A comprehensive evaluation of rodent malaria parasite genomes and gene expression

Abstract

Background: Rodent malaria parasites (RMP) are used extensively as models of human malaria. Draft RMP genomes have been published for Plasmodium yoelii, P. berghei ANKA (PbA) and P. chabaudi AS (PcAS). Although availability of these genomes made a significant impact on recent malaria research, these genomes were highly fragmented and were annotated with little manual curation. The fragmented nature of the genomes has hampered genome wide analysis of Plasmodium gene regulation and function.

Results: We have greatly improved the genome assemblies of PbA and PcAS, newly sequenced the virulent parasite P. yoelii YM genome, sequenced additional RMP isolates/lines and have characterized genotypic diversity within RMP species. We have produced RNA-seq data and utilised it to improve gene-model prediction and to provide quantitative, genome-wide, data on gene expression. Comparison of the RMP genomes with the genome of the human malaria parasite P. falciparum and RNA-seq mapping permitted gene annotation at base-pair resolution. Full-length chromosomal annotation permitted a comprehensive classification of all subtelomeric multigene families including the 'Plasmodium interspersed repeat genes' (pir). Phylogenetic classification of the pir family, combined with pir expression patterns, indicates functional diversification within this family.

Conclusions: Complete RMP genomes, RNA-seq and genotypic diversity data are excellent and important resources for gene-function and post-genomic analyses and to better interrogate Plasmodium biology. Genotypic diversity between P. chabaudi isolates makes this species an excellent parasite to study genotype-phenotype relationships. The improved classification of multigene families will enhance studies on the role of (variant) exported proteins in virulence and immune evasion/modulation.

Figure 1

Organization of subtelomeric regions of RMP chromosomes. A) Organization of subtelomeric regions of chromosomes 13 of PyYM (left region), 5 of PcAS (left region) and 11 of PbA (left region). The order and orientation of the genes are shown, including genes belonging to the pir, fam-a, fam-b and fam-c gene families. Exons are shown in coloured boxes with introns as linking lines. As a comparison, a subtelomeric region of P. falciparum 3D7 chromosome 9 is shown. The shaded/grey areas mark the start of the conserved, syntenic regions. Black angular lines represent gaps. B) Artemis view showing a copy of the PbA-specific 2.3 kb repeat element containing a fragmented and ‘pseudogenised’ bir gene (PBANKA_000720). Also shown is the location of three 27 bp telomeric repeat units. The HMM Pfam match ‘Cir_Bir_Yir PF06022’ (grey box) spans amino acid 6 to 239 with an e-value of 2.5e-77. RPM, rodent malaria parasites.

Figure 2

Gene expression (RNAseq) in multiple RMP life cycle stages. A) Spearman correlation of FPKM values of orthologous genes between life cycle stages of PbA, PcAS and PyYM. PbA: ring (RI), trophozoite (Tr), schizont (Sch), gametocyte (Gct) and 16 and 24 hour ookinetes (Ook). PcAS: trophozoites (trophozoites of blood (Pc-bl)-and vector-transmitted (Pc-vec) PcAS; PyYM; blood stages (2 lines PyYM_WT and PyYM_MUT). B) Heat maps of expression (FPKM normalized by gene) of PbA genes in different life cycle stages. Left panel, all PbA genes ordered based on P. berghei expression pattern (FPKM values >21; in total 4,733 genes). Right panel, 2,236 PbA genes with orthologs in P. falciparum and FPKM values >63, ordered according to the temporal expression levels (in asexual blood stages) of their P. falciparum orthologs as shown in [34]. FPKM, fragments per kilo base of exon per million fragments mapped; RMP, rodent malaria parasites.

Figure 3

Expression of members of three large RMP multigene families in different life cycle stages. Temporal expression patterns of members of the three largest PbA multigene familes (pir, fam-a and fam-b) in different life cycle stages as visualized by heat maps of RNA-seq data. The expression (FKPM) values of genes over the life-cycle stages (yellow-red) are normalised per gene. The min/max column values are the log minimal and log maximal FPKM values for each gene. Only genes with an FPKM above 21, in all conditions, were included. FKPM, fragments per kilo base of exon per million fragments mapped; RMP, rodent malaria parasites.

Figure 4

Features of the RMP pir multigene family A) Phylogenetic tree of RMP pir s , showing the different clades (S1 to 8, L1 to 4) and separation of the ‘long’ (L) and ‘short’ form (S) pir s. B) Features of the different RMP pir clades. For each clade we show the total number of RMP pirs followed by their distribution (pie charts) in the three species and the distribution of gene lengths (box plots). In addition, pie charts show the relative abundance of clades in the different isolates/lines of P. chabaudi and P. yoelii. The expression bar plots (red bars) visualise the expression of the pirs of different clades in the different life cycle stages (except for the ookinete stage since expression/FPKM values are below the cut of level of 21). A pir is assigned to a life cycle stage based on the highest FPKM value. The height of the expression bar represents the percentage of all pirs in that clade. FKPM, fragments per kilo base of exon per million fragments mapped; RMP, rodent malaria parasites.