Matthias Marti

Background Malaria remains a global health challenge, with most fatal cases caused by Plasmodium falciparum in sub-Saharan Africa. A critical bottleneck in the Plasmodium life cycle is the transmission from human to mosquito, which requires a subset of blood stage parasites to switch from asexual to sexual replication in a process known as stage conversion. In P. falciparum , stage conversion is known to be environmentally sensitive, notably to limiting levels of the immunomodulatory lipid lysophosphatidylcholine (LysoPC). Methods Here, we use single cell RNA sequencing (scRNAseq) to deconvolve the transcriptional signature of parasites upon limiting LysoPC levels under controlled in vitro conditions. Results Our data validate previous findings from bulk RNA sequencing, demonstrating a general compensatory metabolic response and reduced nutrient transport triggered by reduced LysoPC levels. Further, we define an initial transcriptional signature of sexual commitment, consisting of 22 genes conserved across two genetically diverse parasite strains. This signature includes an entire genetic locus encoding three proteins putatively involved in host-parasite interactions. Interestingly, the proportion of parasites expressing the transcriptional signature of sexual commitment was about twofold higher than the sexual conversion rate experimentally measured in the subsequent gametocyte cycle. Conclusions Together with recent data demonstrating that sexual commitment and conversion rates as measured by fluorescent reporter lines match closely, these data suggest that sexually committed schizonts may be in a transcriptionally bipotential state and sexual development becomes irreversible only at the post transcriptional level. Our study provides a rationale for systematic functional dissection of this elusive yet essential phase in the parasite cycle.

Sexual commitment and stage conversion are two crucial steps in the blood stage cycle of the human malaria parasite, as they mark its initial decision and subsequent differentiation in preparation for transmission to the mosquito vector. Here we present a Protocol Update that allows tracking and quantification of sexual commitment (in the asexual replicative parent, schizont stage) and conversion (in the nonreplicative sexual progeny, gametocyte stage) rates in the human malaria parasite Plasmodium falciparum. For this purpose, we combine three critical tools we recently established and validated. First, we use a standardized minimal medium to efficiently and reproducibly increase sexual commitment. Second, we use Tubulin Tracker Deep Red to label developing gametocytes and quantify the sexual conversion rate in reporter and reporter-free lines. Third, we use a fluorescent transgenic reporter line for consecutive quantification of sexual commitment and stage conversion in the same experimental procedure. These tools enable multiple experimental readouts including flow cytometry, fixed and live cell microscopy (including high content imaging) and other applications such as fluorescence-activated cell sorting. We anticipate that the presented sexual commitment-conversion assay will enable novel screening approaches for the identification of transmission blocking antimalarials and critical knowledge gaps in the Plasmodium transmission cycle to be closed. The Protocol can be completed in 7 d from start to finish.

Background One of the major challenges for malaria elimination is combating the highly efficient spread of the disease. Despite progress in understanding the development of malaria transmission stages, there remain many unanswered questions about how gametocytes transition from being immature to infectious. In Plasmodium falciparum, immature gametocytes are rigid and sequester outside of circulation in the extravascular space of the bone marrow, while deformable, mature stages are found in circulation and transmitted to mosquitoes. It is currently unclear whether deformable gametocytes are immediately infectious to mosquitoes, or whether they undergo activation upon release into circulation. Methods We used a combination of phenotypic assays and transcriptional analysis to define the transition from immature non-infectious to mature infectious gametocyte. Specifically, we associated gene expression with distinct phenotypic traits: gametocyte deformability assessed by microsphere filtration, and gametocyte infectivity assessed by exflagellation and mosquito feeding assays. Conclusions Our data revealed major transcriptional differences between input and deformable (i.e., filtered) gametocytes, but high similarity between deformable and infectious gametocytes. In combination with exflagellation and transmission results upon mosquito feeding assays, this suggests that deformable gametocytes are immediately infectious upon release from the bone marrow. The transcriptional analysis revealed a comprehensive set of infectivity markers that can be utilized to track gametocytes during their development and serve as diagnostic tools to map the human infectious reservoir.

Background: Malaria poses a significant global health challenge, with over 200 million infections and around 500,000 deaths annually, predominantly affecting children under 5 in sub-Saharan Africa. Severe malarial anaemia (SMA), a major contributor to morbidity and mortality in this demographic, results from various factors including red blood cell destruction and immune-mediated clearance. The role of these mechanisms in SMA differs based on age and infection history. Recent studies indicate increased accumulation of P. falciparum in the bone marrow and spleen, highlighting the need for understanding localized host-parasite interactions. Methods: This study examines the bone marrow response to malarial infection in young children with SMA or mild malarial malaria in Mozambique, contrasting it with responses observed in peripheral blood in the two cohorts. Results and conclusions: The study demonstrated that SMA is associated with increased red blood cell production, iron metabolism and tissue injury, as well as higher total parasite biomass including peripheral and bone marrow parasitaemia. We also demonstrate that direct analysis of bone marrow aspirates provides far more resolution in stratifying host signatures and drivers of malarial anaemia across a spectrum of severity than systemic measures from blood samples.

Background One of the major challenges for malaria elimination is combating the highly efficient spread of the disease. Despite progress in understanding the development of malaria transmission stages, there remain many unanswered questions about how gametocytes transition from being immature to infectious. In Plasmodium falciparum, immature gametocytes are rigid and sequester outside of circulation in the extravascular space of the bone marrow, while deformable, mature stages are found in circulation and transmitted to mosquitoes. It is currently unclear whether deformable gametocytes are immediately infectious to mosquitoes, or whether they undergo activation upon release into circulation. Methods We used a combination of phenotypic assays and transcriptional analysis to define the transition from immature non-infectious to mature infectious gametocyte. Specifically, we associated gene expression with distinct phenotypic traits: gametocyte deformability assessed by microsphere filtration, and gametocyte infectivity assessed by exflagellation and mosquito feeding assays. Conclusions Our data revealed major transcriptional differences between input and deformable (i.e., filtered) gametocytes, but high similarity between deformable and infectious gametocytes. In combination with exflagellation and transmission results upon mosquito feeding assays, this suggests that deformable gametocytes are immediately infectious upon release from the bone marrow. The transcriptional analysis revealed a comprehensive set of infectivity markers that can be utilized to track gametocytes during their development and serve as diagnostic tools to map the human infectious reservoir.

Background One of the major challenges for malaria elimination is combating the highly efficient spread of the disease. Despite progress in understanding the development of malaria transmission stages, there remain many unanswered questions about how gametocytes progress from being immature to infectious. In Plasmodium falciparum, immature gametocytes are rigid and sequester outside of circulation in the extravascular space of the bone marrow, while deformable, mature stages are found in circulation and transmitted to mosquitoes. It is currently unclear whether deformable gametocytes are immediately infectious to mosquitoes, or whether they undergo activation upon release into circulation. Methods We used a combination of phenotypic assays and transcriptional analysis to define the transition from immature non-infectious to mature infectious gametocyte. Specifically, we associated gene expression with distinct phenotypic traits: gametocyte deformability assessed by microsphere filtration, and gametocyte infectivity assessed by exflagellation and mosquito feeding assays. Conclusions Our data revealed major transcriptional differences between unfiltered and deformable gametocytes, but high similarity between deformable and infectious gametocytes. In combination with exflagellation and transmission results upon mosquito feeding assays, this suggests that deformable gametocytes are immediately infectious upon release from the bone marrow. The transcriptional analysis revealed a comprehensive set of infectivity markers that can be utilized to track gametocytes during their development and serve as diagnostic tools to map the human infectious reservoir.

Plasmodium parasites, the causative agents of malaria, undergo crucial developments within the mosquito vector, initiated by the formation of male and female gametes. Male gametogenesis involves three rapid rounds of mitosis without nuclear or cell division, followed by a single round of DNA segregation and nuclear division during gamete budding. How the cell organizes the segregation of eight genomes from a single octoploid nucleus into eight haploid gametes is currently unknown. Here we discovered an atypical Arp2/3 complex in Plasmodium important for DNA segregation during male gametogenesis. Unlike the canonical Arp2/3 complex found in other eukaryotes, Plasmodium Arp2/3 localizes to endomitotic spindles and interacts with a kinetochore-associated protein. Disruption of Arp2/3 subunits or actin polymerization interferes with kinetochore–spindle association, causes the formation of subhaploid gametes, and blocks transmission. Our work identified an evolutionary divergent Arp2/3 complex in malaria parasites, provides insights into gametogenesis, and reveals potential targets for transmission-blocking interventions.

Reducing malaria transmission has been a major pillar of control programmes and is considered crucial for achieving malaria elimination. Gametocytes, the transmissible forms of the P. falciparum parasite, arise during the blood stage of the parasite and develop through 5 morphologically distinct stages. Immature gametocytes (stage I-IV) sequester and develop in the extravascular niche of the bone marrow and possibly spleen. Only mature stage V gametocytes re-enter peripheral circulation to be taken up by mosquitoes for successful onward transmission. We have recently shown that immature, but not mature gametocytes are targets of host immune responses and identified putative target surface antigens. We hypothesize that these antigens play a role in gametocyte sequestration and contribute to acquired transmission-reducing immunity. Here we demonstrate that surface antigen expression, serum reactivity by human IgG, and opsonic phagocytosis by macrophages all show similar dynamics during gametocyte maturation, i.e., peaking in the immature stages and tapering off in mature gametocytes. Moreover, the switch in surface reactivity coincides with reversal in phosphatidylserine (PS) surface exposure, a marker for red blood cell age and clearance. PS is exposed on the surface of a proportion of immature gametocyte-infected RBCs (as well as in late asexual stages) but is removed from the surface in later gametocyte stages (IV-V). Using parasite reverse genetics and drug perturbations, we confirm that parasite protein export into the host cell and phospholipid scramblase activity are required for the observed surface modifications in asexual and sexual P. falciparum stages. Based on these findings we propose that the reversible surface remodelling allows (i) immature gametocyte sequestration in bone marrow followed by (ii) mature gametocyte release into peripheral circulation (and immune evasion due to loss of surface antigens), therefore contributing to mature gametocyte survival in vivo and onward transmission to mosquitoes. Importantly, blocking scramblase activity during gametocyte maturation results in efficient clearance of mature gametocytes, revealing a potential path for transmission blocking interventions. Our studies have important implications for our understanding of parasite biology and form a starting point for novel intervention strategies to simultaneously reduce parasite burden and transmission.

The complex life cycle of the malaria parasite Plasmodium falciparum involves several major differentiation stages, each requiring strict control of gene expression. Fundamental changes in chromatin structure and epigenetic modifications during life cycle progression suggest a central role for these mechanisms in regulating the transcriptional program of malaria parasite development^1–6. P. falciparum chromatin is distinct from other eukaryotes, with an extraordinarily high AT content (>80%)⁷ and highly divergent histones resulting in atypical DNA packaging properties⁸. Moreover, the chromatin remodellers that are critical for shaping chromatin structure are not conserved and are unexplored in P. falciparum. Here we identify P. falciparum Snf2L (PfSnf2L, encoded by PF3D7_1104200) as an ISWI-related ATPase that actively repositions P. falciparum nucleosomes in vitro. Our results demonstrate that PfSnf2L is essential, regulating both asexual development and sexual differentiation. PfSnf2L globally controls just-in-time transcription by spatiotemporally determining nucleosome positioning at the promoters of stage-specific genes. The unique sequence and functional properties of PfSnf2L led to the identification of an inhibitor that specifically kills P. falciparum and phenocopies the loss of correct gene expression timing. The inhibitor represents a new class of antimalarial transmission-blocking drugs, inhibiting gametocyte formation.