) Proportion of genes that have a dN/dS value amongst the top 5% of highest values (green) in for each expression breath category (i.e., number of life stages each gene is expressed in).

Figure 3:. The protein-coding sequences of genes expressed exclusively in the mammalian stages evolve significantly faster, on average, than genes expressed in the mosquito stages.

(A) Range and distribution of dN/dS for genes expressed in the mosquito and mouse stages of the P. berghei life cycle. (B) Proportion of genes expressed in the mosquito or mouse stages that display a dN/dS value amongst the top 5% of all P. berghei genes.

Figure 4:. The protein-coding sequences of genes specifically expressed in sexual stages of the P. berghei and P. vivax life cycles evolve faster than genes specifically expressed in asexual stages.

(A) Distribution of dN/dS for rodent (top) and primate Plasmodium genes (bottom) that are expressed in asexual-, sexual blood stages, or both (based on gene expression determined in P. berghei and P. vivax, respectively). (B) Proportion of P. berghei (left) or P. vivax (right) genes expressed in asexual stages, sexual stages, or both with a dN/dS value in the top 5% for P. berghei or P. vivax genes, respectively.

Figure 5:. There is extensive variation in selective constraints across the P. berghei life cycle.

(A) Distribution of dN/dS values for genes expressed at a given stage. Each stage was compared to all other stages using a Kruskal-Wallis test corrected for multiple comparisons. Genes expressed in male gametocytes, oocysts, sporozoites and trophozoites tend to evolve faster than genes specific to other life cycle stages. Genes specific to rings and female gametocytes appear to evolve slower. Mean dN/dS values: Female: 0.16, Male: 0.20, Oocyst: 0.16, Ookinete: 0.17, Sporozoite: 0.21, Merozoite: 0.15, Liver: 0.16, Ring: 0.079 Trophozoite: 0.15, Schizont: 0.18 (B) Proportion of genes expressed in each stage with a dN/dS value in the top 5% of values for all P. berghei genes. There is significant enrichment for quickly evolving genes in female gametocytes (p=0.014) and sporozoites (0.00042). Proportion of genes among those with highest 5% of dN/dS values per stage: Liver: 5.0%, Female: 6.5%, Male: 8.6%, Merozoite: 7.1%, Oocyst: 4.6%, Ookinete: 5.8%, Ring: 3.3%, Schizont: 7.0%, Sporozoite: 10.8%, Trophozoite: 4.7%.

Figure 6:. Genes with the fastest evolving protein-coding sequences contribute a larger proportion of the P. berghei and P. vivax mRNAs late in the IDC.

(A) Each point represents a single P. berghei asexual blood-stage parasite and is displayed according to its proportion of mRNA molecules transcribed from genes with the 5% highest dN/dS values (y-axis) and its developmental pseudotime (x-axis). Note than the proportion of mRNAs derived from genes with the fastest evolving protein-coding sequences increases late in the IDC and peak in early to mid-schizonts. (B) Each point represents a single P. vivax asexual blood-stage parasite and is displayed according to its proportion of mRNA molecules transcribed from genes with the 5% highest dN/dS values (y-axis) and its developmental pseudotime (x-axis).