Molecular Characterization of Toxoplasma gondii Formin 3, an Actin Nucleator Dispensable for Tachyzoite Growth and Motility

Abstract

bToxoplasma gondii belongs to the phylum Apicomplexa, a group of obligate intracellular parasites that rely on gliding motility to enter host cells. Drugs interfering with the actin cytoskeleton block parasite motility, host cell invasion, and egress from infected cells. Myosin A, profilin, formin 1, formin 2, and actin-depolymerizing factor have all been implicated in parasite motility, yet little is known regarding the importance of actin polymerization and other myosins for the remaining steps of the parasite lytic cycle. Here we establish that T. gondii formin 3 (TgFRM3), a newly described formin homology 2 domain (FH2)-containing protein, binds to Toxoplasma actin and nucleates rabbit actin assembly in vitro. TgFRM3 expressed as a transgene exhibits a patchy localization at several distinct structures within the parasite. Disruption of the TgFRM3 gene by double homologous recombination in a ku80-ko strain reveals no vital function for tachyzoite propagation in vitro, which is consistent with its weak level of expression in this life stage. Conditional stabilization of truncated forms of TgFRM3 suggests that different regions of the molecule contribute to distinct localizations. Moreover, expression of TgFRM3 lacking the C-terminal domain severely affects parasite growth and replication. This work provides a first insight into how this specialized formin, restricted to the group of coccidia, completes its actin-nucleating activity.T he phylum Apicomplexa contains important protozoan parasites such as Plasmodium, Toxoplasma, Babesia, Eimeria, Neospora, Theileria, and Cryptosporidium spp., that are responsible for severe diseases in humans and farm animals. As obligate intracellular organisms, these parasites are reliant on active invasion of host cells to complete their complex life cycle. Their ability to cross host biological barriers and infect a diversity of tissues requires a unique mode of locomotion called gliding motility, which is powered by the parasite actomyosin system (reviewed in references 8 and 42). While the contributions of actin polymerization and the myosin A motor in motility and invasion are well documented, mainly in Toxoplasma and Plasmodium, the importance of actin and the function of other myosins in the biology of these parasites are still poorly understood.Toxoplasma gondii tachyzoites replicate by endodyogeny, a process in which two daughter parasites grow within an intact, fully polarized mother parasite (44). The internal daughter cells are delimited by the inner membrane complex and associated subpellicular microtubules. Inheritance of organelles by daughter cells during parasite cell division happens in a highly synchronized fashion, starting with the centriole and Golgi apparatus, followed by the apicoplast, nucleus, and endoplasmic reticulum, and ending with the mitochondrion and de novo synthesis of the micronemes and rhoptries (32). When the daughter cells are fully mature, the maternal apical complex is disassembled and the daughters bud from the mother, adopting her plas...

Regulation of ATG8 membrane association by ATG4 in the parasitic protistToxoplasma gondii

“…To investigate directly the role of TgATG8 in T. gondii, we attempted to interrupt the open reading frame of the corresponding gene by a single homologous crossover 14 resulting in the integration of a plasmid bearing a 5´ TgATG8 cassette, as diagrammed in Figure S1A. Despite repeated attempts in the RH-derived Ku80Δ strain, which is more amenable to targeted gene deletion, 15 this approach did not yield viable knock-in parasites.…”

“…A 1,665 bp fragment corresponding to the 3´ of TgATG4 was amplified from genomic DNA using primers ML1233/ML1234 and cloned into pTUB8MycGFPPfMyoAtail3Ty-HX vector 14 to yield the p3´-TgATG4-3Ty plasmid. 60 µg of this plasmid were digested by SphI prior to transfection.…”

Plasticity and redundancy among AMA–RON pairs ensure host cell entry of Toxoplasma parasites

“…Disruption of AMA1 gene was performed using the plasmid pKO-AMA1, derived from the p3Ty-HXGPRT vector 38 in the following manner. An internal AMA1 fragment was PCR amplified using the couple of primers ML883/ML884, and subsequently cloned in the p3Ty-HXGPRT using the restriction sites KpnI and NsiI.…”

Toxoplasma gondii Profilin Acts Primarily To Sequester G-Actin While Formins Efficiently Nucleate Actin Filament Formation in Vitro

“…There are three formins in T. gondii and two of them, TgFRM1 and TgFRM2, have been shown to act as nucleators of rabbit actin in vitro and contribute to parasite motility (30). Recently TgFRM3 was shown to bind TgACTI and nucleate rabbit actin assembly in vitro , yet it is not required for parasite survival in culture (31). P. falciparum also encodes three proteins with similarity to formins, and PfFormin1 and PfFormin2 have been shown to act as barbed end nucleators of chicken actin in vitro (32).…”