Genome comparison of human and non-human malaria parasites reveals species subset-specific genes potentially linked to human disease

Abstract

Genes underlying important phenotypic differences between Plasmodium species, the causative agents of malaria, are frequently found in only a subset of species and cluster at dynamically evolving subtelomeric regions of chromosomes. We hypothesized that chromosome-internal regions of Plasmodium genomes harbour additional species subset-specific genes that underlie differences in human pathogenicity, human-to-human transmissibility, and human virulence. We combined sequence similarity searches with synteny block analyses to identify species subset-specific genes in chromosome-internal regions of six published Plasmodium genomes, including Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. To improve comparative analysis, we first revised incorrectly annotated gene models using homology-based gene finders and examined putative subset-specific genes within syntenic contexts. Confirmed subset-specific genes were then analyzed for their role in biological pathways and examined for molecular functions using publicly available databases. We identified 16 genes that are well conserved in the three primate parasites but not found in rodent parasites, including three key enzymes of the thiamine (vitamin B1) biosynthesis pathway. Thirteen genes were found to be present in both human parasites but absent in the monkey parasite P. knowlesi, including genes specifically upregulated in sporozoites or gametocytes that could be linked to parasite transmission success between humans. Furthermore, we propose 15 chromosome-internal P. falciparum-specific genes as new candidate genes underlying increased human virulence and detected a currently uncharacterized cluster of P. vivax-specific genes on chromosome 6 likely involved in erythrocyte invasion. In conclusion, Plasmodium species harbour many chromosome-internal differences in the form of protein-coding genes, some of which are potentially linked to human disease and thus promising leads for future laboratory research.

Figure 1. Proteome comparison reveals 30 proteins conserved in primate malaria parasites but absent in rodent malaria parasites.

Genes were considered specific to a group of parasites if conserved in all in-group species (global protein sequence PID> = 40) but not in any of the out-group species (PID< = 15). In particular, primate-parasite specific genes are genes conserved in the three primate parasite proteomes but not in any of the three rodent parasite proteomes. The Venn diagram shows numbers of species subset-specific genes identified for all possible species combinations. Putative primate parasite-specific genes (30) are shown in bold. Note that gene numbers do not add up to species totals because genes with PIDs between 15% and 40% are not included.

Figure 2. Rodent malaria parasites likely deficient in de novo synthesis of thiamine (vitamin B1).

The diagram illustrates catalytic steps of the thiamine biosynthesis pathway in P. falciparum, with 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole as start products and thiamine phosphate as the end product. The three enzymes predicted to be absent in rodent malaria parasites catalyze subsequent reactions in this pathway, suggesting that rodent malaria parasites are deficient in de novo synthesis of vitamin B1. Gene identifiers correspond to P. falciparum genes (bold) and their P. vivax and P. knowlesi orthologs (below). PFL1920c has a predicted but severely truncated ortholog in P. yoelii (PY04023). Figure based on pathway shown in the Malaria Parasite Metabolic Pathways (MPMP) database .

Figure 3. Syntenic orthologs of thiamine biosynthesis genes present in primate but not rodent parasites.

Each panel shows one of the three P. falciparum thiamine biosynthesis gene on top (gene identifier in bold) and syntenic genomic regions in P. vivax, P. knowlesi, P. yoelii, P. berghei, and P. chabaudi below. Shaded areas indicate orthology. Thiamine biosynthesis genes displayed in yellow. Flanking genes and their orthologs on forward and reverse strand are shown in blue and red, respectively. The three P. falciparum genes are located on three different chromosomes and in all three cases syntenic orthologs are present in primate but not rodent parasite genomes. The syntenic P. yoelii ortholog of PFL1920c (PY04023, Panel C) is an exception but appears to be a truncated gene relic that should be annotated as pseudogene. Orthologs of PFF0683c and PFF0685c (Panel B) are merged into single genes in P. vivax and P. yoelii and should be split. Images adapted from PlasmoDB 8.0.

Figure 4. P. falciparum and P. vivax share extensive synteny with hundreds of putative parasite-specific genes in chromosome-internal regions.

Outer segments depict the 14 nuclear chromosomes of P. falciparum (left semicircle, counter-clockwise) and P. vivax (right semicircle, clockwise). Each chromosome is assigned a different color. Ribbons indicate the 29 identified imperfect synteny blocks (28 non-nested and 1 nested) colored according to connected P. vivax chromosomes. Black tick marks underneath chromosomes indicate putative parasite-specific genes located at synteny gap regions (SGR) and synteny breakpoint regions (SBR) (see inset). Parasite-specific genes in subtelomeric regions (STRs) not shown. Text labels within chromosomes indicate parasite-specific genes mentioned in the text, including the newly identified putative MSP3 gene cluster on P. vivax chromosome 6. Black lines within chromosomes indicate putative centromeres. In both species, chromosome-internal regions contain hundreds of putative parasite-specific genes (388 in both species). Image created with Circos .

Figure 5. P. vivax and P. knowlesi share almost perfect 1-to-1 chromosomal synteny but also harbor hundreds of putative parasite-specific genes in chromosome-internal regions.

Outer segments depict the 14 nuclear chromosomes of P. vivax (left semicircle, counter-clockwise) and P. knowlesi (right semicircle, clockwise). Ribbons represent the 20 identified imperfect synteny blocks (both nested and non-nested) colored according to connected P. vivax chromosomes. Black tick marks underneath chromosomes indicate putative P. vivax-specific genes (281) and P. knowlesi-specific genes (364) located at SGRs and SBRs. Parasite-specific genes in subtelomeric regions (STRs) not shown. Text labels within chromosomes indicate parasite-specific genes mentioned in the text. The inset shows the largest identified SGR in P. vivax containing 26 RAD genes. Black lines within chromosomes indicate putative centromeres. Excluding subtelomeres, imperfect synteny blocks span complete chromosomes with only two exceptions (P. vivax chromosomes 3 and 4), but also contain many putative parasite-specific genes, particularly in P. knowlesi. Image created with Circos .

Figure 6. P. falciparum-specific genes in chromosome-internal regions enriched with virulence genes.

Putative parasite-specific genes identified at SGRs and SBRs between P. falciparum (388 genes) and P. vivax (388 genes) were examined within their syntenic context (upper two diagrams). Differences considered as non-reliable were excluded from further analysis, including positional orthologs (PO), potential missing genes (MG), and potential split or merged genes (SM). Confirmed parasite-specific (CPS) genes were examined for annotated functions (lower two diagrams). P. falciparum-specific genes (lower left diagram) were found to be enriched (FDR-adjusted p-value<0.05) for known virulence factors with associated GO biological processes pathogenesis (GO:0009405), adhesion to host (GO:0044406), cell adhesion (GO:0007155), and defense response (GO:0006952), suggesting potential virulence-associated functions for genes currently not implicated in human virulence.

Figure 7. Human parasites P. falciparum and P. vivax share 13 syntenic orthologs that are absent in the monkey parasite P. knowlesi.

Putative chromosome-internal parasite-specific genes between the human parasite P. vivax (281 genes) and the closely related macaque monkey parasite P. knowlesi (364 genes) were examined within their syntenic context. Slices in white and shades of gray in the upper two diagrams show excluded questionable parasite-specific genes, including differences due to positional orthologs (PO), potential missing genes (MG), sequence gaps (SG), and potential split/merged genes (SM). Black slices represent confirmed parasite-specific (CPS) genes examined for their function. Of the 139 confirmed P. vivax-specific genes, 13 genes (9%, shown in red and in Table 2) have a syntenic ortholog in P. falciparum and thus represent genes present in both human parasites but absent in P. knowlesi.

Figure 8. Uncharacterized genes on P. vivax chromosome 6 possibly involved in erythrocyte invasion.

The upper part of the figure shows a genomic region on P. falciparum chromosome 10 with a cluster of 13 P. falciparum-specific genes, including the S-antigen, liver stage antigen 1 (LSA1), and five members of the MSP3 gene family, including MSP6. The lower part of the figure shows the syntenic genomic region on P. vivax chromosome 6 containing a cluster of eight P. vivax hypothetical proteins. Shaded segments indicate orthology. Genes on the forward strand are shown in blue, genes on the reverse strand shown in red. Figure adapted from PlasmoDB 8.0.