The response of Plasmodium falciparum to isoleucine withdrawal is dependent on the stage of progression through the intraerythrocytic cell cycle.

BACKGROUND: A previous study reported that the malaria parasite Plasmodium falciparum enters an altered growth state upon extracellular withdrawal of the essential amino acid isoleucine. Parasites slowed transit through the cell cycle when deprived of isoleucine prior to the onset of S-phase. METHODS: This project was undertaken to study at higher resolution, how isoleucine withdrawal affects parasite growth. Parasites were followed at regular intervals across an extended isoleucine deprivation time course across the cell cycle using flow cytometry. RESULTS: These experiments revealed that isoleucine-deprived parasites never exit the cell cycle, but instead continuously grow at a markedly reduced pace. Moreover, slow growth occurs only if isoleucine is removed prior to the onset of schizogony. After S-phase commenced, the parasite is insensitive to isoleucine depletion and transits through the cell cycle at the normal pace. CONCLUSIONS: The markedly different response of the parasite to isoleucine withdrawal before or after the onset of DNA replication is reminiscent of the nutrient-dependent G1 cell cycle checkpoints described in other organisms.

A transcription factor helps Plasmodium falciparum gametocytogenesis take shape.

The recent study by Campelo Morillo et al. has shown that one of the small number of non-ApiAP2 DNA-binding proteins in the Plasmodium falciparum genome acts as a transcription factor in the gametocytogenesis cascade and is responsible for the gametocyte's distinctive morphology.

Generation of Transmission-Competent Human Malaria Parasites with Chromosomally-Integrated Fluorescent Reporters.

Malaria parasites have a complex life cycle that includes specialized stages for transmission between their mosquito and human hosts. These stages are an understudied part of the lifecycle yet targeting them is an essential component of the effort to shrink the malaria map. The human parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria. Our goal was to generate transgenic P. falciparum lines that could complete the lifecycle and produce fluorescent transmission stages for more in-depth and high-throughput studies. Using zinc-finger nuclease technology to engineer an integration site, we generated three transgenic P. falciparum lines in which tdtomato or gfp were stably integrated into the genome. Expression was driven by either stage-specific peg4 and csp promoters or the constitutive ef1a promoter. Phenotypic characterization of these lines demonstrates that they complete the life cycle with high infection rates and give rise to fluorescent mosquito stages. The transmission stages are sufficiently bright for intra-vital imaging, flow cytometry and scalable screening of chemical inhibitors and inhibitory antibodies.

Identification of Plasmodium GAPDH epitopes for generation of antibodies that inhibit malaria infection.

Plasmodium sporozoite liver infection is an essential step for parasite development in its mammalian host. Previously, we used a phage display library to identify mimotope peptides that bind to Kupffer cells and competitively inhibit sporozoite-Kupffer cell interaction. These peptides led to the identification of a Kupffer cell receptor-CD68-and a Plasmodium sporozoite ligand-GAPDH-that are required for sporozoite traversal of Kupffer cells and subsequent infection of hepatocytes. Here, we report that the C-terminal end of Plasmodium GAPDH interacts with the Kupffer CD68 receptor, and identify two epitopes within this region as candidate antigens for the development of antibodies that inhibit Plasmodium infection.

Plasmodium falciparum Maf1 Confers Survival upon Amino Acid Starvation.

The target of rapamycin complex 1 (TORC1) pathway is a highly conserved signaling pathway across eukaryotes that integrates nutrient and stress signals to regulate the cellular growth rate and the transition into and maintenance of dormancy. The majority of the pathway's components, including the central TOR kinase, have been lost in the apicomplexan lineage, and it is unknown how these organisms detect and respond to nutrient starvation in its absence. Plasmodium falciparum encodes a putative ortholog of the RNA polymerase (Pol) III repressor Maf1, which has been demonstrated to modulate Pol III transcription in a TOR-dependent manner in a number of organisms. Here, we investigate the role of P. falciparum Maf1 (PfMaf1) in regulating RNA Pol III expression under conditions of nutrient starvation and other stresses. Using a transposon insertion mutant with an altered Maf1 expression profile, we demonstrated that proper Maf1 expression is necessary for survival of the dormancy-like state induced by prolonged amino acid starvation and is needed for full recovery from other stresses that slow or stall the parasite cell cycle. This Maf1 mutant is defective in the downregulation of pre-tRNA synthesis under nutrient-limiting conditions, indicating that the function of Maf1 as a stress-responsive regulator of structural RNA transcription is conserved in P. falciparum Recent work has demonstrated that parasites carrying artemisinin-resistant K13 alleles display an enhanced ability to recover from drug-induced growth retardation. We show that one such artemisinin-resistant line displays greater regulation of pre-tRNA expression and higher survival upon prolonged amino acid starvation, suggesting that overlapping, PfMaf1-associated pathways may regulate growth recovery from both artemisinin treatment and amino acid starvation.IMPORTANCE Eukaryote organisms sense changes in their environment and integrate this information through signaling pathways to activate response programs to ensure survival. The TOR pathway is a well-studied signaling pathway found throughout eukaryotes that is known to integrate a variety of signals to regulate organismal growth in response to starvation and other stresses. The human malaria parasite Plasmodium falciparum appears to have lost the TOR pathway over the course of evolution, and it is unclear how the parasite modulates its growth in response to starvation and drug treatment. Here, we show that Maf1, a protein regulated by TOR in other eukaryotes, plays an important role in maintaining the parasite's viability in the face of starvation and other forms of stress. This suggests that PfMaf1 is a component of a yet-to-be-described nutrient and stress response pathway.

Genetic Control Of Malaria Mosquitoes.

Experiments demonstrating the feasibility of genetically modifying mosquito vectors to impair their ability to transmit the malaria parasite have been known for well over a decade. However, means to spread resistance or population control genes into wild mosquito populations remains an unsolved challenge. Two recent reports give hope that CRISPR technology may allow such challenge to be overcome.