Crosstalk between malaria and host proteome during the intraerythrocytic developmental cycle.

Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein relationships in P. falciparum that are important for the correct interpretation of transcriptional data and that may facilitate the improvement and development of malaria diagnostics and drug therapy.

New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.

BackgroundThe malarial parasite Plasmodium falciparum is an auxotroph for purines, which are required for nucleic acid synthesis during the intra-erythrocytic developmental cycle (IDC) of the parasite. The capabilities of the parasite and extent to which it can use compensatory mechanisms to adapt to purine deprivation were studied by examining changes in its metabolism under sub-optimal concentrations of hypoxanthine, the primary precursor utilized by the parasite for purine-based nucleic acid synthesis.MethodsThe concentration of hypoxanthine that caused a moderate growth defect over the course of one IDC was determined. At this concentration of hypoxanthine (0.5 μM), transcriptomic and metabolomic data were collected during one IDC at multiple time points. These data were integrated with a metabolic network model of the parasite embedded in a red blood cell (RBC) to interpret the metabolic adaptation of P. falciparum to hypoxanthine deprivation.ResultsAt a hypoxanthine concentration of 0.5 μM, vacuole-like structures in the cytosol of many P. falciparum parasites were observed after the 24-h midpoint of the IDC. Parasites grown under these conditions experienced a slowdown in the progression of the IDC. After 72 h of deprivation, the parasite growth could not be recovered despite supplementation with 90 µM hypoxanthine. Simulations of P. falciparum metabolism suggested that alterations in ubiquinone, isoprenoid, shikimate, and mitochondrial metabolism occurred before the appearance of these vacuole-like structures. Alterations were found in metabolic reactions associated with fatty acid synthesis, the pentose phosphate pathway, methionine metabolism, and coenzyme A synthesis in the latter half of the IDC. Furthermore, gene set enrichment analysis revealed that P. falciparum activated genes associated with rosette formation, Maurer’s cleft and protein export under two different nutrient-deprivation conditions (hypoxanthine and isoleucine).ConclusionsThe metabolic network analysis presented here suggests that P. falciparum invokes specific purine-recycling pathways to compensate for hypoxanthine deprivation and maintains a hypoxanthine pool for purine-based nucleic acid synthesis. However, this compensatory mechanism is not sufficient to maintain long-term viability of the parasite. Although P. falciparum can complete a full IDC in low hypoxanthine conditions, subsequent cycles are disrupted.

Malaria pathogenicity results from the parasite’s ability to invade, multiply within and then egress from the host red blood cell (RBC). Infected RBCs are remodeled, expressing antigenic variant proteins (such as PfEMP1, coded by the var gene family) for immune evasion and survival. These processes require the concerted actions of many proteins, but the molecular regulation is poorly understood. We have characterized an essential Plasmodium specific Apicomplexan AP2 (ApiAP2) transcription factor in Plasmodium falciparum (PfAP2-MRP; Master Regulator of Pathogenesis) during the intraerythrocytic developmental cycle (IDC). An inducible gene knockout approach showed that PfAP2-MRP is essential for development during the trophozoite stage, and critical for var gene regulation, merozoite development and parasite egress. ChIP-seq experiments performed at 16 hour post invasion (h.p.i.) and 40 h.p.i. matching the two peaks of PfAP2-MRP expression, demonstrate binding of PfAP2-MRP to the promoters of genes controlling trophozoite development and host cell remodeling at 16 h.p.i. and antigenic variation and pathogenicity at 40 h.p.i. Using single-cell RNA-seq and fluorescence-activated cell sorting, we show de-repression of most var genes in Δpfap2-mrp parasites that express multiple PfEMP1 proteins on the surface of infected RBCs. In addition, the Δpfap2-mrp parasites overexpress several early gametocyte marker genes at both 16 and 40 h.p.i., indicating a regulatory role in the sexual stage conversion. Using the Chromosomes Conformation Capture experiment (Hi-C), we demonstrate that deletion of PfAP2-MRP results in significant reduction of both intra-chromosomal and inter-chromosomal interactions in heterochromatin clusters. We conclude that PfAP2-MRP is a vital upstream transcriptional regulator controlling essential processes in two distinct developmental stages during the IDC that include parasite growth, chromatin structure and var gene expression.

Plasmodium falciparum is a deadly human pathogen responsible for the devastating disease called malaria. In this study, we measured the differential accumulation of RNA secondary structures in coding and non-coding transcripts from the asexual developmental cycle in P. falciparum in human red blood cells. Our comprehensive analysis that combined high-throughput nuclease mapping of RNA structures by duplex RNA-seq, SHAPE-directed RNA structure validation, immunoaffinity purification and characterization of antisense RNAs collectively measured differentially base-paired RNA regions throughout the parasite’s asexual RBC cycle. Our mapping data not only aligned to a diverse pool of RNAs with known structures but also enabled us to identify new structural RNA regions in the malaria genome. On average, approximately 71% of the genes with secondary structures are found to be protein coding mRNAs. The mapping pattern of these base-paired RNAs corresponded to all regions of mRNAs, including the 5ʹ UTR, CDS and 3ʹ UTR as well as the start and stop codons. Histone family genes which are known to form secondary structures in their mRNAs and transcripts from genes which are important for transcriptional and post-transcriptional control, such as the unique plant-like transcription factor family, ApiAP2, DNA-/RNA-binding protein, Alba3 and proteins important for RBC invasion and malaria cytoadherence also showed strong accumulation of duplex RNA reads in various asexual stages in P. falciparum. Intriguingly, our study determined stage-specific, dynamic relationships between mRNA structural contents and translation efficiency in P. falciparum asexual blood stages, suggesting an essential role of RNA structural changes in malaria gene expression programs.

The relationships among gene regulatory mechanisms in the malaria parasite Plasmodium falciparum throughout its asexual intraerythrocytic developmental cycle (IDC) remain poorly understood. To investigate the level and nature of transcriptional activity and its role in controlling gene expression during the IDC, we performed nuclear run-on on whole-transcriptome samples from time points throughout the IDC and found a peak in RNA polymerase II-dependent transcriptional activity related to both the number of nuclei per parasite and variable transcriptional activity per nucleus over time. These differential total transcriptional activity levels allowed the calculation of the absolute transcriptional activities of individual genes from gene-specific nuclear run-on hybridization data. For half of the genes analyzed, sense-strand transcriptional activity peaked at the same time point as total activity. The antisense strands of several genes were substantially transcribed. Comparison of the transcriptional activity of the sense strand of each gene to its steady-state RNA abundance across the time points assayed revealed both correlations and discrepancies, implying transcriptional and posttranscriptional regulation, respectively. Our results demonstrate that such comparisons can effectively indicate gene regulatory mechanisms in P. falciparum and suggest that genes with diverse transcriptional activity levels and patterns combine to produce total transcriptional activity levels tied to parasite development during the IDC.

&lt;p dir="ltr"&gt;Malaria, caused by the intracellular protozoan parasite &lt;i&gt;Plasmodium falciparum&lt;/i&gt;, continues to be a significant global health challenge. One of the major difficulties in combating the disease is the parasite's ability to constantly respond and adapt to changes in its human host environment. Alterations in host environment are tightly linked to disease progression in the intraerythrocytic development cycle (IDC), which is why the parasite has developed unique metabolic adaptation strategies. However, it remains unclear how the parasites sense such changes and regulate protein synthesis, particularly given its lack of the canonical TOR pathway. Thus, understanding how the parasite controls its translation under conditions of limited nutrient availability is crucial for gaining deeper insights into host-parasite interactions and adaptation, which may ultimately aid the development of more effective antimalarial therapies.&lt;/p&gt;&lt;p dir="ltr"&gt;Notably, &lt;i&gt;P. falciparum&lt;/i&gt; can serve as a unique model system for studying translational control as it presents the extremely AT-rich genome, skewed codon and amino acid (AA) usage, but a non-redundant set of tRNA genes. These features raise important questions regarding the ways in which the parasite offsets its codon usage bias to maintain efficient translation, and whether these evolutionary outcomes are neutral or represent adaptive strategies of the parasite to its host.&lt;/p&gt;&lt;p dir="ltr"&gt;Therefore, this thesis explores how &lt;i&gt;P. falciparum&lt;/i&gt; adapts to the human host environment through the lens of tRNA regulation, focusing on tRNA expression dynamics, aminoacylation and tRNA modifications. In Project I and II, by optimizing the tRNA sequencing protocol, we systematically explored the tRNA profiles in P. falciparum across various developmental stages and under different stress stimuli. In Project III, using multi-omics analysis, we elucidated a novel feature of host metabolic adaptation which underlies translational control.&lt;/p&gt;&lt;p dir="ltr"&gt;We provide evidence that the biased AA usage in the &lt;i&gt;P. falciparum&lt;/i&gt; genome is adaptive to host hemoglobin (HB), the primary internal source of AAs. Notably, we show that highly expressed transcripts have a lower requirement and hence dependency on AAs that are scarce or entirely absent, such as Isoleucine (Ile), which cannot be obtained through HB digestion and must be acquired from the external host environment.&lt;/p&gt;&lt;p dir="ltr"&gt;Through comprehensive tRNA profiling in &lt;i&gt;P. falciparum&lt;/i&gt;, we discovered a discordance between anticodon and codon pools. Specifically, tRNA responsible for decoding AAs that are scarce in HB exhibit lower expression levels, suggesting varied decoding efficiency for different AAs. Interestingly, genes associated with lipid synthesis and proliferation have adapted to incorporate a high level of HB- rare AAs such as Ile in their encoded proteins. This adaptation enables the parasite to regulate its proliferation by selectively repressing the protein synthesis of these genes during the periods of nutrient scarcity. Furthermore, by exposing parasites to AA deprivation, we revealed a non-canonical stress sensing mechanism facilitated by the regulation of Ile-tRNA aminoacylation . This, in turn, triggers selective ribosome stalling on Ile-rich transcripts, thereby selectively regulating the translation of these genes. Remarkably, this mechanism is unique as it is independent of kinase-mediated signaling cascades, enabling a decentralized resource allocation that is directly governed by the availability and need for nutrients.&lt;/p&gt;&lt;p dir="ltr"&gt;By analyzing other metabolically relevant pathways, we suggest that the adaptative strategies used by &lt;i&gt;P. falciparum&lt;/i&gt; may have evolved similarly in other intracellular parasites. Our study provides insights that metabolic constrains play an essential role in shaping the protein primary sequence and amino acid composition, challenging the prevailing view that functional constrains are the primary evolutionary drivers. This perspective offers new insights into protein and genome evolution, in particular, it may serve as an interesting model to explore the role of mutation bias in adaptive evolution.&lt;/p&gt;&lt;p dir="ltr"&gt;This thesis presents three related projects:&lt;/p&gt;&lt;p dir="ltr"&gt;Project I: We optimized tRNA sequencing techniques to provide a comprehensive view of the tRNA profiling in &lt;i&gt;P. falciparum&lt;/i&gt;, focusing on tRNA abundance, aminoacylation levels as well as a variety of nucleotide modifications.&lt;/p&gt;&lt;p dir="ltr"&gt;Project II: We explored tRNA modifications during stage transitions and under different stress conditions.&lt;/p&gt;&lt;p dir="ltr"&gt;Project III: By employing multi-omics analysis, we uncovered a novel layer of metabolic adaptation in &lt;i&gt;P. falciparum&lt;/i&gt; characterized by its highly biased AA usage in its proteome and is coupled to a regulated tRNA expression program.&lt;/p&gt;&lt;h3&gt;List of scientific papers&lt;/h3&gt;&lt;p dir="ltr"&gt;I. &lt;b&gt;Qian Li&lt;/b&gt;, Leonie Vetter, Ylva Veith, Elena Christ, Ákos Végvári, Cagla Sahin, Ulf Ribacke, Mats Wahlgren, Johan Ankarklev, Ola Larsson, Sherwin Chun-Leung Chan. tRNA regulation and amino acid usage bias reflect a coordinated metabolic adaptation in Plasmodium falciparum (iScience). &lt;a href="https://doi.org/10.1016/j.isci.2024.111167" rel="noreferrer" target="_blank"&gt;https://doi.org/10.1016/j.isci.2024.111167&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;II. &lt;b&gt;Qian Li&lt;/b&gt;, Ylva Veith, Elena Christ, Mats Wahlgren, Johan Ankarklev, Ola Larsson, Sherwin Chun-Leung Chan. tRNA base methylation identification and quantification in Plasmodium falciparum. [Manuscript]&lt;/p&gt;

&lt;p dir="ltr"&gt;Malaria, caused by the intracellular protozoan parasite &lt;i&gt;Plasmodium falciparum&lt;/i&gt;, continues to be a significant global health challenge. One of the major difficulties in combating the disease is the parasite's ability to constantly respond and adapt to changes in its human host environment. Alterations in host environment are tightly linked to disease progression in the intraerythrocytic development cycle (IDC), which is why the parasite has developed unique metabolic adaptation strategies. However, it remains unclear how the parasites sense such changes and regulate protein synthesis, particularly given its lack of the canonical TOR pathway. Thus, understanding how the parasite controls its translation under conditions of limited nutrient availability is crucial for gaining deeper insights into host-parasite interactions and adaptation, which may ultimately aid the development of more effective antimalarial therapies.&lt;/p&gt;&lt;p dir="ltr"&gt;Notably, &lt;i&gt;P. falciparum&lt;/i&gt; can serve as a unique model system for studying translational control as it presents the extremely AT-rich genome, skewed codon and amino acid (AA) usage, but a non-redundant set of tRNA genes. These features raise important questions regarding the ways in which the parasite offsets its codon usage bias to maintain efficient translation, and whether these evolutionary outcomes are neutral or represent adaptive strategies of the parasite to its host.&lt;/p&gt;&lt;p dir="ltr"&gt;Therefore, this thesis explores how &lt;i&gt;P. falciparum&lt;/i&gt; adapts to the human host environment through the lens of tRNA regulation, focusing on tRNA expression dynamics, aminoacylation and tRNA modifications. In Project I and II, by optimizing the tRNA sequencing protocol, we systematically explored the tRNA profiles in P. falciparum across various developmental stages and under different stress stimuli. In Project III, using multi-omics analysis, we elucidated a novel feature of host metabolic adaptation which underlies translational control.&lt;/p&gt;&lt;p dir="ltr"&gt;We provide evidence that the biased AA usage in the &lt;i&gt;P. falciparum&lt;/i&gt; genome is adaptive to host hemoglobin (HB), the primary internal source of AAs. Notably, we show that highly expressed transcripts have a lower requirement and hence dependency on AAs that are scarce or entirely absent, such as Isoleucine (Ile), which cannot be obtained through HB digestion and must be acquired from the external host environment.&lt;/p&gt;&lt;p dir="ltr"&gt;Through comprehensive tRNA profiling in &lt;i&gt;P. falciparum&lt;/i&gt;, we discovered a discordance between anticodon and codon pools. Specifically, tRNA responsible for decoding AAs that are scarce in HB exhibit lower expression levels, suggesting varied decoding efficiency for different AAs. Interestingly, genes associated with lipid synthesis and proliferation have adapted to incorporate a high level of HB- rare AAs such as Ile in their encoded proteins. This adaptation enables the parasite to regulate its proliferation by selectively repressing the protein synthesis of these genes during the periods of nutrient scarcity. Furthermore, by exposing parasites to AA deprivation, we revealed a non-canonical stress sensing mechanism facilitated by the regulation of Ile-tRNA aminoacylation . This, in turn, triggers selective ribosome stalling on Ile-rich transcripts, thereby selectively regulating the translation of these genes. Remarkably, this mechanism is unique as it is independent of kinase-mediated signaling cascades, enabling a decentralized resource allocation that is directly governed by the availability and need for nutrients.&lt;/p&gt;&lt;p dir="ltr"&gt;By analyzing other metabolically relevant pathways, we suggest that the adaptative strategies used by &lt;i&gt;P. falciparum&lt;/i&gt; may have evolved similarly in other intracellular parasites. Our study provides insights that metabolic constrains play an essential role in shaping the protein primary sequence and amino acid composition, challenging the prevailing view that functional constrains are the primary evolutionary drivers. This perspective offers new insights into protein and genome evolution, in particular, it may serve as an interesting model to explore the role of mutation bias in adaptive evolution.&lt;/p&gt;&lt;p dir="ltr"&gt;This thesis presents three related projects:&lt;/p&gt;&lt;p dir="ltr"&gt;Project I: We optimized tRNA sequencing techniques to provide a comprehensive view of the tRNA profiling in &lt;i&gt;P. falciparum&lt;/i&gt;, focusing on tRNA abundance, aminoacylation levels as well as a variety of nucleotide modifications.&lt;/p&gt;&lt;p dir="ltr"&gt;Project II: We explored tRNA modifications during stage transitions and under different stress conditions.&lt;/p&gt;&lt;p dir="ltr"&gt;Project III: By employing multi-omics analysis, we uncovered a novel layer of metabolic adaptation in &lt;i&gt;P. falciparum&lt;/i&gt; characterized by its highly biased AA usage in its proteome and is coupled to a regulated tRNA expression program.&lt;/p&gt;&lt;h3&gt;List of scientific papers&lt;/h3&gt;&lt;p dir="ltr"&gt;I. &lt;b&gt;Qian Li&lt;/b&gt;, Leonie Vetter, Ylva Veith, Elena Christ, Ákos Végvári, Cagla Sahin, Ulf Ribacke, Mats Wahlgren, Johan Ankarklev, Ola Larsson, Sherwin Chun-Leung Chan. tRNA regulation and amino acid usage bias reflect a coordinated metabolic adaptation in Plasmodium falciparum (iScience). &lt;a href="https://doi.org/10.1016/j.isci.2024.111167" rel="noreferrer" target="_blank"&gt;https://doi.org/10.1016/j.isci.2024.111167&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;II. &lt;b&gt;Qian Li&lt;/b&gt;, Ylva Veith, Elena Christ, Mats Wahlgren, Johan Ankarklev, Ola Larsson, Sherwin Chun-Leung Chan. tRNA base methylation identification and quantification in Plasmodium falciparum. [Manuscript]&lt;/p&gt;

The interdependence of chromatin states and transcription factor (TF) binding in eukaryotic genomes is critical for the proper regulation of gene expression. In this study, we explore the connection between TFs and chromatin states in the human malaria parasite, Plasmodium falciparum, throughout its 48-hour asexual intraerythrocytic developmental cycle (IDC). Most P. falciparum genes are expressed in a periodic manner during the IDC, accompanied by dynamic shifts in histone modifications and chromatin accessibility. Leveraging genome-wide profiles of chromatin accessibility, histone modifications, and Heterochromatin Protein 1 (HP1) occupancy, we characterize chromatin state dynamics during the IDC. Our results indicate that several chromatin states remain stable throughout the lifecycle, while others are dynamic and are linked to gene activation or repression. We further characterize chromatin state dynamics at the genome-wide DNA binding sites for a selection of Plasmodium TFs, allowing us to group TFs according to their chromatin preferences. By correlating changes in chromatin accessibility, histone modifications, and TF binding, we provide a global overview of the chromatin state dynamics that coordinate P. falciparum asexual blood stage development.

BackgroundThe malarial parasite Plasmodium falciparum undergoes a complex life cycle, including an intraerythrocytic developmental cycle, during which it is metabolically dependent on the infected human red blood cell (RBC). To describe whole cell metabolic activity within both P. falciparum and RBCs during the asexual reproduction phase of the intraerythrocytic developmental cycle, we developed an integrated host-parasite metabolic modeling framework driven by time-dependent gene expression data.ResultsWe validated the model by reproducing the experimentally determined 1) stage-specific production of biomass components and their precursors in the parasite and 2) metabolite concentration changes in the medium of P. falciparum-infected RBC cultures. The model allowed us to explore time- and strain-dependent P. falciparum metabolism and hypothesize how host cell metabolism alters in response to malarial infection. Specifically, the metabolic analysis showed that uninfected RBCs that coexist with infected cells in the same culture decrease their production of 2,3-bisphosphoglycerate, an oxygen-carrying regulator, reducing the ability of hemoglobin in these cells to release oxygen. Furthermore, in response to parasite-induced oxidative stress, infected RBCs downgraded their glycolytic flux by using the pentose phosphate pathway and secreting ribulose-5-phosphate. This mechanism links individually observed experimental phenomena, such as glycolytic inhibition and ribulose-5-phosphate secretion, to the oxidative stress response.ConclusionsAlthough the metabolic model does not incorporate regulatory mechanisms per se, alterations in gene expression levels caused by regulatory mechanisms are manifested in the model as altered metabolic states. This provides the model the capability to capture complex multicellular host-pathogen metabolic interactions of the infected RBC culture. The system-level analysis revealed complex relationships such as how the parasite can reduce oxygen release in uninfected cells in the presence of infected RBCs as well as the role of different metabolic pathways involved in the oxidative stress response of infected RBCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0291-2) contains supplementary material, which is available to authorized users.

A mathematical model of the intraerythrocytic developmental cycle identifies a delay between subtelomeric and central chromosomal gene activities in the malaria parasite, Plasmodium falciparum.

Editor's evaluation: A coordinated transcriptional switching network mediates antigenic variation of human malaria parasites

Malaria parasites follow a complex life cycle that consists of multiple stages that span from the human host to the mosquito vector. Among the species causing malaria, Plasmodium falciparum is the most lethal, with clinical symptoms manifesting during the intraerythrocytic developmental cycle (IDC). During the IDC, P. falciparum progresses through a synchronous and continuous cascade of transcriptional programming previously established using population analyses. While individual parasites are known to exhibit transcriptional variations to evade the host immune system or commit to a sexual fate, such rare expression heterogeneity is largely undetectable on a population level. Therefore, we combined single-cell RNA-sequencing (scRNA-seq) on a microfluidic platform and fluorescence imaging to delineate the transcriptional variations among individual parasites during late asexual and sexual stages. The comparison between asexual and sexual parasites uncovered a set of previously undefined sex-specific genes. Asexual parasites were segregated into three distinct clusters based on the differential expression of genes encoding SERAs, rhoptry proteins, and EXP2 plus transporters. Multiple pseudotime analyses revealed that these stage-specific transitions are distinct. RNA fluorescent in situ hybridization of cluster-specific genes validated distinct stage-specific expression and transitions during the IDC and defined the highly variable transcriptional pattern of EXP2. Additionally, these analyses indicated huge variations in the stage-specific transcript levels among parasites. Overall, scRNA-seq and RNA-FISH of P. falciparum revealed distinct stage transitions and unexpected degrees of heterogeneity with potential impact on transcriptional regulation during the IDC and adaptive responses to the host.

To date, malaria caused by Plasmodium falciparum is still a major health threat. It contributes to illness and severe disease and is responsible for up to one million deaths per year. The intra-erythrocytic asexual life cycle stage is responsible for the pathology associated with malaria. The major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1) is exposed at the surface of infected red blood cells (iRBC) and mediates binding to endothelial cells. This leads to sequestration of iRBC in the microvasculature and consequently to evasion of removal in the spleen. PfEMP1 is encoded by the 60-member var gene family, which undergoes antigenic variation by in-situ switching. Importantly, var genes are expressed in a mutually exclusive way, such that only one member is expressed whereas all other copies remain silenced. var genes as well as other gene families such as rif, stevor, phist and pfmc-2tm are located in subtelomeric heterochromatic regions. The function of these additional families is largely unknown, but they are thought to be implicated in host-parasite interactions and to contribute to antigenic variation. With this work, I provide deeper insights into the transcriptional regulation of virulence gene families in P. falciparum by using transfection-based approaches. We functionally identified autonomous cis-acting var promoter elements including an upstream activating sequence that is essential for promoter activation. Notably, an element downstream of the transcriptional start site determines mutually exclusive locus recognition. Further, I used comparative transcriptional profiling to show that mutually exclusive expression is restricted to the var gene family and is not used in the transcription of other subtelomeric gene families. I show for the first time that knock-down of endogenous var gene transcription is also conferred by promoters of a var gene subfamily that is implicated in severe malaria. Taken together, this work provides important insight into the mechanisms involved in the regulation of virulence gene families and antigenic variation in P. falciparum. Moreover, the findings presented here are consistent with a novel mechanism of mutually exclusive gene choice in eukaryotes.