Upon invading the host erythrocyte, the human malaria parasite P. falciparum lives and replicates within a membrane bound compartment referred to as the parasitophorous vacuole. Recently, interest in this compartment and its protein content has grown, due to the important roles these play in parasite egress and protein traffic to the host cell. Surprisingly, the function of many proteins within this compartment has not been experimentally addressed. Here, we study the importance of one of these proteins, termed PfPV1, for intra-erythrocytic parasite survival. Despite numerous attempts to inactivate the gene encoding PfPV1, we were unable to recover deletion mutants. Control experiments verified that the pv1 gene locus was per se open for gene targeting experiments, allowing us to exclude technical limitations in our experimental strategy. Our data provide strong genetic evidence that PfPV1 is essential for survival of blood stage P. falciparum, and further highlight the importance of parasitophorous vacuole proteins in this part of the parasite's life cycle.

Redox balance is essential for the survival, growth and multiplication of malaria parasites and oxidative stress is involved in the mechanism of action of many antimalarial drugs. Hydrogen peroxide (H₂O₂) plays an important role in redox signalling and pathogen-host cell interactions. For monitoring intra- and subcellular redox events, highly sensitive and specific probes are required. Here, we stably expressed the ratiometric H₂O₂redox sensor roGFP2-Orp1 in the cytosol and the mitochondria ofPlasmodium falciparum (P. falciparum)NF54-attBblood-stage parasites and evaluated its sensitivity towards oxidative stress, selected antimalarial drugs, and novel lead compounds. In both compartments, the sensor showed reproducible sensitivity towards H₂O₂in the low micromolar range and towards antimalarial compounds at pharmacologically relevant concentrations. Upon short-term exposure (4 h), artemisinin derivatives, quinine and mefloquine impacted H₂O₂levels in mitochondria, whereas chloroquine and a glucose-6-phosphate dehydrogenase (G6PD) inhibitor affected the cytosol; 24 h exposure to arylmethylamino steroids and G6PD inhibitors revealed oxidation of mitochondria and cytosol, respectively. Genomic integration of an H₂O₂sensor expressed in subcellular compartments ofP. falciparumprovides the basis for studying complex parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity, and sets the stage for high-throughput approaches to identify antimalarial agents perturbing redox equilibrium.

Plasmodium male and female gametocytes are the gatekeepers of human-to-mosquito transmission, therefore essential for propagation of malaria within a population. Whilst dormant in humans, their divergent roles during transmission become apparent soon after mosquito feeding with a rapid transformation into gametes – males forming eight motile sperm-like cells aiming to fertilise a single female gamete. Little is known about how the parasite fuels this abrupt change, and the potential role played by their large and elaborate cristate mitochondrion. Using a sex-specific antibody and functional mitochondrial labelling, we show that the male gametocyte mitochondrion is less active than that of female gametocytes and more sensitive to antimalarials targeting mitochondrial energy metabolism. Rather than a vestigial organelle discarded during male gametogenesis, we demonstrate that mitochondrial ATP synthesis is essential for its completion. Additionally, using a genetically encoded ratiometric ATP sensor, we show that gametocytes can maintain cytoplasmic ATP homeostasis in the absence of mitochondrial respiration, indicating the essentiality of the gametocyte mitochondrion for transmission alone. Together, this reveals how gametocytes responsively balance the conflicting demands of a dormant and active lifestyle, highlighting the mitochondria as a rich source of transmission-blocking targets for future drug development.

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

As part of their life-cycle, malaria parasites undergo rapid cell multiplication and division, with one parasite giving rise to over 20 new parasites within the course of 48 h. To support this, the parasite has an extremely high metabolic rate and level of protein biosynthesis. Underpinning these activities, the parasite encodes a number of chaperone/heat shock proteins, belonging to various families. Research over the past decade has revealed that these proteins are involved in a number of essential processes within the parasite, or within the infected host cell. Due to this, these proteins are now being viewed as potential targets for drug development, and we have begun to characterize their properties in more detail. In this article we summarize the current state of knowledge about one particular chaperone family, that of the HSP70, and highlight their importance, function, and potential co-chaperone interactions. This is then discussed with regard to the suitability of these proteins and interactions for drug development.