Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ, another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.

Plasmodium sporozoites are the product of a complex developmental process in the mosquito vector and are destined to infect the mammalian liver. Attention has been drawn to the mosquito stages and pre-erythrocytic stages owing to recognition that these are bottlenecks in the parasite life cycle and that intervention at these stages can block transmission and prevent infection. Parasite progression in the Anopheles mosquito, sporozoite transmission to the mammalian host by mosquito bite, and subsequent infection of the liver are characterized by extensive migration of invasive stages, cell invasion, and developmental changes. Preparation for the liver phase in the mammalian host begins in the mosquito with an extensive reprogramming of the sporozoite to support efficient infection and survival. Here, we discuss what is known about the molecular and cellular basis of the developmental progression of parasites and their interactions with host tissues in the mosquito and during the early phase of mammalian infection.

The Plasmodium life cycle is a sequence of alternating invasive and replicative stages within the vertebrate and invertebrate hosts. How malarial parasites exit their host cells after completion of reproduction remains largely unsolved. Inhibitor studies indicated a role of Plasmodium cysteine proteases in merozoite release from host erythrocytes. To validate a vital function of malarial cysteine proteases in active parasite egress, we searched for target genes that can be analyzed functionally by reverse genetics. Herein, we describe a complete arrest of Plasmodium sporozoite egress from Anopheles midgut oocysts by targeted disruption of a stage-specific cysteine protease. Our findings show that sporozoites exit oocysts by parasite-dependent proteolysis rather than by passive oocyst rupture resulting from parasite growth. We provide genetic proof that malarial cysteine proteases are necessary for egress of invasive stages from their intracellular compartment and propose that similar cysteine protease–dependent mechanisms occur during egress from liver-stage and blood-stage schizonts.

Falciparum malaria is initiated when Anopheles mosquitoes transmit the Plasmodium sporozoite stage during a blood meal. Irradiated sporozoites confer sterile protection against subsequent malaria infection in animal models and humans. This level of protection is unmatched by current recombinant malaria vaccines. However, the live-attenuated vaccine approach faces formidable obstacles, including development of accurate, reproducible attenuation techniques. We tested whether Plasmodium falciparum could be attenuated at the early liver stage by genetic engineering. The P. falciparum genetically attenuated parasites (GAPs) harbor individual deletions or simultaneous deletions of the sporozoite-expressed genes P52 and P36 . Gene deletions were done by double-cross-over recombination to avoid genetic reversion of the knockout parasites. The gene deletions did not affect parasite replication throughout the erythrocytic cycle, gametocyte production, mosquito infections, and sporozoite production rates. However, the deletions caused parasite developmental arrest during hepatocyte infection. The double-gene deletion line exhibited a more severe intrahepatocytic growth defect compared with the single-gene deletion lines, and it did not persist. This defect was assessed in an in vitro liver-stage growth assay and in a chimeric mouse model harboring human hepatocytes. The strong phenotype of the double knockout GAP justifies its human testing as a whole-organism vaccine candidate using the established sporozoite challenge model. GAPs might provide a safe and reproducible platform to develop an efficacious whole-cell malaria vaccine that prevents infection at the preerythrocytic stage.