Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2-deficient [PPLP2(−)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(−) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(−) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(−) parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2(−) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(−) loss-of-function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm.

The complex life-cycle of the human malaria parasite Plasmodium falciparum requires a high degree of tight coordination allowing the parasite to adapt to changing environments. One of the major challenges for the parasite is the human-to-mosquito transmission, which starts with the differentiation of blood stage parasites into the transmissible gametocytes, followed by the rapid conversion of the gametocytes into gametes, once they are taken up by the blood-feeding Anopheles vector. In order to pre-adapt to this change of host, the gametocytes store transcripts in stress granules that encode proteins needed for parasite development in the mosquito. Here we report on a novel stress granule component, the seven-helix protein 7-Helix-1. The protein, a homolog of the human stress response regulator LanC-like 2, accumulates in stress granules of female gametocytes and interacts with ribonucleoproteins, such as CITH, DOZI, and PABP1. Malaria parasites lacking 7-Helix-1 are significantly impaired in female gametogenesis and thus transmission to the mosquito. Lack of 7-Helix-1 further leads to a deregulation of components required for protein synthesis. Consistently, inhibitors of translation could mimic the 7-Helix-1 loss-of-function phenotype. 7-Helix-1 forms a complex with the RNA-binding protein Puf2, a translational regulator of the female-specific antigen Pfs25, as well as with pfs25-coding mRNA. In accord, gametocytes deficient of 7-Helix-1 exhibit impaired Pfs25 synthesis. Our data demonstrate that 7-Helix-1 constitutes stress granules crucial for regulating the synthesis of proteins needed for life-cycle progression of Plasmodium in the mosquito vector.

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