scholarly journals Apicomplexan motility depends on the operation of an endocytic-secretory cycle

2018 ◽  
Author(s):  
Simon Gras ◽  
Elena Jimenez-Ruiz ◽  
Christen M. Klinger ◽  
Leandro Lemgruber ◽  
Markus Meissner
Keyword(s):  
Gliding Motility ◽  
Surface Proteins ◽  
Host Cells ◽  
Membrane Flow ◽  
Secretory Cycle ◽  
Apicomplexan Parasites ◽  
Open Questions ◽  
Attachment Sites ◽  
Nanogold Particles ◽  
Parasite Motility

ABSTRACTApicomplexan parasites invade host cells in an active process, involving their ability to move by gliding motility and invasion. While the acto-myosin-system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions, such as how the force powering motility is generated. In many eukaryotes a secretory-endocytic cycle leads to recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility and generation of retrograde membrane flow. Here we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, since inhibition of endocytosis blocks retrograde flow and motility. We identified lysophosphatidic acid (LPA) as a potent stimulator of endocytosis and demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as nanogold particles. Furthermore, we show that surface proteins of the parasite are recycled during this process. Interestingly, the endocytic and secretory pathways of the parasite converge, and endocytosed material is subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings and we propose a novel model that reconciles parasite motility with observations in other eukaryotes: the fountain-flow-model for apicomplexan parasite motility.

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 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 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 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 A forward genetic screen identifies a negative regulator of rapid Ca2+-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii

2017 ◽  
Vol 292 (18) ◽  
pp. 7662-7674 ◽  
Author(s):  
James M. McCoy ◽  
Rebecca J. Stewart ◽  
Alessandro D. Uboldi ◽  
Dongdi Li ◽  
Jan Schröder ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Genetic Screen ◽  
Gliding Motility ◽  
Negative Regulator ◽  
Host Cells ◽  
Wild Type ◽  
Forward Genetic Screen ◽  
Apicomplexan Parasites ◽  
Dependent Cell ◽  
Dependent Protein

Toxoplasma gondii, like all apicomplexan parasites, uses Ca2+ signaling pathways to activate gliding motility to power tissue dissemination and host cell invasion and egress. A group of “plant-like” Ca2+-dependent protein kinases (CDPKs) transduces cytosolic Ca2+ flux into enzymatic activity, but how they function is poorly understood. To investigate how Ca2+ signaling activates egress through CDPKs, we performed a forward genetic screen to isolate gain-of-function mutants from an egress-deficient cdpk3 knockout strain. We recovered mutants that regained the ability to egress from host cells that harbored mutations in the gene Suppressor of Ca2+-dependent Egress 1 (SCE1). Global phosphoproteomic analysis showed that SCE1 deletion restored many Δcdpk3-dependent phosphorylation events to near wild-type levels. We also show that CDPK3-dependent SCE1 phosphorylation is required to relieve its suppressive activity to potentiate egress. In summary, our work has uncovered a novel component and suppressor of Ca2+-dependent cell egress during Toxoplasma lytic growth.

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 Functional Analysis of Rhomboid Proteases during Toxoplasma Invasion

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Bang Shen ◽  
Jeffrey S. Buguliskis ◽  
Tobie D. Lee ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Gliding Motility ◽  
Surface Proteins ◽  
Host Cell Invasion ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Transmembrane Segments ◽  
Rhomboid Proteases

ABSTRACT Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.

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

2011 ◽  
Vol 11 (3) ◽  
pp. 343-352 ◽  
Author(s):  
Wassim Daher ◽  
Natacha Klages ◽  
Marie-France Carlier ◽  
Dominique Soldati-Favre
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Polymerization ◽  
Life Stage ◽  
Gliding Motility ◽  
Lytic Cycle ◽  
Host Cells ◽  
Content Type ◽  
Parasite Motility ◽  
Actin Nucleator

ABSTRACT Toxoplasma 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.

scholarly journals Malaria parasite LIMP protein regulates sporozoite gliding motility and infectivity in mosquito and mammalian hosts

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jorge M Santos ◽  
Saskia Egarter ◽  
Vanessa Zuzarte-Luís ◽  
Hirdesh Kumar ◽  
Catherine A Moreau ◽  
...  
Keyword(s):  
Malaria Parasite ◽  
Gliding Motility ◽  
Surface Proteins ◽  
Host Cells ◽  
Extracellular Matrices ◽  
Rodent Malaria ◽  
Maternal Mrna ◽  
Endogenous Protein ◽  
Malaria Model ◽  
Mosquito Infection

Gliding motility allows malaria parasites to migrate and invade tissues and cells in different hosts. It requires parasite surface proteins to provide attachment to host cells and extracellular matrices. Here, we identify the Plasmodium protein LIMP (the name refers to a gliding phenotype in the sporozoite arising from epitope tagging of the endogenous protein) as a key regulator for adhesion during gliding motility in the rodent malaria model P. berghei. Transcribed in gametocytes, LIMP is translated in the ookinete from maternal mRNA, and later in the sporozoite. The absence of LIMP reduces initial mosquito infection by 50%, impedes salivary gland invasion 10-fold, and causes a complete absence of liver invasion as mutants fail to attach to host cells. GFP tagging of LIMP caused a limping defect during movement with reduced speed and transient curvature changes of the parasite. LIMP is an essential motility and invasion factor necessary for malaria transmission.

scholarly journals Apical Organelle Discharge by Cryptosporidium parvum Is Temperature, Cytoskeleton, and Intracellular Calcium Dependent and Required for Host Cell Invasion

2004 ◽  
Vol 72 (12) ◽  
pp. 6806-6816 ◽  
Author(s):  
Xian-Ming Chen ◽  
Steven P. O'Hara ◽  
Bing Q. Huang ◽  
Jeremy B. Nelson ◽  
Jim Jung-Ching Lin ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Host Cell ◽  
Cryptosporidium Parvum ◽  
Cell Invasion ◽  
Cytochalasin D ◽  
Gliding Motility ◽  
Host Cells ◽  
Ionophore A23187 ◽  
Host Cell Invasion ◽  
Apicomplexan Parasites

ABSTRACT The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasite's intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.

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