Regulation of sexual commitment in malaria parasites - a complex affair.

Malaria blood stage parasites commit to either one of two distinct cellular fates while developing within erythrocytes of their mammalian host: they either undergo another round of asexual replication or they differentiate into nonreplicative transmissible gametocytes. Depending on the state of infection, either path may support or impair the ultimate goal of human-to-human transmission via the mosquito vector. Malaria parasites therefore evolved strategies to control investments into asexual proliferation versus gametocyte formation. Recent work provided fascinating molecular insight into shared and unique mechanisms underlying the control and environmental modulation of sexual commitment in the two most widely studied malaria parasite species, Plasmodium falciparum and P. berghei. With this review, we aim at placing these findings into a comparative mechanistic context.

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Repurposing Know-how for Drug Development: Case Studies from the Swiss Tropical and Public Health Institute.

In pursuing novel therapeutic solutions, drug discovery and development rely on efficiently utilising existing knowledge and resources. Repurposing know-how, a strategy that capitalises on previously acquired information and expertise, has emerged as a powerful approach to accelerate drug discovery and development processes, often at a fraction of the costs of de novo developments. For 80 years, collaborating within a network of partnerships, the Swiss Tropical and Public Health Institute (Swiss TPH) has been working along a value chain from innovation to validation and application to combat poverty-related diseases. This article presents an overview of selected know-how repurposing initiatives conducted at Swiss TPH with a particular emphasis on the exploration of drug development pathways in the context of neglected tropical diseases and other infectious diseases of poverty, such as schistosomiasis, malaria and human African trypanosomiasis.

A Plasmodium membrane receptor platform integrates cues for egress and invasion in blood forms and activation of transmission stages.

Critical events in the life cycle of malaria-causing parasites depend on cyclic guanosine monophosphate homeostasis by guanylyl cyclases (GCs) and phosphodiesterases, including merozoite egress or invasion of erythrocytes and gametocyte activation. These processes rely on a single GCα, but in the absence of known signaling receptors, how this pathway integrates distinct triggers is unknown. We show that temperature-dependent epistatic interactions between phosphodiesterases counterbalance GCα basal activity preventing gametocyte activation before mosquito blood feed. GCα interacts with two multipass membrane cofactors in schizonts and gametocytes: UGO (unique GC organizer) and SLF (signaling linking factor). While SLF regulates GCα basal activity, UGO is essential for GCα up-regulation in response to natural signals inducing merozoite egress and gametocyte activation. This work identifies a GC membrane receptor platform that senses signals triggering processes specific to an intracellular parasitic lifestyle, including host cell egress and invasion to ensure intraerythrocytic amplification and transmission to mosquitoes.

The troubled puberty of malaria parasites.

Sexual differentiation of malaria parasites is essential for transmission, yet the underlying mechanisms are poorly understood. Russell et al. elegantly combined a loss-of-function screen with single-cell RNA-sequencing to identify key factors in this process. Gomes et al. further characterized one of them, MD1, as a regulator contributing to male fate determination.

Plasmodium falciparum Sexual Commitment Rate Variation among Clinical Isolates and Diverse Laboratory-Adapted Lines.

Asexual blood-stage malaria parasites must produce sexual progeny to infect mosquitoes. It is important to understand the scope and causes of intraspecific variation in sexual commitment rates, particularly for the major human parasite P. falciparum. First, two alternative assay methods of measuring sexual commitment were compared to test a genetically modified P. falciparum line with elevated commitment rates inducible by overexpression of GDV1. The methods yielded correlated measurements with higher sensitivity and precision being achieved by one employing detection of the early gametocyte differentiation marker Pfs16. Thus, this was used to survey a diverse range of parasite lines and test each in multiple biological replicate assays in a serum-free medium supplemented with Albumax. There were differences among six recent clinical isolates from Ghana in their mean rates of sexual commitment per cycle, ranging from 3.3% to 12.2%. Among 13 diverse long-term laboratory-adapted lines, mean sexual commitment rates for most ranged from 4.7% to 13.4%, a few had lower rates with means from 0.3 to 1.6%, and one with a nonfunctional ap2-g gene always showed zero commitment. Among a subset of lines tested for the effects of exogenous choline to suppress commitment, there were significant differences. As expected, there was no effect in a line that had lost the gdv1 gene and that had generally low commitment, whereas the others showed quantitatively variable but significant responses to choline, suggesting potential trait variation. The results indicated the value of performing multiple replicate assays for understanding the variation of this key reproductive trait that likely affects transmission. IMPORTANCE Only sexual-stage malaria parasites are transmitted from human blood to mosquitoes. Thus, it is vital to understand variations in sexual commitment rates because these may be modifiable or susceptible to blocking. Two different methods of commitment rate measurement were first compared, demonstrating higher sensitivity and precision by the detection of an early differentiation marker, which was subsequently used to survey diverse lines. Clinical isolates from Ghana showed significant variation in mean per-cycle commitment rates and variation among biological replicates. Laboratory-adapted lines of diverse origins had a wider range with most being within the range observed for the clinical isolates, while a minority consistently had lower or zero rates. There was quantitative variation in the effects when adding choline to suppress commitment, indicating differing responsiveness of parasites to this environmental modification. Performing multiple assay replicates and comparisons of diverse isolates was important to understand this trait and its potential effects on transmission.

Revisiting the Effect of Pharmaceuticals on Transmission Stage Formation in the Malaria Parasite Plasmodium falciparum.

Malaria parasites rely on specialized stages, called gametocytes, to ensure human-to-human transmission. The formation of these sexual precursor cells is initiated by commitment of blood stage parasites to the sexual differentiation pathway. Plasmodium falciparum, the most virulent of six parasite species infecting humans, employs nutrient sensing to control the rate at which sexual commitment is initiated, and the presence of stress-inducing factors, including antimalarial drugs, has been linked to increased gametocyte production in vitro and in vivo. These observations suggest that therapeutic interventions may promote gametocytogenesis and malaria transmission. Here, we engineered a P. falciparum reporter line to quantify sexual commitment rates after exposure to antimalarials and other pharmaceuticals commonly prescribed in malaria-endemic regions. Our data reveal that some of the tested drugs indeed have the capacity to elevate sexual commitment rates in vitro. Importantly, however, these effects are only observed at drug concentrations that inhibit parasite survival and only rarely result in a net increase of gametocyte production. Using a drug-resistant parasite reporter line, we further show that the gametocytogenesis-promoting effect of drugs is linked to general stress responses rather than to compound-specific activities. Altogether, we did not observe evidence for mechanistic links between the regulation of sexual commitment and the activity of commonly used pharmaceuticals in vitro. Our data hence does not support scenarios in which currently applied therapeutic interventions would promote the spread of drug-resistant parasites or malaria transmission in general.

The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.

Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum.

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

The ApiAP2 factor PfAP2-HC is an integral component of heterochromatin in the malaria parasite Plasmodium falciparum.

Malaria parasites undergo a complex life cycle in the human host and the mosquito vector. The ApiAP2 family of DNA-binding proteins plays a dominant role in parasite development and life cycle progression. Most ApiAP2 factors studied to date act as transcription factors regulating stage-specific gene expression. Here, we characterized an ApiAP2 factor in Plasmodium falciparum that we termed PfAP2-HC. We demonstrate that PfAP2-HC specifically binds to heterochromatin throughout the genome. Intriguingly, PfAP2-HC does not bind DNA in vivo and recruitment of PfAP2-HC to heterochromatin is independent of its DNA-binding domain but strictly dependent on heterochromatin protein 1. Furthermore, our results suggest that PfAP2-HC functions neither in the regulation of gene expression nor in heterochromatin formation or maintenance. In summary, our findings reveal PfAP2-HC as a core component of heterochromatin in malaria parasites and identify unexpected properties and substantial functional divergence among the members of the ApiAP2 family of regulatory proteins.

A 39-Amino-Acid C-Terminal Truncation of GDV1 Disrupts Sexual Commitment in Plasmodium falciparum.

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite's life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood-stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study, we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase knockout (KO) lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but that both lines harbor the same mutation that leads to a truncation in the extreme C terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild-type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line, we show that the truncation in the GDV1 C terminus does not interfere with the nuclear import of GDV1 or its interaction with HP1 in vitro but appears to be important to sustain GDV1 protein levels and thereby sexual commitment.IMPORTANCE Transmission of malaria-causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the life cycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here, we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C terminus of the gametocyte development 1 protein (GDV1), which abrogates sexual development. The mutation destabilizes the protein but not its interaction with its cognate binding partner HP1. This suggests an important role for the GDV1 C terminus beyond trafficking and protein stability.

The catalytic subunit of Plasmodium falciparum casein kinase 2 is essential for gametocytogenesis.

Casein kinase 2 (CK2) is a pleiotropic kinase phosphorylating substrates in different cellular compartments in eukaryotes. In the malaria parasite Plasmodium falciparum, PfCK2 is vital for asexual proliferation of blood-stage parasites. Here, we applied CRISPR/Cas9-based gene editing to investigate the function of the PfCK2α catalytic subunit in gametocytes, the sexual forms of the parasite that are essential for malaria transmission. We show that PfCK2α localizes to the nucleus and cytoplasm in asexual and sexual parasites alike. Conditional knockdown of PfCK2α expression prevented the transition of stage IV into transmission-competent stage V gametocytes, whereas the conditional knockout of pfck2a completely blocked gametocyte maturation already at an earlier stage of sexual differentiation. In summary, our results demonstrate that PfCK2α is not only essential for asexual but also sexual development of P. falciparum blood-stage parasites and encourage studies exploring PfCK2α as a potential target for dual-active antimalarial drugs.

Investigation of Heterochromatin Protein 1 Function in the Malaria Parasite Plasmodium falciparum Using a Conditional Domain Deletion and Swapping Approach.

The human malaria parasite Plasmodium falciparum encodes a single ortholog of heterochromatin protein 1 (PfHP1) that plays a crucial role in the epigenetic regulation of various survival-related processes. PfHP1 is essential for parasite proliferation and the heritable silencing of genes linked to antigenic variation, host cell invasion, and sexual conversion. Here, we employed CRISPR/Cas9-mediated genome editing combined with the DiCre/loxP system to investigate how the PfHP1 chromodomain (CD), hinge domain, and chromoshadow domain (CSD) contribute to overall PfHP1 function. We show that the 76 C-terminal residues are responsible for targeting PfHP1 to the nucleus. Furthermore, we reveal that each of the three functional domains of PfHP1 are required for heterochromatin formation, gene silencing, and mitotic parasite proliferation. Finally, we discovered that the hinge domain and CSD of HP1 are functionally conserved between P. falciparum and P. berghei, a related malaria parasite infecting rodents. In summary, our study provides new insights into PfHP1 function and offers a tool for further studies on epigenetic regulation and life cycle decision in malaria parasites.IMPORTANCE Malaria is caused by unicellular Plasmodium species parasites that repeatedly invade and replicate inside red blood cells. Some blood-stage parasites exit the cell cycle and differentiate into gametocytes that are essential for malaria transmission via the mosquito vector. Epigenetic control mechanisms allow the parasites to alter the expression of surface antigens and to balance the switch between parasite multiplication and gametocyte production. These processes are crucial to establish chronic infection and optimize parasite transmission. Here, we performed a mutational analysis of heterochromatin protein 1 (HP1) in P. falciparum We demonstrate that all three domains of this protein are indispensable for the proper function of HP1 in parasite multiplication, heterochromatin formation, and gene silencing. Moreover, expression of chimeric proteins revealed the functional conservation of HP1 proteins between different Plasmodium species. These results provide new insight into the function and evolution of HP1 as an essential epigenetic regulator of parasite survival.

PfMAP-2 is essential for male gametogenesis in the malaria parasite Plasmodium falciparum.

In malaria parasites, male gametogenesis is a proliferative stage essential for parasite transmission to the mosquito vector. It is a rapid process involving three rounds of genome replication alternating with closed endomitoses, and assembly of axonemes to produce eight flagellated motile microgametes. Studies in Plasmodium berghei have highlighted tight regulation of gametogenesis by a network of kinases. The P. berghei MAPK homologue PbMAP-2 is dispensable for asexual development but important at the induction of axoneme motility. However, in P. falciparum, causing the most severe form of human malaria, PfMAP-2 was suggested to be essential for asexual proliferation indicating distinct functions for MAP-2 in these two Plasmodium species. We here show that PfMAP-2 is dispensable for asexual growth but important for male gametogenesis in vitro. Similar to PbMAP-2, PfMAP-2 is required for initiating axonemal beating but not for prior DNA replication or axoneme formation. In addition, single and double null mutants of PfMAP-2 and the second P. falciparum MAPK homologue PfMAP-1 show no defect in asexual proliferation, sexual commitment or gametocytogenesis. Our results suggest that MAPK activity plays no major role in the biology of both asexual and sexual blood stage parasites up until the point of male gametogenesis.

Molecular Confirmation of a Fasciola gigantica × Fasciola hepatica Hybrid in a Chadian Bovine.

Fascioliasis is a zoonotic infection of humans and, more commonly, ruminants. It is caused by 2 liver fluke species, Fasciola hepatica and Fasciola gigantica, which differ in size. The traditional morphological methods used to distinguish the 2 species can be unreliable, particularly in the presence of hybrids between the 2 species. The development of advanced molecular methods has allowed for more definitive identification of Fasciola species, including their hybrids. Hybrids are of concern, as it is thought that they could acquire advantageous traits such as increased pathogenicity and host range. In 2013, we collected flukes from Fasciola-positive cattle, sheep, and goats slaughtered in 4 Chadian abattoirs. DNA from 27 flukes was extracted, amplified, and analyzed to identify species using the ITS1+2 locus. Twenty-six of the 27 flukes were identified as F. gigantica, while the remaining fluke showed heterozygosity at all variable sites that distinguish F. hepatica and F. gigantica. Cloning and sequencing of both alleles confirmed the presence of 1 F. hepatica and 1 F. gigantica allele. To our knowledge, this is the first unambiguous, molecular demonstration of the presence of such a hybrid in a bovine in sub-Saharan Africa.

The PHIST protein GEXP02 targets the host cytoskeleton during sexual development of Plasmodium falciparum.

A hallmark of the biology of Plasmodium falciparum blood stage parasites is their extensive host cell remodelling, facilitated by parasite proteins that are exported into the erythrocyte. Although this area has received extensive attention, only a few exported parasite proteins have been analysed in detail, and much of this remodelling process remains unknown, particularly for gametocyte development. Recent advances to induce high rates of sexual commitment enable the production of large numbers of gametocytes. We used this approach to study the Plasmodium helical interspersed subtelomeric (PHIST) protein GEXP02, which is expressed during sexual development. We show by immunofluorescence that GEXP02 is exported to the gametocyte-infected host cell periphery. Co-immunoprecipitation revealed potential interactions between GEXP02 and components of the erythrocyte cytoskeleton as well as other exported parasite proteins. This indicates that GEXP02 targets the erythrocyte cytoskeleton and is likely involved in its remodelling. GEXP02 knock-out parasites show no obvious phenotype during gametocyte maturation, transmission through mosquitoes, and hepatocyte infection, suggesting auxiliary or redundant functions for this protein. In summary, we performed a detailed cellular and biochemical analysis of a sexual stage-specific exported parasite protein using a novel experimental approach that is broadly applicable to study the biology of P. falciparum gametocytes.

Mapping and functional analysis of heterochromatin protein 1 phosphorylation in the malaria parasite Plasmodium falciparum.

Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted; in particular, several phosphorylated residues were embedded in motifs rich in acidic residues, reminiscent of targets for P. falciparum casein kinase 2 (PfCK2). Secondly, we tested recombinant PfCK2 and a number of additional protein kinases for their ability to phosphorylate PfHP1 in in vitro kinase assays. These experiments validated our prediction that PfHP1 acts as a substrate for PfCK2. Furthermore, LC-MS/MS analysis showed that PfCK2 phosphorylates three clustered serine residues in an acidic motif within the central hinge region of PfHP1. To study the role of PfHP1 phosphorylation in live parasites we used CRISPR/Cas9-mediated genome editing to generate a number of conditional PfHP1 phosphomutants based on the DiCre/LoxP system. Our studies revealed that neither PfCK2-dependent phosphorylation of PfHP1, nor phosphorylation of the hinge domain in general, affect PfHP1's ability to localize to heterochromatin, and that PfHP1 phosphorylation in this region is dispensable for the proliferation of P. falciparum blood stage parasites.

A multiplex assay for the sensitive detection and quantification of male and female Plasmodium falciparum gametocytes.

BACKGROUND: The transmission of malaria to mosquitoes depends on the presence of gametocytes that circulate in the peripheral blood of infected human hosts. Sensitive estimates of the densities of female gametocytes (FG) and male gametocytes (MG) may allow the prediction of infectivity to mosquitoes and thus a molecular estimate of the human infectious reservoir for transmission. METHODS: A novel multiplex qRT-PCR assay with intron-spanning primers was developed for the parallel quantification of FG and MG. CCp4 (PF3D7_0903800) transcripts specific for FG and PfMGET (PF3D7_1469900) transcripts specific for MG were quantified in total nucleic acids. The assay was validated on sex-sorted gametocytes from culture material and on samples from clinical trials with gametocytocidal drugs. Synthetic RNA standards were generated for the two targets genes and calibrated against known gametocyte quantities. RESULTS: The limit of detection was determined at 0.1 male and 0.1 female gametocyte/µL, which was equal to the limit of quantification (LOQ) for MG, while the LOQ for FG was 1 FG/µL. Results from previously reported clinical trials that used separate gametocyte qRT-PCR assays for FG (targeting Pfs25) and MG (targeting PfMGET) were reproduced with the multiplex assay. High levels of agreement between separate assays and the multiplex approach were observed (R2 = 0.9473, 95% CI 0.9314-0.9632, for FG measured by transcript levels of Pfs25 in qRT-PCR or CCp4 in multiplex; R2 = 0.8869, 95% CI 0.8541-0.9197, for MG measured by PfMGET in either single or multiplex qRT-PCR). FG and MG transcripts were detected in pure ring stage parasites at 10,000- and 100,000-fold reduced frequency for CCp4 and PfMGET, respectively. The CCp4 and PfMGET transcripts were equally stable under suboptimal storage conditions. CONCLUSIONS: Gametocyte densities and their sex ratios can be determined in the presented one-step multiplex assay with higher throughput than single assays. The interpretation of low gametocyte densities at asexual parasite densities above 1000 parasites/µL requires caution to avoid false positive gametocyte signals from spurious transcript levels in ring stage parasites.

The origins of malaria artemisinin resistance defined by a genetic and transcriptomic background.

The predisposition of parasites acquiring artemisinin resistance still remains unclear beyond the mutations in Pfk13 gene and modulation of the unfolded protein response pathway. To explore the chain of casualty underlying artemisinin resistance, we reanalyze 773 P. falciparum isolates from TRACI-study integrating TWAS, GWAS, and eQTL analyses. We find the majority of P. falciparum parasites are transcriptomically converged within each geographic site with two broader physiological profiles across the Greater Mekong Subregion (GMS). We report 8720 SNP-expression linkages in the eastern GMS parasites and 4537 in the western. The minimal overlap between them suggests differential gene regulatory networks facilitating parasite adaptations to their unique host environments. Finally, we identify two genetic and physiological backgrounds associating with artemisinin resistance in the GMS, together with a farnesyltransferase protein and a thioredoxin-like protein which may act as vital intermediators linking the Pfk13 C580Y mutation to the prolonged parasite clearance time.