Conservation of gene essentiality in Apicomplexa and its application for prioritization of anti-malarial drug targets

Fitness data for knockout mutants

The genome-wide CRISPR screening data on the relative fitness of T. gondii genes during infection of human fibroblasts cells was obtained from Sidik et al.⁹. The authors defined log₂ fold change in abundance of single guide RNA (sgRNA) targeting a given gene as the “phenotype” score for that gene⁹. It was found that for a previously determined set of 81 essential and non-essential genes, a phenotype score of less than -2 identified most of the essential genes, but none of the non-essential genes⁹. We thus defined all genes with a phenotype score of less than -2 as essential (2870 genes). Genes with a phenotype score greater than 0 were defined as non-essential (3071 genes), while those with a phenotype score between 0 and -2 were not classified (2210 genes). The in vivo relative growth rate data for 2574 genes of P. berghei were obtained from the PlasmoGEM database^7,8 (http://plasmogem.sanger.ac.uk/phenotypes). The authors generated knockout mutants by transfection with large pools of barcoded gene knockout vectors. The in vivo growth rate in Balb/c mice was obtained by counting barcodes by next generation sequencing daily between days 4 and 8 post transfection⁷. Essential genes were defined as genes with a growth rate not significantly different from 0.1 (growth rate of the wild type taken as 1), while non-essential genes were defined as genes with growth rate not significantly different from 1⁷.

Proteome data and sequence analyses

Proteome sequences of P. falciparum 3D7, P. berghei ANKA, P. chabaudi chabaudi, P. cynomolgi B, P. knowlesi H, P. reichenowi CDC, P. vivax Sal1, P. yoelii 17X were downloaded from the PlasmoDB database¹⁰ (http://plasmodb.org/common/downloads/release-27/). The Proteome sequences for six apicomplexan species were obtained from EuPathDB¹¹: Cryptosporidium hominis TU502 (http://cryptodb.org/common/downloads/release-29/ChominisTU502/); T. gondii GT1 (http://toxodb.org/common/downloads/release-29/TgondiiGT1/); Eimeria brunetti Houghton (http://toxodb.org/common/downloads/release-29/EbrunettiHoughton/); Babesia bovis T2Bo (http://piroplasmadb.org/common/downloads/release-29/BbovisT2Bo/); Theileria annulata Ankara (http://piroplasmadb.org/common/downloads/release-29/TannulataAnkara/); and Gregarina niphandrodes (http://cryptodb.org/common/downloads/release-29/GniphandrodesUnknown/). Proteome sequences for Homo sapiens were downloaded from EBI (http://www.ebi.ac.uk/reference_proteomes). Homologs of P. berghei genes in H. sapiens were identified with E-value cut-off of 1e-6, with soft mask set as true. Orthologous sequences were identified using best bidirectional hit algorithm¹².

Functional data

RNA-seq data (FPKM values) for different stages of P. berghei was obtained from Otto et al.¹³. Proteomics data on different stages of P. berghei and dN, dN/S values were obtained from Hall et al.¹⁴. Gene Ontology information for P. falciparum was obtained from PlasmoDB¹⁰, and these functions were assigned to their orthologous proteins in P. berghei. Enzyme Commission (EC) numbers for P. berghei and P. falciparum were also obtained from PlasmoDB. Trans-membrane regions were identified using TMHMM¹⁵. All statistical analyses were performed in the R software version 3.3.1 (https://www.r-project.org/).

Conservation of gene essentiality in apicomplexan parasites

The relative in vivo growth rate of knockout mutants for 2574 P. berghei genes (out of total 5076 genes in P. berghei) has recently been measured, of which 1198 genes (46%) with very low growth rate were classified as essential^7,8. Similarly, in vivo relative fitness of knockout mutants for 8151 T. gondii genes have been measured⁹, of which 2870 genes (35%) with very low relative fitness values were classified as essential (see Methods). Of the 2574 P. berghei genes with fitness data, 1617 genes have an ortholog in T. gondii. P. berghei genes with an ortholog in T. gondii were significantly more likely to be essential, compared to P. berghei genes without an ortholog in T. gondii (53% vs. 36%; Fisher test p = 7e-18; Figure 1A). P. berghei genes with an essential ortholog in T. gondii were significantly more likely to be essential, compared to P. berghei genes with a non-essential ortholog in T. gondii (71% vs. 17%; Fisher test p = 6e-59; Figure 1A). There was a significant correlation in relative fitness values of P. berghei and T. gondii (Spearman correlation coefficient 0.47; p = 3e-89; n =1617; Figure 1B). The essentiality of 2502 P. berghei genes was not tested, but the essentiality information of T. gondii orthologs may be used to predict their essentiality in P. berghei. There were 687 genes in P. berghei with an essential ortholog in T. gondii, and thus may be predicted as essential in P. berghei (Dataset 1¹⁶).

Figure 1. Conservation of essentiality between Plasmodium berghei and Toxoplasma gondii.

(A) P. berghei genes with an ortholog in T. gondii were more likely to be essential, compared to P. berghei genes without an ortholog in T. gondii (Fisher test p = 7e-18). P. berghei genes with an essential ortholog in T. gondii were significantly more likely to be essential compared to P. berghei genes with a non-essential ortholog in T. gondii (Fisher test p = 6e-59). (B) There was a significant correlation in relative fitness values of P. berghei and T. gondii (Spearman correlation coefficient 0.47; p = 3e-89; n =1617). Genes classified as essential in both species are colored red. Genes classified as non-essential in both species are colored blue. Genes that are essential in only one of the species are colored green.

Prioritization of anti-malarial drug targets

We argue that genes identified as essential in both the apicomplexan parasites could be more useful drug targets for the following reasons: 1) Genome-scale fitness screens often involve significant false positives and false negatives⁷, thus genes identified as essential in independent experiments in different parasites could be more confidently assigned as essential; 2) the substantial conservation of gene essentiality between the two parasites demonstrates that essentiality information in T. gondii offers relevant information about gene essentiality in P. berghei; 3) genes that are essential in both P. berghei and T. gondii should be more likely to be essential in human malarial species, such as P. falciparum and P. vivax; 4) genes that are essential in both P. berghei and T. gondii should be more likely to be essential across different developmental stages of Plasmodium, which is a highly desirable property of Plasmodium drug targets¹⁷. We thus identified 710 genes that were essential in both species. A total of 289 of these 710 genes encode enzymes, which are typically used as drug targets against pathogens. Of these 289 genes, 245 had an ortholog in all Plasmodium species and did not have more than one trans-membrane segment. We removed proteins with more than one trans-membrane segments, as these are often difficult to purify for in vitro assays. Of the 245 proteins, 30 showed no significant sequence similarity to any human proteins (listed in Table 1), and 83 showed less than 30% identity and 151 showed less than 40% identity to any human protein (Dataset 1¹⁶). Figure 2 shows the flow chart of the selection process.

Figure 2. Selection of potential drug targets in Plasmodium.

Among the P. berghei enzymes that were not tested for essentiality, 186 had an essential ortholog in T. gondii and thus may be predicted as essential in P. berghei. To increase the confidence of these genes to be essential in Plasmodium, we considered 53 genes that were conserved across Plasmodium and apicomplexan species. Among the enzymes tested for essentiality, such a criteria led to a set with 77% enzymes as essential, suggesting high enrichment for essentiality among predicted essential enzymes. In total, 28 of these enzymes had low sequence similarity (<40% identity) with human proteins and thus may also be considered as potential drug targets (Dataset 1¹⁶).

Properties of essential P. berghei genes

Essential genes show different expression, evolutionary and functional properties⁹. We thus tested whether similar patterns would be observed for P. berghei. Essential P. berghei genes showed higher mRNA expression levels in asexual stages, but lower expression levels in sexual stages compared to non-essential genes (Figure 3A). Proteins encoded by essential genes were more likely to be detected by mass-spectrometry in different developmental stages compared to non-essential genes (Figure 3B). Essential genes showed a lower evolutionary rate (dN and dN/dS) and higher conservation in apicomplexan species (Figure 3C). Essential genes were significantly enriched in functional classes, such as “Translation”, “Ribosome”, “DNA replication”, “Intracellular protein transport”, “Cytoplasm”, and “Nucleus” (Figure 4).

Figure 3. Properties of essential Plasmodium berghei genes.

(A) Essential P. berghei genes showed higher mRNA expression levels in asexual stages, but lower mRNA expression levels in sexual stages. The mean FPKM values for the essential and non-essential genes were calculated for different development stages and their log₂ ratio was taken. All stages except ‘ookinete 24h’ showed a statistically significant difference between essential and non-essential genes (t-test; p < 0.05). The RNA-seq data was taken from Otto et al.¹³. (B) Proteins encoded by essential genes were more likely to be detected by mass-spectrometry in different stages compared to non-essential genes. All stages except ‘sporozoites’ showed a significant difference between essential and non-essential genes (Chi-square test; p < 0.05). Overall 47% of the tested genes were essential. The proteomics data was obtained from Hall et al.¹⁴ (C) Essential genes showed a lower evolutionary rate and higher conservation across apicomplexan species. The mean dN and dN/dS values for essential and non-essential genes was calculated and their log₂ ratio was taken. This data was taken from Hall et al.¹⁴. The mean number of apicomplexan species (out of six), in which an ortholog was identified, was calculated for essential and non-essential genes and their log₂ ratio was taken. dN and conservation in apicomplexan species showed a statistically significant difference between essential and non-essential genes (t-test; p < 0.05), but not dN/dS.

Figure 4. Prevalence of essential genes in different functional classes.

The Gene Ontology information for Plasmodium falciparum genes was obtained from PlasmoDB¹⁰ and assigned to their P. berghei orthologs. Classes with a significant difference (Chi-square test; p < 0.05) in essential genes are marked with *.