Competitive outcomes between co-infecting malaria parasite lines can reveal fitness disparities in blood stage growth. Blood stage fitness costs often accompany the evolution of drug resistance, with the expectation that relatively fitter parasites will be more likely to spread in populations. With the recent emergence of artemisinin resistance, it is important to understand the relative competitive fitness of the metabolically active asexual blood stage parasites. Genetically distinct drug resistant parasite clones with independently evolved sets of mutations are likely to vary in asexual proliferation rate, contributing to their chance of transmission to the mosquito vector.

Determining the genetic basis of fitness is central to understanding evolution and transmission of microbial pathogens. In human malaria parasites (Plasmodium falciparum), most experimental work on fitness has focused on asexual blood stage parasites, because this stage can be easily cultured, although the transmission of malaria requires both female Anopheles mosquitoes and vertebrate hosts. We explore a powerful approach to identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-cycle stages of P. falciparum. This combines experimental genetic crosses using humanized mice, with selective whole genome amplification and pooled sequencing to determine genome-wide allele frequencies and identify genomic regions under selection across multiple lifecycle stages. We applied this approach to genetic crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, kelch13-WT) parasites, recently isolated from Southeast Asian patients. Two striking results emerge: we observed (i) a strong genome-wide skew (>80%) towards alleles from the ART-R parent in the mosquito stage, that dropped to ~50% in the blood stage as selfed ART-R parasites were selected against; and (ii) repeatable allele specific skews in blood stage parasites with particularly strong selection (selection coefficient (s) ≤ 0.18/asexual cycle) against alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 containing ARPS10. This approach robustly identifies selected loci and has strong potential for identifying parasite genes that interact with the mosquito vector or compensatory loci involved in drug resistance.

Genetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses-(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels.

Plasmodium sporozoites invade hepatocytes and transform into liver stages within a parasitophorous vacuole (PV). The parasites then grow and replicate their genome to form exoerythrocytic merozoites that infect red blood cells. We report that the human malaria parasite Plasmodium falciparum (Pf) expresses a C-type ATP-binding cassette transporter, Pf ABCC2, which marks the transition from invasive sporozoite to intrahepatocytic early liver stage. Using a humanized mouse infection model, we show that Pf ABCC2 localizes to the parasite plasma membrane in early and mid-liver stage parasites but is not detectable in late liver stages. Pf abcc2 - sporozoites invade hepatocytes, form a PV, and transform into liver stage trophozoites but cannot transition to exoerythrocytic schizogony and fail to transition to blood stage infection. Thus, Pf ABCC2 is an expression marker for early phases of parasite liver infection and plays an essential role in the successful initiation of liver stage replication.

We present an optimized probe design for copy number variation (CNV) and SNP genotyping in the Plasmodium falciparum genome. We demonstrate that variable length and isothermal probes are superior to static length probes. We show that sample preparation and hybridization conditions mitigate the effects of host DNA contamination in field samples. The microarray and workflow presented can be used to identify CNVs and SNPs with 95% accuracy in a single hybridization, in field samples containing up to 92% human DNA contamination.

Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to pathogenesis of severe malaria. To determine whether this balance is maintained by classical regulatory T cells (CD4(+) FOXP3(+) CD127(-/low); Tregs) we compared cellular responses between Gambian children (n = 124) with severe Plasmodium falciparum malaria or uncomplicated malaria infections. Although no significant differences in Treg numbers or function were observed between the groups, Treg activity during acute disease was inversely correlated with malaria-specific memory responses detectable 28 days later. Thus, while Tregs may not regulate acute malarial inflammation, they may limit memory responses to levels that subsequently facilitate parasite clearance without causing immunopathology. Importantly, we identified a population of FOXP3(-), CD45RO(+) CD4(+) T cells which coproduce IL-10 and IFN-gamma. These cells are more prevalent in children with uncomplicated malaria than in those with severe disease, suggesting that they may be the regulators of acute malarial inflammation.

Multiple kelch13 alleles conferring artemisinin resistance (ART-R) are currently spreading through Southeast Asian malaria parasite populations, providing a unique opportunity to observe an ongoing soft selective sweep, investigate why resistance alleles have evolved multiple times and determine fundamental population genetic parameters for Plasmodium We sequenced kelch13 (n = 1,876), genotyped 75 flanking SNPs, and measured clearance rate (n = 3,552) in parasite infections from Western Thailand (2001-2014). We describe 32 independent coding mutations including common mutations outside the kelch13 propeller associated with significant reductions in clearance rate. Mutations were first observed in 2003 and rose to 90% by 2014, consistent with a selection coefficient of ∼0.079. ART-R allele diversity rose until 2012 and then dropped as one allele (C580Y) spread to high frequency. The frequency with which adaptive alleles arise is determined by the rate of mutation and the population size. Two factors drive this soft sweep: (1) multiple kelch13 amino-acid mutations confer resistance providing a large mutational target-we estimate the target is 87-163 bp. (2) The population mutation parameter (Θ = 2Neμ) can be estimated from the frequency distribution of ART-R alleles and is ∼5.69, suggesting that short term effective population size is 88 thousand to 1.2 million. This is 52-705 times greater than Ne estimated from fluctuation in allele frequencies, suggesting that we have previously underestimated the capacity for adaptive evolution in Plasmodium Our central conclusions are that retrospective studies may underestimate the complexity of selective events and the Ne relevant for adaptation for malaria is considerably higher than previously estimated.

Cell fusion of female and male gametes is the climax of sexual reproduction. In many organisms, the Hapless 2 (HAP2) family of proteins play a critical role in gamete fusion. We find that Plasmodium falciparum, the causative agent of human malaria, expresses two HAP2 proteins: PfHAP2 and PfHAP2p. These proteins are present in stage V gametocytes and localize throughout the flagellum of male gametes. Gene deletion analysis and genetic crosses show that PfHAP2 and PfHAP2p individually are essential for male fertility and thereby, parasite transmission to the mosquito. Using a cell fusion assay, we demonstrate that PfHAP2 and PfHAP2p are both authentic plasma membrane fusogens. Our results establish nonredundant essential roles for PfHAP2 and PfHAP2p in mediating gamete fusion in Plasmodium and suggest avenues in the design of novel strategies to prevent malaria parasite transmission from humans to mosquitoes.

Serine/arginine-rich protein kinases (SRPKs) are cell cycle-regulated serine/threonine protein kinases and are important regulators of splicing factors. In this study, we functionally characterize SRPK1 of the human malaria parasite Plasmodium falciparum. P. falciparum SRPK1 (PfSRPK1) was expressed in asexual blood-stage and sexual-stage gametocytes. Pfsrpk1- parasites formed asexual schizonts that generated far fewer merozoites than wild-type parasites, causing reduced replication rates. Pfsrpk1- parasites also showed a severe defect in the differentiation of male gametes, causing a complete block in parasite transmission to mosquitoes. RNA sequencing (RNA-seq) analysis of wild-type PfNF54 and Pfsrpk1- stage V gametocytes suggested a role for PfSRPK1 in regulating transcript splicing and transcript abundance of genes coding for (i) microtubule/cilium morphogenesis-related proteins, (ii) proteins involved in cyclic nucleotide metabolic processes, (iii) proteins involved in signaling such as PfMAP2, (iv) lipid metabolism enzymes, (v) proteins of osmophilic bodies, and (vi) crystalloid components. Our study reveals an essential role for PfSRPK1 in parasite cell morphogenesis and suggests this kinase as a target to prevent malaria transmission from humans to mosquitoes. IMPORTANCE Plasmodium sexual stages represent a critical bottleneck in the parasite life cycle. Gametocytes taken up in an infectious blood meal by female anopheline mosquito get activated to form gametes and fuse to form short-lived zygotes, which transform into ookinetes to infect mosquitoes. In the present study, we demonstrate that PfSRPK1 is important for merozoite formation and critical for male gametogenesis and is involved in transcript homeostasis for numerous parasite genes. Targeting PfSRPK1 and its downstream pathways may reduce parasite replication and help achieve effective malaria transmission-blocking strategies.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and four copies of pm2/3. We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC50 (LP-IC50). We find that inheritance of the KH004 pfcrt allele is required for PPQ resistance, whereas copy number variation in pm2/3 further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations in pfcrt. Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance.

We explored the potential of pooled sequencing to swiftly and economically identify selective sweeps due to emerging artemisinin (ART) resistance in a South-East Asian malaria parasite population. ART resistance is defined by slow parasite clearance from the blood of ART-treated patients and mutations in the kelch gene (chr. 13) have been strongly implicated to play a role. We constructed triplicate pools of 70 slow-clearing (resistant) and 70 fast-clearing (sensitive) infections collected from the Thai-Myanmar border and sequenced these to high (∼ 150-fold) read depth. Allele frequency estimates from pools showed almost perfect correlation (Lin's concordance = 0.98) with allele frequencies at 93 single nucleotide polymorphisms measured directly from individual infections, giving us confidence in the accuracy of this approach. By mapping genome-wide divergence (FST) between pools of drug-resistant and drug-sensitive parasites, we identified two large (>150 kb) regions (on chrs. 13 and 14) and 17 smaller candidate genome regions. To identify individual genes within these genome regions, we resequenced an additional 38 parasite genomes (16 slow and 22 fast-clearing) and performed rare variant association tests. These confirmed kelch as a major molecular marker for ART resistance (P = 6.03 × 10(-6)). This two-tier approach is powerful because pooled sequencing rapidly narrows down genome regions of interest, while targeted rare variant association testing within these regions can pinpoint the genetic basis of resistance. We show that our approach is robust to recurrent mutation and the generation of soft selective sweeps, which are predicted to be common in pathogen populations with large effective population sizes, and may confound more traditional gene mapping approaches.

Most malaria infections contain complex mixtures of distinct parasite lineages. These multiple-genotype infections (MGIs) impact virulence evolution, drug resistance, intra-host dynamics, and recombination, but are poorly understood. To address this we have developed a single-cell genomics approach to dissect MGIs. By combining cell sorting and whole-genome amplification (WGA), we are able to generate high-quality material from parasite-infected red blood cells (RBCs) for genotyping and next-generation sequencing. We optimized our approach through analysis of >260 single-cell assays. To quantify accuracy, we decomposed mixtures of known parasite genotypes and obtained highly accurate (>99%) single-cell genotypes. We applied this validated approach directly to infections of two major malaria species, Plasmodium falciparum, for which long term culture is possible, and Plasmodium vivax, for which no long-term culture is feasible. We demonstrate that our single-cell genomics approach can be used to generate parasite genome sequences directly from patient blood in order to unravel the complexity of P. vivax and P. falciparum infections. These methods open the door for large-scale analysis of within-host variation of malaria infections, and reveal information on relatedness and drug resistance haplotypes that is inaccessible through conventional sequencing of infections.

Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4- parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4- parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4- late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.

Sexual reproduction of Plasmodium falciparum parasites is critical to the spread of malaria in the human population. The factors that regulate gene expression underlying formation of fertilization-competent gametes, however, remain unknown. Here, we report that P. falciparum expresses a protein with an AT-rich interaction domain (ARID) which, in other organisms, is part of chromatin remodeling complexes. P. falciparum ARID (PfARID) localized to the parasite nucleus and is critical for the formation of male gametes and fertility of female gametes. PfARID gene deletion (Pfarid-) gametocytes showed downregulation of gene expression important for gametogenesis, antigenic variation, and cell signaling and for parasite development in the mosquito. Our study identifies PfARID as a critical nuclear protein involved in regulating the gene expression landscape of mature gametocytes. This establishes fertility and also prepares the parasite for postfertilization events that are essential for infection of the mosquito vector. IMPORTANCE Successful completion of the Plasmodium life cycle requires formation of mature gametocytes and their uptake by the female Anopheles mosquito vector in an infected blood meal. Inside the mosquito midgut the parasite undergoes gametogenesis and sexual reproduction. In the present study, we demonstrate that PfARID is essential for male gametogenesis and female fertility and, thereby, transmission to the mosquito vector. PfARID possibly regulates the chromatin landscape of stage V gametocytes and targeting PfARID function may provide new avenues into designing interventions to prevent malaria transmission.

In high-transmission regions, we expect parasite lineages within complex malaria infections to be unrelated due to parasite inoculations from different mosquitoes. This project was designed to test this prediction. We generated 485 single-cell genome sequences from fifteen P. falciparum malaria patients from Chikhwawa, Malawi-an area of intense transmission. Patients harbored up to seventeen unique parasite lineages. Surprisingly, parasite lineages within infections tend to be closely related, suggesting that superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Both closely and distantly related parasites comprise an infection, suggesting sequential transmission of complex infections between multiple hosts. We identified tetrads and reconstructed parental haplotypes, which revealed the inbred ancestry of infections and non-Mendelian inheritance. Our analysis suggests strong barriers to secondary infection and outbreeding amongst malaria parasites from a high transmission setting, providing unexpected insights into the biology and transmission of malaria.

Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.

Malaria infections containing multiple parasite genotypes are ubiquitous in nature, and play a central role in models of recombination, intra-host dynamics, virulence, sex ratio, immunity and drug resistance evolution in Plasmodium. While these multiple infections (MIs) are often assumed to result from superinfection (bites from multiple infected mosquitoes), we know remarkably little about their composition or generation. We isolated 336 parasite clones from eight patients from Malawi (high transmission) and six from Thailand (low transmission) by dilution cloning. These were genotyped using 384 single-nucleotide polymorphisms, revealing 22 independent haplotypes in Malawi (2-6 per MI) and 15 in Thailand (2-5 per MI). Surprisingly, all six patients from Thailand and six of eight from Malawi contained related haplotypes, and haplotypes were more similar within- than between-infections. These results argue against a simple superinfection model. Instead, the observed kinship patterns may be explained by inoculation of multiple related haploid sporozoites from single mosquito bites, by immune suppression of parasite subpopulations within infections, and serial transmission of related parasites between people. That relatedness is maintained in endemic areas in the face of repeated bites from infected mosquitoes has profound implications for understanding malaria transmission, immunity and intra-host dynamics of co-infecting parasite genotypes.

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.

Artemisinin-based combination therapies are the first line of treatment for Plasmodium falciparum infections worldwide, but artemisinin resistance has risen rapidly in Southeast Asia over the past decade. Mutations in the kelch13 gene have been implicated in this resistance. We used longitudinal genomic surveillance to detect signals in kelch13 and other loci that contribute to artemisinin or partner drug resistance. We retrospectively sequenced the genomes of 194 P. falciparum isolates from five sites in Northwest Thailand, over the period of a rapid increase in the emergence of artemisinin resistance (2001-2014).

Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.