Defining species specific genome differences in malaria parasites

Abstract

Background: In recent years a number of genome sequences for different plasmodium species have become available. This has allowed the identification of numerous conserved genes across the different species and has significantly enhanced our understanding of parasite biology. In contrast little is known about species specific differences between the different genomes partly due to the lower sequence coverage and therefore relatively poor annotation of some of the draft genomes particularly the rodent malarias parasite species.

Results: To improve the current annotation and gene identification status of the draft genomes of P. berghei, P. chabaudi and P. yoelii, we performed genome-wide comparisons between these three species. Through analyses via comparative genome hybridizations using a newly designed pan-rodent array as well as in depth bioinformatics analysis, we were able to improve on the coverage of the draft rodent parasite genomes by detecting orthologous genes between these related rodent parasite species. More than 1,000 orthologs for P. yoelii were now newly associated with a P. falciparum gene. In addition to extending the current core gene set for all plasmodium species this analysis also for the first time identifies a relatively small number of genes that are unique to the primate malaria parasites while a larger gene set is uniquely conserved amongst the rodent malaria parasites.

Conclusions: These findings allow a more thorough investigation of the genes that are important for host specificity in malaria.

Figure 1

The overall design schematics of the pan-rodent chip. (A) Methodology of the chip design. Firstly, all possible oligonucleotides of P. yoelii were used to search in the homologous region of the other two species using NCBI blastn and were scored and ranked accordingly. The oligonucleotides were then filtered using three rules such they must have: (i) at least 90% homology to target sequences, (ii) less than 37.5% to non-target sequences and (iii) GC% tolerance of ± 5%. Oligonucleotides for all three species were selected followed by oligonucleotides for P. yoelii and P. berghei and then for P. yoelii and P. chabaudi. Next, the remaining oligonucleotides were selected to be specific for both P. berghei and P. chabaudi. The remaining sequences unaccounted for were then used to design oligonucleotides specific either to P. yoelii, P. berghei or P. chabaudi. (B) Rank-sum strategy. Oligonucleotides were scored accordingly to (i) first and second BLAST hits, (ii) GC content (Tm) and (iii) Smith-Waterman score (self-binding). The oligonucleotides are then ranked based on each parameter and ordinal rank number is given to all oligonucleotides in each parameter rank independently. The final weighted rank-sum (RS) is calculated for all oligonucleotides using multiple weight sets (not indicated) and the lowest value is considered. Finally, the optimal candidate is selected based on the lowest RS(k) amongst all oligonucleotides in the locus of interest [18]

Figure 2

Venn diagram showing distribution of target rodent parasite gene hits of all oligonucleotides. All oligonucleotides are 60 bases long and the GC content is targeted at 30% and the allowable deviation is 5% for overlapping oligonucleotides. Complementary oligonucleotides to each rodent malaria parasite species was calculated from the sum of all possible combinations, i.e. oligonucleotides specific to itself and those that can hybridize to itself and to other rodent malaria parasite species. (Legend: Pb = P. berghei specific oligonucleotides only; Pc = P. chabaudi specific olinucleotides only; Py = P. yoelii specific oligonucleotides only; Pbc = P. berghei &P. chabaudi specific oligonucleotides; Pby = P. berghei &P. yoelii specific oligonucleotides; Pcy = P. chabaudi &P. yoelii specific oligonucleotides; Pbcy = oligonucleotides specific to all 3 rodent parasite species)

Figure 3

PCR screening of a random sample of newly discovered genes. Screenings were performed pair-wise with the PCR products of the species containing the known gene of interest loaded in odd-numbered wells while the corresponding PCR screen of the other species whereby sequence is absent or the gene is not predicted are in the even-numbered wells. The Genbank accession numbers of these novel orthologs are indicated in parentheses in the following description. (1&2): PY00632 screen with Py and Pb gDNA (GU390534); (3&4): PY03414 screen with Py and Pb gDNA (GU390535); (5&6): PY04600 screen with Py and Pb gDNA (GU390540); (7&8): PY04485 screen with Py and Pb gDNA (GU390538); (9&10): PY02086 screen with Py and Pc gDNA (GU390539); (11&12): PY04869 with Py and Pc gDNA; (13&14): PY06972 screen with Py and Pc gDNA (GU390536); (15&16): PY05482 screen with Py and Pc gDNA (GU390537). (Legend: M = 100 bp DNA ladder)

Figure 4

PCR screening of bioinformatics-filtered dataset with contig information. (A) Schematic depicting the scenario whereby a contig from 1 species containing the gene of interest (shown as a hatched block) is aligned with the best corresponding hit contig from another species. In this case, the gene of interest is lost in 1 species while the flanking sequences are still present. (B) PFI0535w-PB104921.00.0 orthologs screened with (1)Pb, (2)Pc and (3)Py gDNA. PF14 0473-PC0001359.02.0 orthologs screened with (4)Pb, (5)Pc and (6)Py gDNA. MAL13P1.345-PY04218 orthologs genes screened with (7)Pb, (8)Pc and (9)Py gDNA. PFL0595c-PB301230.00.0-PC000699.01.0 orthologs screened with (10)Pb, (11)Pc and (12)Py gDNA. PFF1480w(3'end)-PB000730.00.0PY03519 orthologs screened with (13)Pb, (14)Pc and (15)Py gDNA. PF13_0131PC000708.04.0-PY04599 orthologs screened with (16)Pb, (17)Pc and (18)Py gDNA. (Legend: M = 100 bp DNA ladder)

Figure 5

PCR screening of bioinformatics-filtered dataset without contig information. (A) Schematic depicting the scenario whereby a contig from 1 species containing the gene of interest (shown as a hatched block) is aligned with the best corresponding hit contig from another species. In this case, the gene of interest is lost in 1 species due to missing sequence information while the adjacent flanking contig sequences are still present. (B) PFC0095c-PB000276.02.0 orthologs screened with (1)Pb, (2)Pc and (3)Py gDNA. PFL2450c-PC000344.03.0 orthologs screened with (4)Pb, (5)Pc and (6)Py gDNA. MAL8P1.310-PY06565 orthologs screened with (7)Pb, (8)Pc and (9)Py gDNA. PFB0645c-PB000193.00.0-PC000452.02.0 orthologs screened with (10)Pb, (11)Pc and (12)Py gDNA. PFF1480w(5'-end)-PB000730.00.0-PY03519 orthologs screened with (13)Pb, (14)Pc and (15)Py gDNA. PF13_0278-PC000860.02.0-PY04659 orthologs screened with (16)Pb, (17)Pc and (18)Py gDNA. (Legend: M = 100 bp DNA ladder)

Figure 6

Graphical plot showing the re-distribution of the rodent malaria parasite genes after removing the PIR genes. Most of the reduction occurs in the group Py-Pb-Pc in which all the three rodent malaria parasite species share a common gene. (Legend: Py = genes specific to P. yoelii; Py-Pb = genes common to both P. yoelii and P. berghei; Py-Pc = genes common to both P. yoelii and P. chabaudi; Py-Pb-Pc = genes common to all three species)