A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

Plasmodium GPI-anchored micronemal antigen is essential for parasite transmission through the mosquito host.

Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.

A Plasmodium falciparum genetic cross reveals the contributions of pfcrt and plasmepsin II/III to piperaquine drug resistance.

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.

Sporozoite immunization: innovative translational science to support the fight against malaria.

INTRODUCTION: Malaria, a devastating febrile illness caused by protozoan parasites, sickened 247,000,000 people in 2021 and killed 619,000, mostly children and pregnant women in sub-Saharan Africa. A highly effective vaccine is urgently needed, especially for Plasmodium falciparum (Pf), the deadliest human malaria parasite. AREAS COVERED: Sporozoites (SPZ), the parasite stage transmitted by Anopheles mosquitoes to humans, are the only vaccine immunogen achieving >90% efficacy against Pf infection. This review describes >30 clinical trials of PfSPZ vaccines in the U.S.A., Europe, Africa, and Asia, based on first-hand knowledge of the trials and PubMed searches of 'sporozoites,' 'malaria,' and 'vaccines.' EXPERT OPINION: First generation (radiation-attenuated) PfSPZ vaccines are safe, well tolerated, 80-100% efficacious against homologous controlled human malaria infection (CHMI) and provide 18-19 months protection without boosting in Africa. Second generation chemo-attenuated PfSPZ are more potent, 100% efficacious against stringent heterologous (variant strain) CHMI, but require a co-administered drug, raising safety concerns. Third generation, late liver stage-arresting, replication competent (LARC), genetically-attenuated PfSPZ are expected to be both safe and highly efficacious. Overall, PfSPZ vaccines meet safety, tolerability, and efficacy requirements for protecting pregnant women and travelers exposed to Pf in Africa, with licensure for these populations possible within 5 years. Protecting children and mass vaccination programs to block transmission and eliminate malaria are long-term objectives.

Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1.

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.

Global gene expression of human malaria parasite liver stages throughout intrahepatocytic development.

Plasmodium falciparum (Pf) is causing the greatest malaria burden, yet the liver stages (LS) of this most important parasite species have remained poorly studied. Here, we used a human liver-chimeric mouse model in combination with a novel fluorescent PfNF54 parasite line (PfNF54cspGFP) to isolate PfLS-infected hepatocytes and generate transcriptomes that cover the major LS developmental phases in human hepatocytes. RNA-seq analysis of early Pf LS trophozoites two days after infection, revealed a central role of translational regulation in the transformation of the extracellular invasive sporozoite into intracellular LS. The developmental time course gene expression analysis indicated that fatty acid biosynthesis, isoprenoid biosynthesis and iron metabolism are sustaining LS development along with amino acid metabolism and biosynthesis. Countering oxidative stress appears to play an important role during intrahepatic LS development. Furthermore, we observed expression of the variant PfEMP1 antigen-encoding var genes, and we confirmed expression of PfEMP1 protein during LS development. Transcriptome comparison of the late Pf liver stage schizonts with P. vivax (Pv) late liver stages revealed highly conserved gene expression profiles among orthologous genes. A notable difference however was the expression of genes regulating sexual stage commitment. While Pv schizonts expressed markers of sexual commitment, the Pf LS parasites were not sexually committed and showed expression of gametocytogenesis repression factors. Our results provide the first comprehensive gene expression profile of the human malaria parasite Pf LS isolated during in vivo intrahepatocytic development. This data will inform biological studies and the search for effective intervention strategies that can prevent infection.

Malaria parasites utilize two essential plasma membrane fusogens for gamete fertilization.

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.

PfSRPK1 Regulates Asexual Blood Stage Schizogony and Is Essential for Male Gamete Formation.

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.

A genetically engineered Plasmodium falciparum parasite vaccine provides protection from controlled human malaria infection.

Genetically engineered live Plasmodium falciparum sporozoites constitute a potential platform for creating consistently attenuated, genetically defined, whole-parasite vaccines against malaria through targeted gene deletions. Such genetically attenuated parasites (GAPs) do not require attenuation by irradiation or concomitant drug treatment. We previously developed a P. falciparum (Pf) GAP with deletions in P52, P36, and SAP1 genes (PfGAP3KO) and demonstrated its safety and immunogenicity in humans. Here, we further assessed safety, tolerability, and immunogenicity of the PfGAP3KO vaccine and tested its efficacy against controlled human malaria infection (CHMI) in malaria-naïve subjects. The vaccine was delivered by three (n = 6) or five (n = 8) immunizations with ~200 PfGAP3KO-infected mosquito bites per immunization. PfGAP3KO was safe and well tolerated with no breakthrough P. falciparum blood stage infections. Vaccine-related adverse events were predominately localized urticaria related to the numerous mosquito bites administered per vaccination. CHMI via bites with mosquitoes carrying fully infectious Pf NF54 parasites was carried out 1 month after the last immunization. Half of the study participants who received either three or five PfGAP3KO immunizations remained P. falciparum blood stage negative, as shown by a lack of detection of Plasmodium 18S rRNA in the blood for 28 days after CHMI. Six protected study participants received a second CHMI 6 months later, and one remained completely protected. Thus, the PfGAP3KO vaccine was safe and immunogenic and was capable of inducing protection against sporozoite infection. These results warrant further evaluation of PfGAP3KO vaccine efficacy in dose-range finding trials with an injectable formulation.

A Malaria Parasite Cross Reveals Genetic Determinants of Plasmodium falciparum Growth in Different Culture Media.

What genes determine in vitro growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between NF54, clone 3D7, a lab-adapted African parasite, and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: 3D7 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes 3D7 in media containing AlbuMAX, a commercial lipid-rich bovine serum formulation. To determine the basis for this polymorphism, we conducted parasite genetic crosses using humanized mice and compared genome-wide allele frequency changes in three independent progeny populations cultured in media containing human serum or AlbuMAX. This bulk segregant analysis detected three quantitative trait loci (QTL) regions [on chromosome (chr) 2 containing aspartate transaminase AST; chr 13 containing EBA-140; and chr 14 containing cysteine protease ATG4] linked with differential growth in serum or AlbuMAX in each of the three independent progeny pools. Selection driving differential growth was strong (s = 0.10 - 0.23 per 48-hour lifecycle). We conducted validation experiments for the strongest QTL on chr 13: competition experiments between ΔEBA-140 and 3D7 wildtype parasites showed fitness reversals in the two medium types as seen in the parental parasites, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of bulk segregant analysis for dissecting fitness traits in P. falciparum genetic crosses, and (ii) reveal intimate links between red blood cell invasion and nutrient composition of growth media. Use of parasite crosses combined with bulk segregant analysis will allow systematic dissection of key nutrient acquisition/metabolism and red blood cell invasion pathways in P. falciparum.

A Plasmodium falciparum ATP-binding cassette transporter is essential for liver stage entry into schizogony.

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.

PfARID Regulates P. falciparum Malaria Parasite Male Gametogenesis and Female Fertility and Is Critical for Parasite Transmission to the Mosquito Vector.

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.

A Putative Plasmodium RNA-Binding Protein Plays a Critical Role in Female Gamete Fertility and Parasite Transmission to the Mosquito Vector.

Plasmodium falciparum sexual stage gametocytes are critical for parasite transmission from the human host to the mosquito vector. Mature gametocytes generate fertile male (micro-) or female (macro-) gametes upon activation inside the mosquito midgut. While a number of parasite genes have been described that are critical for P. falciparum gametogenesis and fertility, no parasite gene has been shown to have a unique function in macrogametes. The genome of P. falciparum encodes numerous RNA-binding proteins. We identified a novel protein containing a putative RNA-binding domain, which we named Macrogamete-Contributed Factor Essential for Transmission (MaCFET). This protein is expressed in the asexual and sexual stages. Parasites that carry a deletion of MaCFET (Pfmacfet¯), developed normally as asexual stages, indicating that its function is not essential for the asexual proliferation of the parasite in vitro. Furthermore, Pfmacfet¯ male and female gametocytes developed normally and underwent activation to form microgametes and macrogametes. However, by utilizing genetic crosses, we demonstrate that Pfmacfet¯ parasites suffer a complete female-specific defect in successful fertilization. Therefore, PfMaCFET is a critical female-contributed factor for parasite transmission to the mosquito. Based on its putative RNA-binding properties, PfMaCFET might be in involved in the regulation of mRNAs that encode female-specific functions for fertilization or female-contributed factors needed post fertilization.

Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice.

Classical malaria parasite genetic crosses involve isolation, genotyping, and phenotyping of progeny parasites, which is time consuming and laborious. We tested a rapid alternative approach-bulk segregant analysis (BSA)-that utilizes sequencing of bulk progeny populations with and without drug selection for rapid identification of drug resistance loci. We used dihydroartemisinin (DHA) selection in two genetic crosses and investigated how synchronization, cryopreservation, and the drug selection regimen impacted BSA success. We detected a robust quantitative trait locus (QTL) at kelch13 in both crosses but did not detect QTLs at four other candidate loci. QTLs were detected using synchronized, but not unsynchronized progeny pools, consistent with the stage-specific action of DHA. We also successfully applied BSA to cryopreserved progeny pools, expanding the utility of this approach. We conclude that BSA provides a powerful approach for investigating the genetic architecture of drug resistance in Plasmodium falciparum.

Plasmodium falciparum Cysteine Rich Secretory Protein uniquely localizes to one end of male gametes.

Fertilization is a central event during the life cycle of most eukaryotic organisms and involves gamete recognition and fusion, ultimately resulting in zygote formation. Gamete fertilization in the malaria-causing Plasmodium parasites occurs inside the mosquito midgut and represents a major bottleneck in the life cycle. Cysteine Rich Secretory Proteins (CRISPs) are key molecules involved in fertilization in vertebrates and the presence of a CRISP ortholog in human malaria infective Plasmodium falciparum suggested a possible role in fertilization. Strikingly, P. falciparum CRISP exhibited a unique terminal localization in the male microgamete. Parasites with a CRISP gene deletion (P. falciparum crisp-) proliferated asexually similar to wildtype NF54 parasites and differentiated into gametocytes. Further analysis showed that Plasmodium falciparum crisp- gametocytes underwent exflagellation to form male gametes and no apparent defect in transmission to the mosquito vector was observed. These data show that P. falciparum CRISP is a marker for the apical end of the microgamete and that it might only have an ancillary or redundant function in the male sexual stages.

Plasmodium falciparum Calcium-Dependent Protein Kinase 4 is Critical for Male Gametogenesis and Transmission to the Mosquito Vector.

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.

Plasmodium vivax pre-erythrocytic stages and the latent hypnozoite.

Plasmodium vivax is the most geographically widespread malaria parasite on the planet. This is largely because after mosquito transmission, P. vivax sporozoites can invade hepatocytes and form latent liver stages known as hypnozoites. These persistent liver stages can activate weeks, months or even years after an infected individual suffers a primary clinical infection. Activation then leads to replication and liver stage schizont maturation that ultimately cause relapse of blood stage infection, disease, and onward transmission. Thus, the latent hypnozoite can lie in wait during times when onward transmission is unlikely due to conditions that do not favor the mosquito. For example, in temperate climates where mosquito prevalence is only seasonal. Furthermore, the elimination of hypnozoites is challenging since the hypnozoite reservoir is currently undetectable and not killed by most antimalarial drugs. Here, we review our current knowledge of the pre-erythrocytic stages of the malaria parasite - the sporozoite and liver stages, including the elusive and enigmatic hypnozoite. We focus on our understanding of sporozoite biology, the novel animal models that are available to study the hypnozoite and hypnozoite activation and the ongoing efforts to understand the biological makeup of the hypnozoite that allow for its persistence in the human host.

Plasmodium vivax Latent Liver Stage Infection and Relapse: Biological Insights and New Experimental Tools.

Plasmodium vivax is the most widespread human malaria parasite, in part because it can form latent liver stages known as hypnozoites after transmission by female anopheline mosquitoes to human hosts. These persistent stages can activate weeks, months, or even years after the primary clinical infection; replicate; and initiate relapses of blood stage infection, which causes disease and recurring transmission. Eliminating hypnozoites is a substantial obstacle for malaria treatment and eradication since the hypnozoite reservoir is undetectable and unaffected by most antimalarial drugs. Importantly, in some parts of the globe where P. vivax malaria is endemic, as many as 90% of P. vivax blood stage infections are thought to be relapses rather than primary infections, rendering the hypnozoite a major driver of P. vivax epidemiology. Here, we review the biology of the hypnozoite and recent discoveries concerning this enigmatic parasite stage. We discuss treatment and prevention challenges, novel animal models to study hypnozoites and relapse, and hypotheses related to hypnozoite formation and activation.

The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice.

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.

Humanized Mice and the Rebirth of Malaria Genetic Crosses.

The first experimental crosses carried out with the human malaria parasite Plasmodium falciparum played a key role in determining the genetic loci responsible for drug resistance, virulence, invasion, growth rate, and transmission. These crosses relied on splenectomized chimpanzees to complete the liver stage of the parasite's life cycle and the subsequent transition to asexual blood stage culture followed by cloning of recombinant progeny in vitro. Crosses can now be routinely carried out using human-liver-chimeric mice infused with human erythrocytes to generate hundreds of unique recombinant progeny for genetic linkage mapping, bulk segregant analysis, and high-throughput 'omics readouts. The high number of recombinant progeny should allow for unprecedented power and efficiency in the execution of a systems genetics approach to study P. falciparum biology.