Post-Genomic Approaches to Understanding Malaria Parasite Biology

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Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX

Post-Genomic Approaches to Understanding Malaria Parasite Biology: Linking Genes to Biological Functions Anna E. Sexton,† Christian Doerig,‡ Darren J. Creek,*,†,∥ and Teresa G. Carvalho*,§,∥

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Drug Delivery Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia ‡ Centre for Chronic, Inflammatory and Infectious Diseases, Biomedical Sciences Cluster, School of Health and Biomedical Sciences, RMIT University, 264 Plenty Road, Bundoora, VIC 3083, Australia § Molecular Parasitology Laboratory, Department of Physiology, Anatomy and Microbiology, La Trobe University, Kingsbury Drive, Bundoora, VIC 3086, Australia ABSTRACT: Plasmodium species are evolutionarily distant from model eukaryotes, and as a consequence they exhibit many non-canonical cellular processes. In the postgenomic era, functional “omics” disciplines (transcriptomics, proteomics, and metabolomics) have accelerated our understanding of unique aspects of the biology of malaria parasites. Functional “omics” tools, in combination with genetic manipulations, have offered new opportunities to investigate the function of previously uncharacterized genes. Knowledge of basic parasite biology is fundamental to understanding drug modes of action, mechanisms of drug resistance, and relevance of vaccine candidates. This Perspective highlights recent “omics”-based discoveries in basic biology and gene function of the most virulent human malaria parasite, Plasmodium falciparum. KEYWORDS: Plasmodium falciparum, malaria, systems biology, “omics”, putative genes, genetic manipulation

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processes challenging. In addition, the prediction of gene function is close to impossible when addressing the noncanonical cellular processes that dominate much of P. falciparum biology. Functional “omics” technologies (transcriptomics, proteomics, and metabolomics) have been, and continue to be, central to overcoming such challenges. In this Perspective, we outline the progress achieved in the malaria “omics” field over the past 20 years. Further, we highlight key “omics”-centered discoveries that have unraveled the molecular processes that underpin unique aspects of P. falciparum biology. Finally, we outline recent successes that combined “omics” technologies and genetic manipulation techniques to characterize gene function.

alaria remains a major burden in global public health, with an estimated 219 million cases and 435 000 deaths in 2017.1 Ongoing initiatives to reduce malaria transmission (such as insecticide-treated bednets and indoor insecticide spraying), and the success of artemisinin-based combinations therapies to treat malaria, have helped to lower this burden over the last two decades. However, progress is threatened by the emergence of mosquitoes and malaria parasites that are resistant to these current treatments. Decades of effort have resulted in the first malaria vaccine (RTS,S/AS01) to complete phase 3 clinical trials, and pilot implementation studies are underway.2 Its efficacy is, at best, 39% in children aged 5−17 months.3 Consequently, there is a persistent need to fuel the therapeutics pipeline with drugs that have novel mechanisms of action and vaccine candidates that can offer higher levels of protection. A deeper comprehension of basic malaria parasite biology is a pathway toward identifying and characterizing novel therapeutic targets and understanding mechanisms of drug resistance. In recent years, the advent of systems biology, in particular the “omics” disciplines, has shed considerable light on malaria parasite biology. Within the last 16 years, the malaria genome, transcriptome, proteome, and metabolome have been described for the most virulent human malaria parasite, Plasmodium falciparum. However, the evolutionary distance of Plasmodium species from model organisms has seriously impaired homology-based genome annotation: over a third of P. falciparum genes remain without an assigned function, and over a third have only “putative” functions. This has made the identification of some of the genes that mediate core cellular © XXXX American Chemical Society

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THE “OMES” P. falciparum has a complex life cycle in both the mosquito and human hosts, with striking morphological differences between its different developmental stages. Underpinning this biology is the ability of the parasite to respond to environmental cues and to undergo a developmental program, both of which prompt transcription of genes and their translation into proteins at specific times during the life cycle. Many proteins then use small molecules (“metabolites”) to carry out their functions. Changes in parasite development can be assessed comprehensively by “omics” approaches (Figure 1). The core “omes” (genome, transcriptome, proteome, and metabolome) have been characterized across many of the P. falciparum Received: March 6, 2019 Published: June 6, 2019 A

DOI: 10.1021/acsinfecdis.9b00093 ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

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Figure 1. The “omics” cascade and analytical techniques used to characterize each “ome”. Abbreviations: NMR, nuclear magnetic resonance; npc RNA, non-protein coding RNA; PTMs, post-translational modifications; RNA-Seq, RNA-sequencing; SNPs, single nucleotide polymorphisms.

annotation is a current challenge to investigations of fundamental Plasmodium biology, drug mechanisms of action, vaccine candidates, and host−parasite interactions. Since 2002, the genomes of many P. falciparum laboratory strains and field isolates have been sequenced with the goal of identifying genes and genetic traits associated with drug resistance and virulence (see refs 5−7 for recent examples). Genomics has enabled surveillance8,9 and epidemiological modeling10 of parasite population dynamics in malaria endemic regions. Advances in single-cell genome sequencing11,12 have recently been applied to investigate transmission dynamics and genetic diversity in polygenomic P. falciparum infections.13 Altogether, these studies reveal the remarkable genetic diversity of P. falciparum populations, and in the future, this information will undoubtedly inform targeted therapeutics for patients and aid surveillance of drug resistance adaptations in parasite populations. These population genomics studies have provided an efficient approach to identify associations between various parasite characteristics and specific genes, but the mechanistic interpretation of these relationships is often hindered when significant genes of interest have no functional annotation. The Transcriptome. Initial P. falciparum transcriptome studies relied on DNA microarrays to characterize the transcriptional signatures of distinct developmental stages of P. falciparum.14−17 More recently, RNA-Seq has enabled global characterization of P. falciparum transcription at multiple developmental stages, has defined 5′ and 3′ untranslated regions of genes, and has identified mRNA splicing events and antisense transcripts.18−21 Notably, these population-based studies suggest that P. falciparum employs a highly ordered and continuous cascade of gene expression during the asexual blood-stage cycle. However, the recent application of single cell RNA-Seq to individual P. falciparum-infected red blood cells has updated this model and unmasked abrupt changes to gene expression whereby groups of genes are simultaneously

developmental stages, and have revealed several new functions that explain how the parasite supports the unique biological needs of each life cycle stage. The Genome. The P. falciparum genome was first published in 2002 after several years of international effort.4 The nuclear genome is a haploid set of 14 chromosomes and is exceptionally AT-rich (80.6% AT content on average and approaching 90% in non-coding regions). The current version (2015-06-18) defines the nuclear genome as 23.33 Mb in size, encoding 5712 genes (www.plasmodb.org). In addition to the nuclear genome, P. falciparum parasites possess a 6 kb mitochondrial genome and a 35 kb apicoplast genome. Genome annotation has revealed important insights into parasite biology. For example, the P. falciparum genome comprises fewer genes encoding transporters and metabolic enzymes compared to other unicellular, non-parasitic eukaryotes; it lacks genes that encode enzymes for de novo amino acid and purine synthesis; and 1.3% of its genes are related to immune evasion and cell adhesion.4 In the first draft of the genome, approximately 60% of genes did not have sufficient homology to characterized genes from model organisms to be assigned predicted functional annotation and were therefore annotated as “hypothetical”.4 This terminology has mostly been abandoned, and there are now distinct annotations for genes of “unknown” function and “putative” function (where function is predicted, but experimental validation is required). Advances in homology matching, comparative genomics, and metabolic modeling have improved genome annotation. Nonetheless, a text search against the current version of the nuclear genome for annotations of “putative” yields 36% of all genes, and a text search for annotations of “unknown function” yields a further 35% of genes. Further, 41% of the predicted protein-encoding genes in the apicoplast genome are annotated “putative”, “probable”, or “hypothetical” (www.plasmodb.org). The substantial incompleteness of the P. falciparum genome B



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switched on and off during asexual blood-stage development.22 Such distinct gene expression transitions in single cells were previously masked in population-based studies and likely constitute developmental checkpoints that were previously elusive. Transcriptomic studies have further revealed genes whose expression is not reliant on programmed developmental control but varies according to other cues, including antigenic variation mechanisms,23 host cell exposure,17 and drug exposure.24 Indeed, single-cell RNA-seq identified 56 genes whose expression varied independently of the cell cycle, many with putative or unknown functions.22 In addition to the transcriptome of protein-coding mRNA, the complement of non-protein coding (npc) RNA has been characterized and includes small structural npc-RNA, long npc-RNA, circular RNA, as well as npc-RNA of unknown function.25−27 It is suggested that P. falciparum npc-RNAs regulate networks that play a role in antigenic variation,28 sexual commitment,29 chromatin assembly,30 and novel parasite-specific processes.26 Notably, no microRNA has been identified in P. falciparum,31,32 and this is in line with the absence of RNAinterference machinery in Plasmodium parasites.33 The Proteome. Two independent seminal studies of the P. falciparum proteome were published concurrently with the first genome annotation in 2002. Florens et al. identified over 2400 proteins across the proteomes of the sporozoite, merozoite, trophozoite, and gametocyte stages.34 Lasonder et al. identified 1289 proteins across the proteomes of gametocytes, gametes, and late asexual blood stages (trophozoite and schizonts combined).35 Less than 30% of the identified proteins were identified across all parasite stages, and stage-specific proteins have shed light on the different biological functions operating across the parasite life cycle.34,35 For example, proteins of invasion machinery were abundant in merozoites; proteases involved in hemoglobin digestion were abundant in trophozoites; and a number of dynein proteins were expressed exclusively in sexual stages, likely contributing to male gamete motility. Since then, proteomic studies have extensively surveyed stage-specific differences, including the identification of sex-specific proteins that could be targeted for transmission blocking strategies.20,36,37 Advanced proteomics studies have characterized subsets of proteins that carry post-translational modifications (e.g., phosphorylation,38−40 acetylation,41,42 prenylation,43 glycosylation,44 and methylation45). These studies have revealed potential regulatory mechanisms for many cellular processes and are reviewed elsewhere.46 Organelle-specific proteins of the nucleus,47,48 rhoptries,49 apicoplast,50 and parasitophorous vacuole51 provide useful insight into the function of such cellular structures. Proteomics studies have also surveyed proteins on the surface of P. falciparum-infected red blood cells identifying novel antigens for rational vaccine design.52,53 The Metabolome. Metabolomics of P. falciparum-infected red blood cells has provided insights into how the parasite modulates metabolism over its developmental stages.54−56 For example, to support the rapid growth during the asexual blood stage, asexual parasites rely mostly on aerobic glycolysis for rapid energy production. In contrast, non-replicative gametocytes rely more heavily on the TCA cycle to produce energy through oxidative phosphorylation.57 The global metabolome of P. falciparum mosquito stages remains to be characterized. However, genetic studies have revealed metabolic processes essential for development in the mosquito, including the FASII

pathway for de novo fatty acid synthesis58 and the TCA cycle.59 Unlike the macromolecules that form the basis of sequencebased “omics”, metabolites cannot be easily delineated as arising from the host or the parasite; however, comparisons between infected and non-infected red blood cells are starting to describe important host-parasite interactions.54,56,60 Metabolic profiling has been key to functional gene annotation because it provides biochemical evidence for the function of putative enzymes, and it has also led to the annotation of new metabolic pathways in the parasite.61,62 Metabolomics has also been useful in surveying drug mechanisms of action,63,64 as drug-specific metabolic perturbations can elucidate the inhibition of particular enzymes (e.g., atovaquone65 and fosmidimycin66 ). Metabolomics-based screens have predicted the functions of many antimalarial compounds in the Malaria Box resource,67,68 and therefore shed light on several pathways that are essential for parasite growth in the asexual blood stage.

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FROM “OMES” TO PARASITE BIOLOGY The characterization of each “ome” has formed a strong foundation for further “omics” studies of P. falciparum biology and applications of functional “omics” techniques have enabled, for the first time, the identification of genes associated with parasite-specific processes. For example, transcriptomics of parasites under high-density conditions has linked the expression of stress-related and cell death-related genes to density-dependent control of population size.69 Transcriptomics and proteomics have linked genes specific to male and female gametocytes, and have revealed large-scale translational repression as a hallmark of female gametocyte biology.20 Metabolomics has identified novel metabolites in the plasma of blood from patients, which revealed that a plant-like, αlinolenic acid (ALA) pathway is active in P. falciparum. Homology-based searches have identified P. falciparum genes that are likely to fulfill these metabolic functions.61 As functional “omics” techniques have become more widespread, it is now possible to simultaneously access multiple “omics” techniques for comprehensive analyses. These multi-“omics” analyses begin to shed light on how each level of the “omics” cascade integrates to perform parasite functions. For example, merozoites that were selected for their extended viability and increased invasion efficiency displayed extensive changes at the proteome level but few changes at the genome and transcriptome levels. Increased abundance of known invasion proteins, protein kinase A, and several merozoite surface proteins confirmed known mechanisms of invasion, while proteins of unknown function offer new opportunities to understand the molecular events underpinning invasion. This multi-“omics” approach was essential for indicating the important role of post-transcriptional and post-translational mechanisms in the regulation of invasion.70 Perhaps one of the greatest successes of multi-“omics” has been the major advances in our knowledge of gametocytogenesis (the process by which parasites commit to sexual development and convert into gametocytes). For a long time, the search for traditional transcription regulators in Plasmodium produced few results, and the regulatory mechanisms behind sexual commitment remained elusive. However, homology searches for DNA binding domains and motifs within the P. falciparum genome ultimately identified the AP2 family of transcription factors (formerly found only in plants and algae), and transcriptomics analysis elucidated the C



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differential expression of AP2 members across the life cycle.71 Subsequent transcriptomics studies revealed close association of the transcription factor, AP2-G, with known markers of gametocyte development23 and led to the identification of AP2-G as an essential regulator of gametocytogenesis.72 Singlecell RNA-Seq analysis of sexually committed schizonts (AP2-G +) has shown promising results in characterizing the transcriptional program of gametocytogenesis.73 Further, functional assays and single-cell RNA-Seq have recently revealed that sexual conversion of parasites into gametocytes can occur either within the same replicative cycle as initial AP2-G expression or in the subsequent replicative cycle.74 Single-cell RNA-Seq shows that these routes have different transcriptional signatures74 and provides a basis for investigation of how these routes are induced. Various epigenetic mechanisms control the expression of AP2-G, including the removal of epigenetic silencing from the ap2-g locus, which requires gametocyte development 1 protein.29 The environmental cues that operate upstream of this process are beginning to be elucidated, and lysophosphatidylcholine (LysoPC) depletion was the first cue to be linked with the induction of AP2-G expression using “omics” techniques.75 Brancucci et al. observed that serum-free media stimulated gametocyte commitment in vitro and therefore fractionated the serum on the basis of polarity to empirically identify gametocyte-repressing fractions using functional assays.75 Metabolic profiling of the gametocyte-repressing fractions identified the phospholipid LysoPC as the main component of those fractions. Indeed, addition of LysoPC to serum-free media repressed gametocyte production. Stable isotope-labeling of LysoPC showed that it was metabolized through the Kennedy pathway and inhibiting this pathway induced gametocytogenesis. LysoPC depletion led to transcriptional changes (measured using RNASeq) in late asexual blood-stage parasites, including the induction of AP2-G. Undoubtedly, “omics” will be useful for elucidating the links between LysoPC depletion and the induction of epigenetic changes required for AP2-G expression. “Omics” approaches have been pivotal to understanding many aspects of parasite biology (Figure 2). However, many of these studies identify candidate genes putatively involved in such processes, and these require functional validation. Many candidate genes do not have a functional annotation, or have only “putative” annotation. A combination of “omics” techniques alongside advanced genetic manipulation strategies is forging the way toward understanding the function of such candidate genes and the systems within which they operate (Table 1).

Figure 2. Discovery of new P. falciparum biology using “omics” technology. The blood stages of the P. falciparum life cycle and selected examples of “omics” studies are illustrated. Colored circles indicated the technology used: red (transcriptomics), green (proteomics), and blue (metabolomics).

protein kinases,81 DNA-binding proteins,82 transcription factors,71 and metabolic enzymes.83 However, even for those genes that contain annotated functional domains, the phylogenetic distance from model organisms impairs prediction of exact physiological interactions. For example, many Plasmodium protein kinases have unknown functions, even in the case of the numerous kinases that have been assigned to a specific kinase group. Furthermore, function and signaling pathway organization cannot be predicted for the kinases that belong to “orphan” groups or families, such as the FIKKs, an apicomplexan-specific group that comprises 20 paralogues in P. falciparum.81 The combination of functional “omics” and genetic manipulation approaches has proven a productive strategy for probing gene function. Kinomics. While a kinome-wide reverse genetics screen has informed on which protein kinases are essential for completion of the asexual blood-stage cycle, phosphoproteomics has been key to identifying protein kinase substrates and the parasite processes that they regulate.40 For example, phosphoproteomic analysis of protein kinase knock-out and knock-down parasites has revealed roles for PfCRK4 in DNA replication84 and PfCDPK1 in parasite invasion.85 PfPK7 knock-out parasites have reduced proliferation during the asexual blood stage,86 and phosphoproteomics revealed that PfPK7 may regulate the phosphorylation of 146 different proteins, including three uncharacterized proteins which it can directly phosphorylate in vitro.87 Studying essential proteins is a particular challenge for P. falciparum research, and innovative approaches have been applied to study essential protein kinases. Alam et al. characterized the function of the essential protein kinase, PfPKG, by genetically altering the sensitivity of PfPKG to the inhibitor called “Compound 2” via a mutation of the so-called “gatekeeper” residue that prevents inhibitor binding but does not interfere with the activity of the enzyme.88 Phosphoproteomics of wild-type and PfPKG mutant parasites that had

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USING “OMICS” TO UNDERSTAND GENE FUNCTION The publication of the P. falciparum genome sequence enabled the identification of whole complements of genes with targeting motifs, such as the apicoplast-targeting sequence76 and the PEXEL motif for directing protein export beyond the parasitophorous vacuole (the membranous vacuole within which the parasite resides inside infected red blood cells).77,78 Genetic ablation of some hypothetical genes with PEXEL motifs indeed leads to defects in cytoadhesion, rigidity, and protein trafficking (for example, in the case of the major virulence protein, PfEMP1).79 More generally, homologybased approaches to identify particular functional domains have successfully uncovered putative protein transporters,80 D



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Table 1. “Omics” Analysis of Gene Function Using Genetically Altered Parasites gene

genetic manipulation

PfRhopH2 PfHsp70-x

knock-down knock-out

PfSEMP1 Pfg377 PfCRK4 PfPKG

knock-down knock-out knock-down amino acid substitution

PfCDPK1

knock-down knock-out knock-out sensitive and resistant alleles

PfPK7 PfCRT PfKelch13

single-point mutation (sensitive and resistant)

“omics” technique metabolomics transcriptomics, proteomics transcriptomics proteomics phosphoproteomics phosphoproteomics phosphoproteomics transcriptomics phosphoproteomics peptidomics metabolomics peptidomics metabolomics

findings

reference

PfRhopH2 implicated in nutrient uptake increased expression and abundance of chaperones and other exported proteins increased expression of other exported proteins identification of proteins in osmiophilic bodies PfCRK4 implicated in the regulation of DNA synthesis PfPKG identified as a key regulator of several cellular processes PfCDPK1 regulates invasion-related proteins increased expression of gametocyte-specific genes novel proteins substrates identified native PfCRT function is related to normal hemoglobin digestion PfKelch13 function is related to hemoglobin digestion and glutathione metabolism

Counihan et al.89 Charnaud et al.93 Dietz et al.94 Suarez-Cortes et al.109 Ganter et al.84 Alam et al.88 Kumar et al.85 Bansal et al.110 Pease et al.87 Lewis et al.99 and Lee et al.100 Siddiqui et al.101

peptidomics), suggesting that one aspect of PfCRT function is to regulate parasite access to peptides or to maintain the physiological conditions in the digestive vacuole such that enzymes can function normally. This observation was recapitulated in a recent study using parasites of a different genetic background.100 Similarly, Siddiqui et al. performed a multi-“omics” analysis of artemisinin-resistant and -sensitive parasites with PfKelch13 mutations on the same genetic background.101 Metabolomics and peptidomics revealed that mutations in PfKelch13 affect hemoglobin digestion and glutathione production, two pathways intimately involved with the free-radical mediated mechanisms of action of artemisinin. The widespread application of “omics” workflows to explore gene function has provided unprecedented information on the global networks and parasite processes within which individual genes operate. However, the availability of genetic mutants to underpin these studies has previously been limited. More recently, the application of CRISPR-Cas9 technology has markedly improved the efficiency by which P. falciparum mutants can be generated,102,103 and there are now several conditional knock-down approaches available to systematically dissect gene function in P. falciparum (reviewed elsewhere104). Furthermore, transposon-based mutagenesis approaches have enabled the first comprehensive forward-genetic screen of the P. falciparum genome and have uncovered genes essential for asexual blood-stage replication.105 These screens are amenable to probing genes that are essential for stress responses, drug exposure, and other developmental stages of P. falciparum. The application of single-cell based “omics” workflows to analyze mutants generated by these screens en masse is an exciting future direction. Importantly, the diligent curation of “omics” and other functional data into Web-based resources such as Malaria Parasite Metabolic Pathways,106 PlasmoDB,107 and PhenoPlasm108 has secured the accessibility of these invaluable data sets for all malaria researchers.

been treated with Compound 2 revealed 69 proteins whose phosphorylation was dependent on PfPKG activity. These proteins were involved in several cellular processes (including invasion, protein export, and gene regulation) and revealed PfPKG as a key signaling hub. Protein Trafficking. Upon infection of red blood cells, P. falciparum scavenges a number of nutrients from its host environment by establishing new permeability pathways in the infected red blood cell plasma membrane. The molecular components of these pathways require identification. Following the observation that knock-down of PfRhopH2 resulted in sorbitol lysis-resistant parasites (and hence is likely involved in the transport of solutes across the red blood cell plasma membrane), metabolomics was used to investigate whether PfRhopH2 was involved in nutrient import.89 This revealed depletion of vitamins and cofactors (normally imported from the host) in the PfRhopH2 knock-down parasites, supporting the hypothesis that PfRhopH2 is involved in new permeability pathways. The establishment of new permeability pathways requires the export of parasite proteins beyond the parasitophorous vacuole, many of which are transported across by a proteinaceous pore, the “Plasmodium translocon of exported proteins” (PTEX) apparatus.90,91 PfHsp70-x was identified as associating with the PTEX,92 and PfHsp70-x knock-out parasites showed reduced adhesion and slower export of PfEMP1 to the red blood cell surface.93 Proteomics and transcriptomics analysis of PfHsp70-x knock-out parasites demonstrated the upregulation of other chaperone and exported proteins, indicating a compensatory mechanism could be at play.93 Export processes may have in-built redundancy, as knock-down of the exported protein, PfSEMP1, leads to the upregulation of several exported proteins (including PfHsp70-x), as shown by transcriptomics analyses.94 Drug Resistance. In vitro evolution and whole-genome sequencing have illuminated genetic determinants of resistance for large cohorts of anti-malarial compounds.64,95 However, in some cases the native function of these genes remains unclear, even for well-known resistance determinants: chloroquine resistance transporter (PfCRT)96 and PfKelch13 (a vesicleassociated protein97 linked to artemisinin resistance98). “Omics” characterization of parasites with variant PfCRT alleles (that were either sensitive or resistant to chloroquine) were used to interrogate the native function of PfCRT.99 The chloroquine-resistant parasites exhibited increased levels of peptides from hemoglobin (shown by metabolomics and

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CONCLUDING REMARKS The age of “omics” technology has fundamentally transformed malaria research, and has vastly expanded and improved the means by which P. falciparum biology can be interrogated. In particular, these “omics” studies have contributed significantly to elucidating non-canonical process of P. falciparum biology. Genes that remain without functional annotation and that are unique to P. falciparum are a particular challenge for the field, E



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(3) RTS,S Clinical Trials Partnership (2015) Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet 386 (9988), 31−45. (4) Gardner, M. J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R. W., Carlton, J. M., Pain, A., Nelson, K. E., Bowman, S., Paulsen, I. T., James, K., Eisen, J. A., Rutherford, K., Salzberg, S. L., Craig, A., Kyes, S., Chan, M. S., Nene, V., Shallom, S. J., Suh, B., Peterson, J., Angiuoli, S., Pertea, M., Allen, J., Selengut, J., Haft, D., Mather, M. W., Vaidya, A. B., Martin, D. M., Fairlamb, A. H., Fraunholz, M. J., Roos, D. S., Ralph, S. A., McFadden, G. I., Cummings, L. M., Subramanian, G. M., Mungall, C., Venter, J. C., Carucci, D. J., Hoffman, S. L., Newbold, C., Davis, R. W., Fraser, C. M., and Barrell, B. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419 (6906), 498−511. (5) Amambua-Ngwa, A., Jeffries, D., Amato, R., Worwui, A., Karim, M., Ceesay, S., Nyang, H., Nwakanma, D., Okebe, J., Kwiatkowski, D., Conway, D. J., and D’Alessandro, U. (2018) Consistent signatures of selection from genomic analysis of pairs of temporal and spatial Plasmodium falciparum populations from The Gambia. Sci. Rep. 8 (1), 9687. (6) Duffy, C. W., Amambua-Ngwa, A., Ahouidi, A. D., Diakite, M., Awandare, G. A., Ba, H., Tarr, S. J., Murray, L., Stewart, L. B., D’Alessandro, U., Otto, T. D., Kwiatkowski, D. P., and Conway, D. J. (2018) Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development. Sci. Rep. 8 (1), 15763. (7) Demas, A. R., Sharma, A. I., Wong, W., Early, A. M., Redmond, S., Bopp, S., Neafsey, D. E., Volkman, S. K., Hartl, D. L., and Wirth, D. F. (2018) Mutations in Plasmodium falciparum actin-binding protein coronin confer reduced artemisinin susceptibility. Proc. Natl. Acad. Sci. U. S. A. 115 (50), 12799−12804. (8) Bankole, B. E., Kayode, A. T., Nosamiefan, I. O., Eromon, P., Baniecki, M. L., Daniels, R. F., Hamilton, E. J., Durfee, K., MacInnis, B., Okafor, H., Sowunmi, A., Volkman, S. K., Sabeti, P., Wirth, D., Happi, C. T., and Folarin, O. A. (2018) Characterization of Plasmodium falciparum structure in Nigeria with malaria SNPs barcode. Malar. J. 17 (1), 472. (9) Bei, A. K., Niang, M., Deme, A. B., Daniels, R. F., Sarr, F. D., Sokhna, C., Talla, C., Faye, J., Diagne, N., Doucoure, S., Mboup, S., Wirth, D. F., Tall, A., Ndiaye, D., Hartl, D. L., Volkman, S. K., and Toure-Balde, A. (2018) Dramatic Changes in Malaria Population Genetic Complexity in Dielmo and Ndiop, Senegal, Revealed Using Genomic Surveillance. J. Infect. Dis. 217 (4), 622−627. (10) Chang, H. H., Worby, C. J., Yeka, A., Nankabirwa, J., Kamya, M. R., Staedke, S. G., Dorsey, G., Murphy, M., Neafsey, D. E., Jeffreys, A. E., Hubbart, C., Rockett, K. A., Amato, R., Kwiatkowski, D. P., Buckee, C. O., and Greenhouse, B. (2017) THE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites. PLoS Comput. Biol. 13 (1), No. e1005348. (11) Trevino, S. G., Nkhoma, S. C., Nair, S., Daniel, B. J., Moncada, K., Khoswe, S., Banda, R. L., Nosten, F., and Cheeseman, I. H. (2017) High-Resolution Single-Cell Sequencing of Malaria Parasites. Genome Biol. Evol. 9 (12), 3373−3383. (12) Nair, S., Nkhoma, S. C., Serre, D., Zimmerman, P. A., Gorena, K., Daniel, B. J., Nosten, F., Anderson, T. J., and Cheeseman, I. H. (2014) Single-cell genomics for dissection of complex malaria infections. Genome Res. 24 (6), 1028−38. (13) Nkhoma, S. C., Trevino, S. G., Gorena, K. M., Nair, S., Khoswe, S., Jett, C., Garcia, R., Daniel, B., Dia, A., Terlouw, D. J., Ward, S. A., Anderson, T. J. C., and Cheeseman, I. H. (2018) Resolving withinhost malaria parasite diversity using single-cell sequencing. Bio Rxiv, 391268. (14) Bozdech, Z., Llinas, M., Pulliam, B. L., Wong, E. D., Zhu, J., and DeRisi, J. L. (2003) The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol. 1 (1), E5. (15) Le Roch, K. G., Zhou, Y., Blair, P. L., Grainger, M., Moch, J. K., Haynes, J. D., De La Vega, P., Holder, A. A., Batalov, S., Carucci, D. J.,

but are especially important to characterize as not only do they have the potential to reveal unique parasite biology, they also represent untapped potential in the search for novel vaccine candidates and selective drug targets. Undoubtedly, the ongoing collection of functional “omics” data over the entire P. falciparum life cycle will continue to update genome annotation. Future challenges also lie in the integration of multiple “omics” data sets, and advances in this area will provide a more sophisticated understanding of parasite physiology. Ultimately, understanding fundamental parasite biology provides an important basis for rational drug design and vaccine candidate development, and provides a foundation for elucidating drug action and parasite resistance mechanisms.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Darren J. Creek: 0000-0001-7497-7082 Author Contributions ∥

D.J.C. and T.G.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS D.J.C. is supported by a NHMRC Career Development Fellowship (II), APP1148700. GLOSSARY malaria, infectious disease caused by apicomplexan parasites of the genus Plasmodium; Plasmodium, a genus of parasitic organisms that are transmitted to humans and other organisms via the bite of an infected anopheline mosquito; systems biology, comprehensive study of a biological system on a large scale by examining all constituents within specific pools of biomolecules, includes mathematical modeling, bioinformatic analyses, and advanced analytical (“omics”) techniques; “omics”, colloquial term referring to a collection of analytical techniques used in systems biology that includes genomics, transcriptomics, proteomics, and metabolomics (and subsets of these, such as phosphoproteomics or lipidomics); transcriptomics, the characterization of all mRNA transcripts in a particular biological system; proteomics, the characterization of all proteins in a particular biological system; metabolomics, the characterization of all small molecules (“metabolites”) in a particular biological system; genetic manipulation, the modification of genomic sequences via the introduction of DNA sequences into cells, the results of which include permanent disruption of genes (“knock-out”) and conditional disruption of genes/proteins (“knock-down”); putative annotation, a gene that has a predicted function (often from homology-matching approaches) but which requires experimental validation; uncharacterized gene, a gene with an annotation of “unknown function”, sometimes referred to as a “hypothetical” gene

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