Defining the Essential Exportome of the Malaria Parasite

The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.

Severe malaria is caused by the apicomplexan parasite Plasmodium falciparum. Despite decades of research the unique biology of these parasites has made it challenging to establish high throughput genetic approaches for identification of therapeutic targets. Using transposon mutagenesis of P. falciparum in an approach that exploited its AT-rich genome we generated >38,000 mutants, saturating the genome and defining fitness costs for 95% of genes. Of 5,399 genes we found ~3,000 genes are essential for optimal growth of asexual blood-stages in vitro . Our study defines ∼1000 essential genes, including genes associated with drug resistance, vaccine candidates, and conserved proteins of unknown function. We validated this approach by testing proteasome pathways for individual mutants associated with artemisinin sensitivity. Transposon mutagenesis of Plasmodium falciparum was used to generate >38,000 mutants, saturating the genome and defining fitness costs for 95% of genes. We functionally define the relative fitness cost of disruption for 5,399 genes, and find that ~3,000 genes, ~62% of the genome, are essential for optimal asexual blood-stage in vitro growth. Our study defines ∼1000 essential genes, including genes associated with drug resistance, leading vaccine candidates, and hundreds of Plasmodium- conserved proteins of unknown function that are now potential therapeutic intervention targets. We experimentally validated the essentiality of proteasome pathways with drug studies of individual mutants associated with artemisinin sensitivity. This study defines high-priority targets and pathways and points the way for the future of P. falciparum high throughput genetics. Saturation-scale mutagenesis of Plasmodium falciparum reveals a core set of genes essential for asexual blood-stage growth in vitro . INTRODUCTION: Malaria remains a devastating global parasitic disease, with the majority of malaria deaths caused by the highly virulent Plasmodium falciparum . The extreme AT-bias of the P. falciparum genome has hampered genetic studies through targeted approaches such as homologous recombination or CRISPR-Cas9, and only a few hundred P. falciparum mutants have been experimentally generated in the past decades. In this study, we have used high throughput piggyBac transposon insertional mutagenesis and Quantitative Insertion Site Sequencing (QIseq) to reach saturation-level mutagenesis of this parasite. RATIONALE: Our study exploits the AT-richness of P. falciparum genome, which provides numerous piggyBac transposon insertion targets within both gene coding and non-coding flanking sequences, to generate over 38,000 P. falciparum mutants. At this level of mutagenesis, we could distinguish essential genes as non-mutable and dispensable genes as mutable. Subsequently, we identified 3,357 genes essential for in vitro asexual blood-stage growth. RESULTS: We calculated Mutagenesis Index Scores (MIS) and Mutagenesis Fitness Scores (MFS) to functionally define the relative fitness cost of disruption for 5,399 genes. A competitive growth phenotype screen confirmed that MIS and MFS were predictive of the fitness cost for in vitro asexual growth. Genes predicted to be essential included genes implicated in drug resistance, such as the “ K13 ” Kelch propeller, mdr and dhfr-ts , as well as targets considered to be high-value for drugs development such as pkg , and cdpk5 . The screen revealed essential genes that are specific to human Plasmodium parasites but absent from rodent-infective species, such as lipid metabolic genes that may be crucial to transmission commitment in human infections. MIS and MFS profiling provides a clear ranking of the relative essentiality of gene ontology (GO) functions in P. falciparum . GO pathways associated with translation, RNA metabolism, and cell cycle control are more essential, whereas genes associated with protein phosphorylation, virulence factors, and transcription are more likely to be dispensable. Finally, we confirm that the proteasome-degradation pathway is a high-value druggable target based on its high ratio of essential:dispensable genes, and by functionally confirming its link to the mode of action of artemisinin, the current front-line antimalarial. CONCLUSION: Saturation-scale mutagenesis allows prioritization of intervention targets in the genome of the most important cause of malaria. The identification of the essential genome, consisting of over 3000 genes, will be valuable for antimalarial therapeutic research.

Summary The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.