scholarly journals Blocking palmitoylation of Toxoplasma gondii myosin light chain 1 disrupts glideosome composition but has little impact on parasite motility

2020 ◽  
Author(s):  
Pramod K. Rompikuntal ◽  
Ian T. Foe ◽  
Bin Deng ◽  
Matthew Bogyo ◽  
Gary E. Ward
Keyword(s):  
Toxoplasma Gondii ◽  
Current Model ◽  
Severe Disease ◽  
Gliding Motility ◽  
The Body ◽  
Phase Partitioning ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Animal Pathogens ◽  
Parasite Motility

AbstractToxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin-based motor consisting of a Class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. The binding of TgMLC1 to TgGAP45 is therefore considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation disrupt the association of TgMLC1 with the membrane fraction of the parasite in phase partitioning experiments and completely block the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete.ImportanceGliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention and treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely-held “linear motor” model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of and between cells of the hosts they infect.

scholarly journals Blocking Palmitoylation of Toxoplasma gondii Myosin Light Chain 1 Disrupts Glideosome Composition but Has Little Impact on Parasite Motility

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Pramod K. Rompikuntal ◽  
Robyn S. Kent ◽  
Ian T. Foe ◽  
Bin Deng ◽  
Matthew Bogyo ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Current Model ◽  
Severe Disease ◽  
Gliding Motility ◽  
The Body ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Myosin Motor ◽  
Animal Pathogens ◽  
Parasite Motility

ABSTRACT Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of substrate-dependent gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin motor consisting of a class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. Therefore, the binding of TgMLC1 to TgGAP45 is considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation completely abrogate the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete. IMPORTANCE Gliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention or treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely held “linear motor” model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of, and between cells of the hosts they infect.

scholarly journals Toxoplasma gondii myosins B/C

2001 ◽  
Vol 155 (4) ◽  
pp. 613-624 ◽  
Author(s):  
Frédéric Delbac ◽  
Astrid Sänger ◽  
Eva M. Neuhaus ◽  
Rolf Stratmann ◽  
James W. Ajioka ◽  
...  
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Daughter Cell ◽  
Gliding Motility ◽  
Apicomplexan Parasites ◽  
Daughter Cells ◽  
Membrane Complex ◽  
Alternatively Spliced ◽  
Butanedione Monoxime ◽  
Inner Membrane Complex

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

scholarly journals Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii

2004 ◽  
Vol 165 (3) ◽  
pp. 383-393 ◽  
Author(s):  
Elizabeth Gaskins ◽  
Stacey Gilk ◽  
Nicolette DeVore ◽  
Tara Mann ◽  
Gary Ward ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Complex ◽  
Integral Membrane Protein ◽  
Gliding Motility ◽  
Host Cells ◽  
Inner Membrane ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Two Stages

Apicomplexan parasites exhibit a unique form of substrate-dependent motility, gliding motility, which is essential during their invasion of host cells and during their spread between host cells. This process is dependent on actin filaments and myosin that are both located between the plasma membrane and two underlying membranes of the inner membrane complex. We have identified a protein complex in the apicomplexan parasite Toxoplasma gondii that contains the class XIV myosin required for gliding motility, TgMyoA, its associated light chain, TgMLC1, and two novel proteins, TgGAP45 and TgGAP50. We have localized this complex to the inner membrane complex of Toxoplasma, where it is anchored in the membrane by TgGAP50, an integral membrane glycoprotein. Assembly of the protein complex is spatially controlled and occurs in two stages. These results provide the first molecular description of an integral membrane protein as a specific receptor for a myosin motor, and further our understanding of the motile apparatus underlying gliding motility in apicomplexan parasites.

scholarly journals A Novel Actin-Related Protein Is Associated with Daughter Cell Formation in Toxoplasma gondii

2008 ◽  
Vol 7 (9) ◽  
pp. 1500-1512 ◽  
Author(s):  
Jennifer L. Gordon ◽  
Wandy L. Beatty ◽  
L. David Sibley
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Daughter Cell ◽  
Cell Formation ◽  
The Body ◽  
Apicomplexan Parasites ◽  
Daughter Cells ◽  
Membrane Complex ◽  
Transport Vesicles ◽  
Inner Membrane Complex

ABSTRACT Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.

scholarly journals Two Essential Light Chains Regulate the MyoA Lever Arm To Promote Toxoplasma Gliding Motility

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Melanie J. Williams ◽  
Hernan Alonso ◽  
Marta Enciso ◽  
Saskia Egarter ◽  
Lilach Sheiner ◽  
...  
Keyword(s):  
Light Chain ◽  
Therapeutic Potential ◽  
Point Mutations ◽  
Gliding Motility ◽  
Host Cells ◽  
Light Chains ◽  
Apicomplexan Parasites ◽  
Essential Light Chain ◽  
Lever Arm ◽  
Parasite Motility

ABSTRACT Key to the virulence of apicomplexan parasites is their ability to move through tissue and to invade and egress from host cells. Apicomplexan motility requires the activity of the glideosome, a multicomponent molecular motor composed of a type XIV myosin, MyoA. Here we identify a novel glideosome component, essential light chain 2 (ELC2), and functionally characterize the two essential light chains (ELC1 and ELC2) of MyoA in Toxoplasma. We show that these proteins are functionally redundant but are important for invasion, egress, and motility. Molecular simulations of the MyoA lever arm identify a role for Ca2+ in promoting intermolecular contacts between the ELCs and the adjacent MLC1 light chain to stabilize this domain. Using point mutations predicted to ablate either the interaction with Ca2+ or the interface between the two light chains, we demonstrate their contribution to the quality, displacement, and speed of gliding Toxoplasma parasites. Our work therefore delineates the importance of the MyoA lever arm and highlights a mechanism by which this domain could be stabilized in order to promote invasion, egress, and gliding motility in apicomplexan parasites. IMPORTANCE Tissue dissemination and host cell invasion by apicomplexan parasites such as Toxoplasma are pivotal to their pathogenesis. Central to these processes is gliding motility, which is driven by an actomyosin motor, the MyoA glideosome. Others have demonstrated the importance of the MyoA glideosome for parasite motility and virulence in mice. Disruption of its function may therefore have therapeutic potential, and yet a deeper mechanistic understanding of how it works is required. Ca2+-dependent and -independent phosphorylation and the direct binding of Ca2+ to the essential light chain have been implicated in the regulation of MyoA activity. Here we identify a second essential light chain of MyoA and demonstrate the importance of both to Toxoplasma motility. We also investigate the role of Ca2+ and the MyoA regulatory site in parasite motility and identify a potential mechanism whereby binding of a divalent cation to the essential light chains could stabilize the myosin to allow productive movement.

scholarly journals A Focused Small-Molecule Screen Identifies 14 Compounds with Distinct Effects on Toxoplasma gondii

2012 ◽  
Vol 56 (11) ◽  
pp. 5581-5590 ◽  
Author(s):  
Edwin T. Kamau ◽  
Ananth R. Srinivasan ◽  
Mark J. Brown ◽  
Matthew G. Fair ◽  
Erin J. Caraher ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Kinase Inhibitors ◽  
Severe Disease ◽  
Content Type ◽  
Cellular Target ◽  
Small Molecule Screen ◽  
Parasite Motility ◽  
Future Work

ABSTRACTToxoplasma gondiiis a globally ubiquitous pathogen that can cause severe disease in immunocompromised humans and the developing fetus. Given the proven role ofToxoplasma-secreted kinases in the interaction ofToxoplasmawith its host cell, identification of novel kinase inhibitors could precipitate the development of new anti-Toxoplasmadrugs and define new pathways important for parasite survival. We selected a small (n= 527) but diverse set of putative kinase inhibitors and screened them for effects on the growth ofToxoplasmain vitro. We identified and validated 14 noncytotoxic compounds, all of which had 50% effective concentrations in the nanomolar to micromolar range. We further characterized eight of these compounds, four inhibitors and four enhancers, by determining their effects on parasite motility, invasion, and the likely cellular target (parasite or host cell). Only two compounds had an effect on parasite motility and invasion. All the inhibitors appeared to target the parasite, and interestingly, two of the enhancers appeared to rather target the host cell, suggesting modulation of host cell pathways beneficial for parasite growth. For the four inhibitors, we also tested their efficacy in a mouse model, where one compound proved potent. Overall, these 14 compounds represent a new and diverse set of small molecules that are likely targeting distinct parasite and host cell pathways. Future work will aim to characterize their molecular targets in both the host and parasite.

scholarly journals Reassessing the mechanics of parasite motility and host-cell invasion

2016 ◽  
Vol 214 (5) ◽  
pp. 507-515 ◽  
Author(s):  
Isabelle Tardieux ◽  
Jake Baum
Keyword(s):  
Plasma Membrane ◽  
Host Cell ◽  
Cell Invasion ◽  
Motor System ◽  
Current Model ◽  
Cell Movement ◽  
Host Cell Invasion ◽  
Motor Functions ◽  
Apicomplexan Parasites ◽  
Parasite Motility

The capacity to migrate is fundamental to multicellular and single-celled life. Apicomplexan parasites, an ancient protozoan clade that includes malaria parasites (Plasmodium) and Toxoplasma, achieve remarkable speeds of directional cell movement. This rapidity is achieved via a divergent actomyosin motor system, housed within a narrow compartment that lies underneath the length of the parasite plasma membrane. How this motor functions at a mechanistic level during motility and host cell invasion is a matter of debate. Here, we integrate old and new insights toward refining the current model for the function of this motor with the aim of revitalizing interest in the mechanics of how these deadly pathogens move.

scholarly journals Daughter Cell Assembly in the Protozoan ParasiteToxoplasma gondii

2002 ◽  
Vol 13 (2) ◽  
pp. 593-606 ◽  
Author(s):  
Ke Hu ◽  
Tara Mann ◽  
Boris Striepen ◽  
Con J. M. Beckers ◽  
David S. Roos ◽  
...  
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Molecular Genetic ◽  
Inner Membrane ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Ideal System ◽  
Animal Pathogens ◽  
Inner Membrane Complex

The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii.Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites.

scholarly journals C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence

2019 ◽  
Vol 295 (4) ◽  
pp. 1066-1076 ◽  
Author(s):  
Andreia Albuquerque-Wendt ◽  
Damien Jacot ◽  
Nicolas Dos Santos Pacheco ◽  
Carla Seegers ◽  
Patricia Zarnovican ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protective Immunity ◽  
Host Cells ◽  
Apicomplexan Parasites ◽  
Tryptophan Residues ◽  
Parasite Survival ◽  
Parasite Motility ◽  
Parasite Egress ◽  
Thrombospondin Type 1 Repeats

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase–deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

scholarly journals Identification of PhIL1, a Novel Cytoskeletal Protein of the Toxoplasma gondii Pellicle, through Photosensitized Labeling with 5-[125I]Iodonaphthalene-1-Azide

2006 ◽  
Vol 5 (10) ◽  
pp. 1622-1634 ◽  
Author(s):  
Stacey D. Gilk ◽  
Yossef Raviv ◽  
Ke Hu ◽  
John M. Murray ◽  
Con J. M. Beckers ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Protozoan Parasite ◽  
Integral Membrane Protein ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Complex Integral ◽  
Host Cell Recognition

ABSTRACT The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[125I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [125I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

Sign in / Sign up

Export Citation Format

Share Document