Uncovering the essential genes of the human malaria parasitePlasmodium falciparumby saturation mutagenesis

Abstract

<p class="first" id="P2">Severe malaria is caused by the apicomplexan parasite <i>Plasmodium falciparum. </i> 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 <i>P. falciparum</i> in an approach that exploited its AT-rich genome we generated &gt;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 <i>in vitro </i>. 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. </p><p id="P3">Transposon mutagenesis of <i>Plasmodium falciparum</i> was used to generate &gt;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 <i>in vitro </i> growth. Our study defines ∼1000 essential genes, including genes associated with drug resistance, leading vaccine candidates, and hundreds of <i>Plasmodium-</i>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 <i>P. falciparum</i> high throughput genetics. </p><p id="P4">Saturation-scale mutagenesis of <i>Plasmodium falciparum</i> reveals a core set of genes essential for asexual blood-stage growth <i>in vitro </i>. </p><p id="P5"> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/fce2bb0b-0929-4898-9968-9fb89f2fbc6b/PubMedCentral/image/nihms-1004827-f0007.jpg"/> </div> </p><div class="section"> <a class="named-anchor" id="S1"> <!-- named anchor --> </a> <h5 class="section-title" id="d3165984e297">INTRODUCTION:</h5> <p id="P6">Malaria remains a devastating global parasitic disease, with the majority of malaria deaths caused by the highly virulent <i>Plasmodium falciparum </i>. The extreme AT-bias of the <i>P. falciparum </i> genome has hampered genetic studies through targeted approaches such as homologous recombination or CRISPR-Cas9, and only a few hundred <i>P. falciparum</i> mutants have been experimentally generated in the past decades. In this study, we have used high throughput <i>piggyBac</i> transposon insertional mutagenesis and Quantitative Insertion Site Sequencing (QIseq) to reach saturation-level mutagenesis of this parasite. </p> </div><div class="section"> <a class="named-anchor" id="S2"> <!-- named anchor --> </a> <h5 class="section-title" id="d3165984e314">RATIONALE:</h5> <p id="P7">Our study exploits the AT-richness of <i>P. falciparum</i> genome, which provides numerous <i>piggyBac</i> transposon insertion targets within both gene coding and non-coding flanking sequences, to generate over 38,000 <i>P. falciparum</i> 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 <i>in vitro</i> asexual blood-stage growth. </p> </div><div class="section"> <a class="named-anchor" id="S3"> <!-- named anchor --> </a> <h5 class="section-title" id="d3165984e331">RESULTS:</h5> <p id="P8">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 <i>in vitro</i> asexual growth. Genes predicted to be essential included genes implicated in drug resistance, such as the “ <i>K13</i>” Kelch propeller, <i>mdr</i> and <i>dhfr-ts</i>, as well as targets considered to be high-value for drugs development such as <i>pkg</i>, and <i>cdpk5</i>. The screen revealed essential genes that are specific to human <i>Plasmodium</i> 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 <i>P. falciparum</i>. 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. </p> </div><div class="section"> <a class="named-anchor" id="S4"> <!-- named anchor --> </a> <h5 class="section-title" id="d3165984e361">CONCLUSION:</h5> <p id="P9">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. </p> </div>