Chwen L. Tay

Bio: Chwen L. Tay is an academic researcher from Imperial College London. The author has contributed to research in topics: Actin & Plasmodium falciparum. The author has an hindex of 3, co-authored 4 publications receiving 97 citations.

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

Abstract: 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.

Binding Of A Newly Identified Essential Light Chain To Expressed Plasmodium falciparum Class XIV Myosin Enhances Actin Motility

Abstract: Motility of the apicomplexan parasite Plasmodium falciparum, the causative agent of malaria, is enabled by the glideosome, a multi-protein complex containing the class XIV myosin motor, PfMyoA. Parasite motility is necessary for invasion into host cells and for virulence. Here we show that milligram quantities of functional PfMyoA can be expressed using the baculovirus/Sf9 cell expression system, provided that a UCS (UNC-45/CRO1/She4p) family myosin co-chaperone from Plasmodium spp. is co-expressed with the heavy chain. The homologous chaperone from the apicomplexan Toxoplasma gondii does not functionally substitute. We expressed a functional full-length PfMyoA with bound myosin tail interacting protein (MTIP), the only known light chain of PfMyoA. We then identified an additional essential light chain (PfELC) that co-purified with PfMyoA isolated from parasite lysates. PfMyoA expressed with both light chains moved actin at ~3.8 μm/sec, more than twice that of PfMyoA-MTIP (~1.7 μm/sec), consistent with the light chain binding domain acting as a lever arm to amplify nucleotide-dependent motions in the motor domain. Surprisingly, PfMyoA moved skeletal actin or expressed P. falciparum actin at the same speed. Duty ratio estimates suggest that PfMyoA may be able to move actin at maximal speed with as few as 6 motors. Under unloaded conditions, neither phosphorylation of Ser19 of the heavy chain, phosphorylation of several Ser residues in the N-terminal extension of MTIP, or calcium affected the speed of actin motion. These studies provide the essential framework for targeting the glideosome as a potential drug target to inhibit invasion by the malaria parasite.

Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii.

Abstract: Toxoplasma gondii possesses a limited set of actin-regulatory proteins and relies on only three formins (FRMs) to nucleate and polymerize actin. We combined filamentous actin (F-actin) chromobodies with gene disruption to assign specific populations of actin filaments to individual formins. FRM2 localizes to the apical juxtanuclear region and participates in apicoplast inheritance. Restricted to the residual body, FRM3 maintains the intravacuolar cell-cell communication. Conoidal FRM1 initiates a flux of F-actin crucial for motility, invasion and egress. This flux depends on myosins A and H and is controlled by phosphorylation via PKG (protein kinase G) and CDPK1 (calcium-dependent protein kinase 1) and by methylation via AKMT (apical lysine methyltransferase). This flux is independent of microneme secretion and persists in the absence of the glideosome-associated connector (GAC). This study offers a coherent model of the key players controlling actin polymerization, stressing the importance of well-timed post-translational modifications to power parasite motility.

The Actinomyosin Motor Drives Malaria Parasite Red Blood Cell Invasion but Not Egress.

Abstract: Apicomplexa are obligate intracellular parasites that actively invade, replicate within, and egress from host cells. The parasite actinomyosin-based molecular motor complex (often referred to as the glideosome) is considered an important mediator of parasite motility and virulence. Mature intracellular parasites often become motile just prior to egress from their host cells, and in some genera, this motility is important for successful egress as well as for subsequent invasion of new host cells. To determine whether actinomyosin-based motility is important in the red blood cell egress and invasion activities of the malaria parasite, we have used a conditional genetic approach to delete GAP45, a primary component of the glideosome, in asexual blood stages of Plasmodium falciparum Our results confirm the essential nature of GAP45 for invasion but show that P. falciparum does not require a functional motor complex to undergo egress from the red blood cell. Malarial egress therefore differs fundamentally from induced egress in the related apicomplexan Toxoplasma gondiiIMPORTANCE Clinical malaria results from cycles of replication of single-celled parasites of the genus Plasmodium in red blood cells. Intracellular parasite replication is followed by a highly regulated, protease-dependent process called egress, in which rupture of the bounding membranes allows explosive release of daughter merozoites which rapidly invade fresh red cells. A parasite actinomyosin-based molecular motor (the glideosome) has been proposed to provide the mechanical force to drive invasion. Studies of the related parasite Toxoplasma gondii have shown that induced egress requires parasite motility, mediated by a functional glideosome. However, whether the glideosome has a similar essential role in egress of malaria merozoites from red blood cells is unknown. Here, we show that although a functional glideosome is required for red blood cell invasion by Plasmodium falciparum merozoites, it is not required for egress. These findings place further emphasis on the key role of the protease cascade in malarial egress.

PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection

Abstract: 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.

Motility precedes egress of malaria parasites from oocysts

Abstract: 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.