https://minerva-access.unimelb.edu.au/bitstream/handle/11343/273759/PIIS193131282030113X.pdf

Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle

Efforts to knock out Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) from asexual erythrocytic stage have not been successful, indicating an indispensable role of the enzyme in asexual growth. We recently reported generation of a transgenic parasite with mutant CDPK1 [Bansal A, et al. (2016) MBio 7:e02011-16]. The mutant CDPK1 (T145M) had reduced activity of transphosphorylation. We reasoned that CDPK1 could be disrupted in the mutant parasites. Consistent with this assumption, CDPK1 was successfully disrupted in the mutant parasites using CRISPR/Cas9. We and others could not disrupt PfCDPK1 in the WT parasites. The CDPK1 KO parasites show a slow growth rate compared with the WT and the CDPK1 T145M parasites. Additionally, the CDPK1 KO parasites show a defect in both male and female gametogenesis and could not establish an infection in mosquitoes. Complementation of the KO parasite with full-length PfCDPK1 partially rescued the asexual growth defect and mosquito infection. Comparative global transcriptomics of WT and the CDPK1 KO schizonts using RNA-seq show significantly high transcript expression of gametocyte-specific genes in the CDPK1 KO parasites. This study conclusively demonstrates that CDPK1 is a good target for developing transmission-blocking drugs.

https://www.pnas.org/content/pnas/115/4/774.full.pdf

PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection

Malaria is caused by a parasite transmitted by certain types of mosquito. The parasite lives in different organs within its vertebrate animal and insect hosts and to cope with these different environments it has a complex life cycle with several highly specialized life stages. To move from an infected mosquito into vertebrates the parasite produces spore-like cells called sporozoites that are able to enter different tissues and move very fast. These cells develop inside parasite-made structures called oocysts, which form at the stomach wall of the mosquito. After emerging from the oocyst, sporozoites float through the mosquito’s circulatory system and eventually enter the salivary glands where they can be transmitted to vertebrates when the mosquito bites. Efforts to develop malaria treatments and vaccines have focused on understanding the parasite’s life cycle and identifying ways to control or eradicate key stages. Most researchers focus on the stage where the parasite is living in the vertebrate and actively causing disease, while the events in the mosquito are less intensely investigated. While several parasite proteins have been shown to be important for the release of sporozoites from oocysts, the molecular events leading to this release have not yet been fully resolved. Klug and Frischknecht used time-lapse microscopy to film the release of the sporozoites of a malaria parasite known as Plasmodium berghei. The experiments show that the sporozoites can leave oocysts in several different ways. Furthermore, Klug and Frischknecht identified a new parasite protein named TRP1 that is essential for the sporozoites to leave oocysts and invade the salivary glands. Sporozoites lacking TRP1 were not able to move and they were unable to leave the oocyst or invade the salivary glands. Klug and Frischknecht propose a new working model of the molecular events that govern sporozoite release in which TRP1 is required for sporozoites to move prior to their exit from oocysts. In the future, using the same techniques to analyze genetically modified parasites will help to reveal more details about sporozoite release.

Motility precedes egress of malaria parasites from oocysts

To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite.

/pdf/capturing-in-vivo-rna-transcriptional-dynamics-from-the-x2ira9mv10.pdf

Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum.

The phylum Apicomplexa includes intracellular parasites causing immense global disease burden, the deadliest of them being the human malaria parasite Plasmodium falciparum, which invades and replicates within erythrocytes. The cytoskeletal protein actin is well conserved within apicomplexans but divergent from mammalian actins, and was primarily reported to function during host cell invasion. However, novel invasion mechanisms have been described for several apicomplexans, and specific functions of the acto-myosin system are being reinvestigated. Of the two actin genes in P. falciparum, actin-1 (pfact1) is ubiquitously expressed in all life-cycle stages and is thought to be required for erythrocyte invasion, although its functions during parasite development are unknown, and definitive in vivo characterisation during invasion is lacking. Here we have used a conditional Cre-lox system to investigate the functions of PfACT1 during P. falciparum blood-stage development and host cell invasion. We demonstrate that PfACT1 is crucially required for segregation of the plastid-like organelle, the apicoplast, and for efficient daughter cell separation during the final stages of cytokinesis. Surprisingly, we observe that egress from the host cell is not an actin-dependent process. Finally, we show that parasites lacking PfACT1 are capable of microneme secretion, attachment and formation of a junction with the erythrocyte, but are incapable of host cell invasion. This study provides important mechanistic insights into the definitive essential functions of PfACT1 in P. falciparum, which are not only of biological interest, but owing to functional divergence from mammalian actins, could also form the basis for the development of novel therapeutics against apicomplexans.

/pdf/multiple-essential-functions-of-plasmodium-falciparum-actin-2vl9tiu033.pdf

Multiple essential functions of Plasmodium falciparum actin-1 during malaria blood-stage development.

https://minerva-access.unimelb.edu.au/bitstream/handle/11343/257851/PMC5104695.pdf

Alice Rantz

Papers

Plasmodium Merozoite TRAP Family Protein Is Essential for Vacuole Membrane Disruption and Gamete Egress from Erythrocytes.