Abstract The unicellular malaria parasite Plasmodium falciparum proliferates within red blood cells of its human host, where it generates approximately 20 new parasites within a two-day developmental cycle. Before cellularization and release of the daughter cells, the nuclei multiply in a shared cytoplasm. In stark contrast to highly synchronized nuclear division cycles seen in other developing eukaryotes, Plasmodium nuclear cycles desynchronize rapidly. Combining live-cell imaging with biophysical modeling, we elucidate the mechanism of desynchronization and study its impact on parasite proliferation. We find that standard models of autonomous nuclear cycles cannot account for the experimental data, and therefore desynchronization requires nuclear coupling. Competition for a limiting pool of proteins needed for DNA replication explains the data, provided that they are allocated sequentially to individual nuclei. Sequential allocation can be achieved by reversible but stable association of the resources with DNA. Remarkably, the resultant asynchronous nuclear cycles accelerate parasite proliferation by minimizing idling times of the resource. This mechanism may be a general strategy to maximize proliferation in suboptimal growth conditions. Together, our findings identify nuclear cycle asynchrony as a resource-efficient means to achieve rapid proliferation.

Parasites display many three-dimensional (3D) features that are related to their parasitic lifestyle. Using two-dimensional images alone, such 3D features are difficult to convey during teaching or public outreach. Thus, we provide parasite models for 3D printing to support education and to encourage adoption and adaptation by the parasitology community.

Parasitic protozoa exhibit a high demand for iron, with mitochondrial iron metabolism representing a vulnerable target for chemotherapeutic intervention. We recently demonstrated that mitochondrial targeting of the iron chelator deferoxamine (DFO) via triphenylphosphonium (TPP) conjugation enhances its antiparasitic efficacy. To expand upon this strategy, mitochondrially targeted derivatives of DFO and deferasirox (DFX) were synthesized and evaluated for their activity against important human parasites. The DFX derivative mitoDFX was effective against Trypanosoma spp. and Toxoplasma gondii with remarkable selectivity. The fact that mitoDFX is a promising anticancer agent, which is likely safe to use in the context of human health, highlights the potential for drug repurposing in parasitology. Structure-activity relationship (SAR) studies and iron distribution analyses in trypanosomes revealed that mitochondrial targeting of the compounds, rather than iron chelation per se, is the main driver of the antiparasitic effects, underscoring the critical role of phosphonium salts in bioactivity.

Malaria tropica, caused by Plasmodium falciparum (P. falciparum), remains a global health challenge with limited therapeutic options. In mammalian cells, the small-molecule compound RAS-selective lethal 3 (RSL3) induces ferroptosis via lipid peroxidation. In this study, we demonstrate that RSL3 synergizes with Pyrimethamine, an inhibitor of P. falciparum dihydrofolate reductase (DHFR), to suppress parasite proliferation in red blood cells (RBCs). A similar synergistic effect was observed with Cycloguanil, a structural analog of Pyrimethamine, but not with other DHFR inhibitors or alternative agents that induce ferroptosis in nucleated mammalian cells. Notably, Ferrostatin-1, an antagonist of lipid peroxidation, largely failed to rescue parasite growth in the presence of RSL3, possibly suggesting a mechanism distinct from canonical ferroptosis. These findings suggest that the synergy may involve unidentified targets of RSL3 and Pyrimethamine in P. falciparum, divergent from those described in mammalian systems. Moreover, RSL3 and related compounds could serve as promising adjuvants to enhance the antimalarial efficacy of Pyrimethamine and potentially overcome drug resistance.

Fluorescence microscopy is a powerful tool to analyze subcellular architecture, and long-term live-cell imaging permits the analysis of the dynamics of intracellular structures. Applying this approach to the membranes of Plasmodium spp., the causative agent of malaria, yielded important insight. Fluorescent labelling of Plasmodium ’s plasma membrane via membrane-resident proteins has been described, but proteins, which broadly mark internal membranes are currently elusive. Alternatively, general membrane dyes can be employed to study membrane dynamics, however, we find that the membrane dye BODIPY TR Ceramide has adverse effects on cell viability during live-cell imaging. To overcome this limitation, we present FLUMMI (Fixed-and-Live-cell Universal Membrane Marker for Imaging), a 32-amino acid long peptide derived from P. falciparum PCNA1, which targets (fluorescent) protein tags to internal membranes for detection in live or fixed samples. Importantly, FLUMMI enables non-invasive, long-term live-cell imaging of internal membranes without affecting P. falciparum viability and is functional in blood stages of both, the human malaria parasite P. falciparum and the rodent-infecting parasite P. berghei , as well as in P. berghei mosquito stages. FLUMMI is also compatible with advanced techniques such as expansion microscopy. Together, our results establish FLUMMI as a versatile and non-toxic tool for membrane imaging in Plasmodium . ### Competing Interest Statement The authors have declared no competing interest. European Research Council, 101162759 Deutsche Forschungsgemeinschaft, 240245660, 531930468 Health + Life Science Alliance Heidelberg Mannheim

Malaria tropica remains a major global health challenge, raising the need for new therapeutic strategies against Plasmodium falciparum. While nucleoside analogues are effective against viruses and cancer, their use against P. falciparum is limited by the lack of nucleoside kinases in this species. To overcome this, we generated and tested cell-permeable derivatives of 5-fluorodeoxyuridine triphosphate (cpFdUTP) for antiparasitic activity in infected human red blood cells. cpFdUTP rapidly and potently inhibited the proliferation of P. falciparum, arresting development at the trophozoite-to-schizont transition by stalling DNA replication, as observed in a P. falciparum nuclear cycle sensor line. Although cpFdUTP also impaired the growth of human cells, supplementation with thymidine or cell-permeable deoxythymidine triphosphate (cpdTTP) selectively rescued human cells while maintaining parasite inhibition. This identifies a potential therapeutic window for cpFdUTP in combination with thymidine, outlining a novel approach for malaria treatment.

This May marked the 20th anniversary of the BioMalPar conference at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. The meeting originated as part of a European network of excellence but has evolved to a world-leading conference on the biology and pathology of malaria parasites. Ally Olotu (Ifakara Health Institute, Tanzania) kicked off the conference this year with a keynote presentation on malaria prevention, emphasizing current and emerging vaccine strategies. Another highlight was the presentation of the BioMalPar Lifetime Achievement Award to Andy Waters (University of Glasgow, UK). In his speech, Andy not only reminisced about his distinguished academic career in fundamental Plasmodium research but stressed the importance of supporting such research in endemic communities. These and many other talks showcased the significant progress that has been made in the field and the challenges that remain in eradicating this devastating disease. In this TrendsTalk, we invite both early-career and established Plasmodium researchers to highlight the excellent work presented over six '2005-themed' sessions at BioMalPar XX.

Iron, as an essential micronutrient, plays a crucial role in host-pathogen interactions. In order to limit the growth of the pathogen, a common strategy of innate immunity includes withdrawing available iron to interfere with the cellular processes of the microorganism. Against that, unicellular parasites have developed powerful strategies to scavenge iron, despite the effort of the host. Iron-sequestering compounds, such as the approved and potent chelator deferoxamine (DFO), are considered a viable option for therapeutic intervention. Since iron is heavily utilized in the mitochondrion, targeting iron chelators in this organelle could constitute an effective therapeutic strategy. This work presents mitochondrially targeted DFO, mitoDFO, as a candidate against a range of unicellular parasites with promising in vitro efficiency. Intracellular Leishmania infection can be cleared by this compound, and experimentation with Trypanosoma brucei 427 elucidates its possible mode of action. The compound not only affects iron homeostasis but also alters the physiochemical properties of the inner mitochondrial membrane, resulting in a loss of function. Furthermore, investigating the virulence factors of pathogenic yeasts confirms that mitoDFO is a viable candidate for therapeutic intervention against a wide spectrum of microbe-associated diseases.

The development of redox-sensitive molecular fluorescent probes for the detection of redox changes in Plasmodium falciparum-parasitized red blood cells remains of interest due to the limitations of current genetically encoded biosensors. This study describes the design, screening and synthesis of new pro-fluorophores based on flavylium azido dyes coupled by CuAAC click chemistry to alkynyl analogues of plasmodione oxide, the key metabolite of the potent redox-active antimalarial plasmodione. The photophysical and electrochemical properties of these probes were evaluated, focusing on their fluorogenic responses. The influence of both the redox status of the quinone and the length of the PEG chain separating the fluorophore from the electrophore on the photophysical properties was investigated. The fluorescence quenching by photoinduced electron transfer is reversible and of high amplitude for probes in oxidized quinone forms and fluorescence is reinstated for reduced hydroquinone forms. Our results demonstrate that shortening the PEG chain has the effect of enhancing the fluorogenic response, likely due to non-covalent interactions between the two chromophores. All these systems were evaluated for their antiparasitic activities and fluorescence imaging suggests the efficacy of the fluorescent flavylium dyes in P. falciparum-parasitized red blood cells, paving the way for future parasite imaging studies to monitor cellular redox processes.

Plasmodium falciparum proliferates through schizogony in the clinically relevant blood stage of infection. During schizogony, consecutive rounds of DNA replication and nuclear division give rise to multinucleated stages before cellularization occurs. Although these nuclei reside in a shared cytoplasm, DNA replication and nuclear division occur asynchronously. Here, by mapping the proteomic context of the S-phase-promoting kinase PfCRK4, we show that it has a dual role for nuclear-cycle progression: PfCRK4 orchestrates not only DNA replication, but in parallel also the rearrangement of intranuclear microtubules from hemispindles into early mitotic spindles. Live-cell imaging of a reporter parasite showed that these microtubule rearrangements coincide with the onset of DNA replication. Together, our data render PfCRK4 a key factor for nuclear-cycle progression, linking entry into S-phase with the initiation of mitotic events. In part, such links may compensate for the absence of canonical cell cycle checkpoints in P. falciparum. IMPORTANCE The human malaria parasite Plasmodium falciparum proliferates in erythrocytes through schizogony, forming multinucleated stages before cellularization occurs. In marked contrast to the pattern of proliferation seen in most model organisms, P. falciparum nuclei multiply asynchronously despite residing in a shared cytoplasm. This divergent mode of replication is, thus, a good target for therapeutic interventions. To exploit this potential, we investigated a key regulator of the parasite’s unusual cell cycle, the kinase PfCRK4 and found that this kinase regulated not only DNA replication but also in parallel the rearrangement of nuclear microtubules into early mitotic spindles. Since canonical cell cycle checkpoints have not been described in P. falciparum parasites, linking entry into S-phase and the initiation of mitotic events via a kinase, may be an alternative means to exert control, which is typically achieved by checkpoints.

Malaria-causing parasites achieve rapid proliferation in human blood through multiple rounds of asynchronous nuclear division followed by daughter cell formation. Nuclear divisions critically depend on the centriolar plaque, which organizes intranuclear spindle microtubules. The centriolar plaque consists of an extranuclear compartment, which is connected via a nuclear pore-like structure to a chromatin-free intranuclear compartment. Composition and function of this non-canonical centrosome remain largely elusive. Centrins, which reside in the extranuclear part, are among the very few centrosomal proteins conserved in Plasmodium falciparum. Here we identify a novel centrin-interacting centriolar plaque protein. Conditional knock down of this Sfi1-like protein (PfSlp) caused a growth delay in blood stages, which correlated with a reduced number of daughter cells. Surprisingly, intranuclear tubulin abundance was significantly increased, which raises the hypothesis that the centriolar plaque might be implicated in regulating tubulin levels. Disruption of tubulin homeostasis caused excess microtubules and aberrant mitotic spindles. Time-lapse microscopy revealed that this prevented or delayed mitotic spindle extension but did not significantly interfere with DNA replication. Our study thereby identifies a novel extranuclear centriolar plaque factor and establishes a functional link to the intranuclear compartment of this divergent eukaryotic centrosome.

Malaria remains a significant threat to global health, and despite concerted efforts to curb the disease, malaria-related morbidity and mortality increased in recent years. Malaria is caused by unicellular eukaryotes of the genus Plasmodium, and all clinical manifestations occur during asexual proliferation of the parasite inside host erythrocytes. In the blood stage, Plasmodium proliferates through an unusual cell cycle mode called schizogony. Contrary to most studied eukaryotes, which divide by binary fission, the parasite undergoes several rounds of DNA replication and nuclear division that are not directly followed by cytokinesis, resulting in multinucleated cells. Moreover, despite sharing a common cytoplasm, these nuclei multiply asynchronously. Schizogony challenges our current models of cell cycle regulation and, at the same time, offers targets for therapeutic interventions. Over the recent years, the adaptation of advanced molecular and cell biological techniques have given us deeper insight how DNA replication, nuclear division, and cytokinesis are coordinated. Here, we review our current understanding of the chronological events that characterize the unusual cell division cycle of P. falciparum in the clinically relevant blood stage of infection.

Centrins are small calcium-binding proteins that have a variety of roles and are universally associated with eukaryotic centrosomes. Rapid proliferation of the malaria-causing parasite Plasmodium falciparum in the human blood depends on a particularly divergent and acentriolar centrosome, which incorporates several essential centrins. Their precise mode of action, however, remains unclear. In this study calcium-inducible liquid-liquid phase separation is revealed as an evolutionarily conserved principle of assembly for multiple centrins from P. falciparum and other species. Furthermore, the disordered N-terminus and calcium-binding motifs are defined as essential features for reversible biomolecular condensation, and we demonstrate that certain centrins can form co-condensates. In vivo analysis using live cell STED microscopy shows liquid-like dynamics of centrosomal centrin. Additionally, implementation of an inducible protein overexpression system reveals concentration-dependent formation of extra-centrosomal centrin assemblies with condensate-like properties. The timing of foci formation and dissolution suggests that centrin assembly is regulated. This study thereby provides a new model for centrin accumulation at eukaryotic centrosomes.

Many of the currently available anti-parasitic and anti-fungal frontline drugs have severe limitations, including adverse side effects, complex administration, and increasing occurrence of resistance. The discovery and development of new therapeutic agents is a costly and lengthy process. Therefore, repurposing drugs with already established clinical application offers an attractive, fast-track approach for novel treatment options. In this study, we show that the anti-cancer drug candidate MitoTam, a mitochondria-targeted analog of tamoxifen, efficiently eliminates a wide range of evolutionarily distinct pathogens in vitro, including pathogenic fungi, Plasmodium falciparum, and several species of trypanosomatid parasites, causative agents of debilitating neglected tropical diseases. MitoTam treatment was also effective in vivo and significantly reduced parasitemia of two medically important parasites, Leishmania mexicana and Trypanosoma brucei, in their respective animal infection models. Functional analysis in the bloodstream form of T. brucei showed that MitoTam rapidly altered mitochondrial functions, particularly affecting cellular respiration, lowering ATP levels, and dissipating mitochondrial membrane potential. Our data suggest that the mode of action of MitoTam involves disruption of the inner mitochondrial membrane, leading to rapid organelle depolarization and cell death. Altogether, MitoTam is an excellent candidate drug against several important pathogens, for which there are no efficient therapies and for which drug development is not a priority.

Paracrine ATP release by erythrocytes has been shown to regulate endothelial cell function via purinergic signaling, and this erythoid-endothelial signaling network is pathologically dysregulated in sickle cell disease. We tested the role of extracellular ATP-mediated purinergic signaling in the activation of Psickle, the mechanosensitive Ca2+-permeable cation channel of human sickle erythrocytes (SS RBC). Psickle activation increases intracellular [Ca2+] to stimulate activity of the RBC Gardos channel, KCNN4/KCa3.1, leading to cell shrinkage and accelerated deoxygenation-activated sickling.We found that hypoxic activation of Psickle recorded by cell-attached patch clamp in SS RBC is inhibited by extracellular apyrase, which hydrolyzes extracellular ATP. Hypoxic activation of Psickle was also inhibited by the pannexin-1 inhibitor, probenecid, and by the P2 antagonist, suramin. A Psickle-like activity was also activated in normoxic SS RBC (but not in control red cells) by bath pH 6.0. Acid-activated Psickle-like activity was similarly blocked by apyrase, probenecid, and suramin, as well as by the Psickle inhibitor, Grammastola spatulata mechanotoxin-4 (GsMTx-4).In vitro-differentiated cultured human sickle reticulocytes (SS cRBC), but not control cultured reticulocytes, also exhibited hypoxia-activated Psickle activity that was abrogated by GsMTx-4. Psickle-like activity in SS cRBC was similarly elicited by normoxic exposure to acid pH, and this acid-stimulated activity was nearly completely blocked by apyrase, probenecid, and suramin, as well as by GsMTx-4.Thus, hypoxia-activated and normoxic acid-activated cation channel activities are expressed in both SS RBC and SS cRBC, and both types of activation appear to be mediated or greatly amplified by autocrine or paracrine purinergic signaling.

Malaria-causing parasites proliferate within erythrocytes through schizogony, forming multinucleated stages before cellularization. Nuclear multiplication does not follow a strict geometric 2 n progression, and each proliferative cycle produces a variable number of progeny. Here, by tracking nuclei and DNA replication, we show that individual nuclei replicate their DNA at different times, despite residing in a shared cytoplasm. Extrapolating from experimental data using mathematical modeling, we provide strong indication that a limiting factor exists, which slows down the nuclear multiplication rate. Consistent with this prediction, our data show that temporally overlapping DNA replication events were significantly slower than partially overlapping or nonoverlapping events. Our findings suggest the existence of evolutionary pressure that selects for asynchronous DNA replication, balancing available resources with rapid pathogen proliferation.

Immunofluorescence labeling enables the detection and characterization of various parasite proteins presented on the surface of the infected red blood cell. Several approaches for immunofluorescence detection of red blood cell surface-presented proteins of Plasmodium spp. have been successfully established and published over the years. However, finding the right approach depends on the scientific question, and different protocols have different advantages. Here, we discuss some aspects that should be considered and present an easily applicable protocol for labeling parasite surface antigens, which subsequently can be analyzed by immunofluorescence microscopy (or flow cytometry).

Plasmodium merozoites invade erythrocytes in a stepwise manner through ligand binding, calcium signaling, and membrane deformation. Using a recently developed light-sheet microscope, Geoghegan et al. investigated invasion with unprecedented temporal resolution. Their spectacular footage revealed roles for host cell cholesterol and pore formation at the parasite-host cell interface.