Mirko Singer

Apicomplexan parasites are obligate intracellular pathogens possessing unique organelles but lacking several components of the membrane trafficking machinery conserved in other eukaryotes. While some of these components have been lost during evolution, others remain undetectable by standard bioinformatics approaches. Using a conditional splitCas9 system in Toxoplasma gondii, we previously identified TGGT1_301410, a hypothetical gene conserved among apicomplexans, as a potential trafficking factor. Here, we show that TGGT1_301410 is a distant ortholog of T. gondii tepsin (TgTEP), localized to the trans-Golgi and functioning as an accessory protein of the adaptor protein complex 4 (AP4). We demonstrate that AP4-TgTEP is essential for the actin-dependent transport of vesicles to the plant-like vacuole (PLVAC) and Golgi organization. Notably, our findings reveal that, unlike in metazoans, the AP4 complex in T. gondii utilizes clathrin as a coat protein, a mechanism more closely aligned with that of plants. These results underscore a conserved yet functionally adapted vesicular transport system in Apicomplexa.

Active movement is essential for the survival of microorganisms like bacteria, algae, and unicellular parasites. In three dimensions, both swimming and gliding microorganisms often exhibit helical trajectories. One such case are malaria parasites gliding through 3D hydrogels, for which we find that the internal correlation time for the stochastic process generating propulsion is similar to the time taken for one helical turn. Motivated by this experimental finding, here we theoretically analyze the case of finite internal correlation time for microorganisms with helical trajectories as chiral active particles with an Ornstein-Uhlenbeck process for torque. We present an analytical solution which is in very good agreement with computer simulations. We then show that, for this type of internal noise, chirality and rotation increase the persistence of motion and results in helical trajectories that have a larger long-time mean squared displacement than straight trajectories at the same propulsion speed. Finally, we provide experimental evidence for this prediction for the case of malaria parasites.

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.

Plasmodium sporozoites are the highly polarized and motile forms of the malaria parasite that are transmitted by mosquitoes to the vertebrate hosts. Sporozoites use myosin molecular motors to generate retrograde flow of actin filaments. These are linked to plasma-membrane spanning adhesins, which in turn bind to the extracellular environment, resulting in forward directed gliding motility. The gliding motility machine of sporozoites leads to high speeds in the range of micrometer per second, which are essential for efficient migration in the skin. Yet, it is not clear how the individual parts of the machinery work together to generate force during migration. Sporozoites are elongated and curved cells and move on circular tracks in vitro . Sporozoites lacking the adhesin thrombospondin related anonymous protein (TRAP) like protein, TLP, can still migrate in the skin, but at a lower level. TLP lacking sporozoites generate a lower force on the dorsal (non-substrate facing) surface as measured by laser tweezers. Here we use traction force microscopy to investigate motile sporozoites and the forces they produce during migration on their ventral surface. Both wild type and tlp(-) sporozoites show distinct foci of force generation, but tlp(-) sporozoites generating overall lower forces. Our findings demonstrate that TLP is an important element of the force-generating machinery during sporozoite gliding motility.

Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI‐anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full‐length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP‐GFP. Replacing the N‐terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP‐GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP‐GFP expressing parasites that are blocked in egress with wild‐type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP‐GFP. Expressing CSP‐GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP‐GFP without disruption of oocyst egress and salivary gland invasion.

The malaria-causing parasite Plasmodium has a complex life cycle involving both vertebrate and mosquito hosts. Sexual stages or gametocytes are the only stage competent for transmission to mosquitoes. Formation of flagellated male gametes from gametocytes requires rapid rounds of genome replication. Here we discovered a non-canonical Plasmodium actin-related protein 2/3 (Arp2/3) complex essential for DNA segregation during male gametogenesis. Plasmodium Arp2/3 dynamically localizes within the nucleus to the endomitotic spindles and interacts with a kinetochore protein. Deletion of key Arp2/3 subunits or interfering with actin polymerisation leads to the formation of sub-haploid male gametes and a complete block in transmission through delayed developmental arrest at the oocyst stage. Our work identified an evolutionary divergent protein complex in malaria parasites that offers potential targets for transmission-blocking interventions.