SummaryMalaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.

Ribosome-targeting antibiotics exert their antimalarial activity on the apicoplast of the malaria parasite, an organelle of prokaryote origin having essential metabolic functions. These antibiotics typically cause a delayeddeath phenotype, which manifests in parasite killing during the second replication cycle following administration. As an exception, treatment with the antibiotic thiostrepton results in an immediate killing. We recently demonstrated that thiostrepton and its derivatives interfere with the eukaryotic proteasome, a multimeric protease complex that is important for the degradation of ubiquitinated proteins. Here, we report that the thiostrepton-based compounds are active against chloroquine-sensitive and -resistant Plasmodium falciparum, where they rapidly eliminate parasites before DNA replication. The minor parasite fraction that escapes the fast killing of the first replication cycle is arrested in the schizont stage of the following cycle, displaying a delayed-death phenotype. Thiostrepton further exhibits gametocytocidal activity by eliminating gametocytes, the sexual precursor cells that are crucial for parasite transmission to the mosquito. Compound treatment results in an accumulation of ubiquitinated proteins in the blood stages, indicating an effect on the parasite proteasome. In accordance with these findings, expression profiling revealed that the proteasome is present in the nucleus and cytoplasm of trophozoites, schizonts, and gametocytes. In conclusion, thiostrepton derivatives represent promising candidates for malaria therapy by dually acting on two independent targets, the parasite proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite.

Physical contact is important for the interaction between animal cells, but can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of more than 100 µm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close-ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive “nanotubes” in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.

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