Characterisation of the Acto-MyoA motor complex in Toxoplasma gondii

In apicomplexan parasites, the machinery required for gliding motility is located between the plasma membrane and the Inner Membrane Complex (IMC). This type of motility depends on the regulated polymerisation and depolymerisation of actin and a multi-subunit complex, known as the Myosin A motor complex. This complex consists of the myosin heavy chain A (MyoA), the myosin light chain 1 (MLC1), the essential light chain 1 (ELC1) and three gliding-associated proteins (GAP40, GAP45 and GAP50). Gliding motility is thought to be essential for host cell egress and linked to active, parasite driven penetration of the host cell. Many components of this complex are extensively studied using either the ddFKBP system or the tetracycline-inducible knockdown system (Tet-system). Strikingly, while depletion of myoA has no impact on IMC formation, overexpression of the tail domain of MyoA results in a severe IMC biogenesis phenotype. In order to investigate this issue, conditional knockout (KO) mutants of the interacting partners of MyoA-tail were generated using the conditional site-specific DiCre recombination system. Indeed, GAP40 and GAP50 were identified as being essential for parasite replication and having a crucial role during IMC biogenesis. This is the first evidence showing that components of the MyoA motor complex fulfil essential functions during IMC formation and thus are not exclusively important for gliding motility dependant processes. Several components of the MyoA motor complex were characterised using the Tet-system and showed a complete block in gliding motility, but not in host cell invasion. While it is possible that leaky expression of the gene in the knockdown mutants is responsible for this uncoupling of gliding motility and invasion, it remains feasible that different mechanisms are involved in these two processes. In order to shed light on this issue, conditional KOs for the Acto-MyoA motor complex were generated in this study and their functions during gliding dependent processes thoroughly analysed. Intriguingly, while depletion of individual components of this complex caused a severe block in host cell egress, gliding motility and host cell penetration were decreased, but not blocked, demonstrating an important, but not essential role of the Acto-MyoA motor complex during these processes. Altogether, this study raises questions of our current view of what drives gliding motility and invasion and supports the argument for critical revision of the linear motor model

Two essential light chains regulate the MyoA lever arm to promote Toxoplasma gliding motility

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

Gliding motility protein LIMP promotes optimal mosquito midgut traversal and infection by Plasmodium berghei

© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Substrate-dependent gliding motility is key to malaria transmission. It mediates host cell traversal, invasion and infection by Plasmodium and related apicomplexan parasites. The 110 amino acid-long cell surface protein LIMP is essential for P. berghei sporozoites where it is required for the invasion of the mosquito’s salivary glands and the liver cells of the rodent host. Here we define an additional role for LIMP during mosquito invasion by the ookinete. limp mRNA is provided as a translationally repressed mRNP (messenger ribonucleoprotein) by the female gametocyte and the protein translated in the ookinete. Parasites depleted of limp (Δlimp) develop ookinetes with apparent normal morphology and no defect during in vitro gliding motility, and yet display a pronounced reduction in oocyst numbers; compared to wildtype 82 % more Δlimp ookinetes remain within the mosquito blood meal explaining the decrease in oocysts. As in the sporozoite, LIMP exerts a profound role on ookinete infection of the mosquito.This study was supported by Fundação para a Ciência e a Tecnologia grants to GRM (PTDC/BIA-BCM/105610/2008 and PTDC/SAU-MIC/122082/2010) and JMS (SFRH/BD/63849/2009); the National Institutes of Allergy and Infectious Diseases to GRM (1R21AI139579-01A1); the Horizon 2020 Framework Programme Marie Sklodowska-Curie grant agreement No 660211 to SE; as well as a Human Frontier Science Program Grant (RGY/0071/2011) to FF. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. The authors have no competing interests to declare.info:eu-repo/semantics/publishedVersio

Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B

Apicomplexan parasites belong to a recently recognised group of protozoa referred to as Alveolata. These protists contain membranous sacs (alveoli) beneath the plasma membrane, termed the Inner Membrane Complex (IMC) in the case of Apicomplexa. During parasite replication the IMC is formed de novo within the mother cell in a process described as internal budding. We hypothesized that an alveolate specific factor is involved in the specific transport of vesicles from the Golgi to the IMC and identified the small GTPase Rab11B as an alveolate specific Rab-GTPase that localises to the growing end of the IMC during replication of Toxoplasma gondii. Conditional interference with Rab11B function leads to a profound defect in IMC biogenesis, indicating that Rab11B is required for the transport of Golgi derived vesicles to the nascent IMC of the daughter cell. Curiously, a block in IMC biogenesis did not affect formation of sub-pellicular microtubules, indicating that IMC biogenesis and formation of sub-pellicular microtubules is not mechanistically linked. We propose a model where Rab11B specifically transports vesicles derived from the Golgi to the immature IMC of the growing daughter parasites

Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of <i>Toxoplasma gondii</i>

<div><p>The inner membrane complex (IMC) of apicomplexan parasites is a specialised structure localised beneath the parasite’s plasma membrane, and is important for parasite stability and intracellular replication. Furthermore, it serves as an anchor for the myosin A motor complex, termed the glideosome. While the role of this protein complex in parasite motility and host cell invasion has been well described, additional roles during the asexual life cycle are unknown. Here, we demonstrate that core elements of the glideosome, the gliding associated proteins GAP40 and GAP50 as well as members of the GAPM family, have critical roles in the biogenesis of the IMC during intracellular replication. Deletion or disruption of these genes resulted in the rapid collapse of developing parasites after initiation of the cell cycle and led to redistribution of other glideosome components.</p></div

Conditional deletion of <i>gap40</i> or <i>gap50</i> results in altered localisation of components of the glideosome.

<p>(<b>a</b>) Deletion of <i>gap40</i> leads to loss of MyoA staining from the periphery of the vacuole at 18 and 40 h post induction although some MyoA-positive structures were visible within the vacuole (arrows). Using a specific antibody, MLC1 localisation was also significantly affected at both time points tested. GAP45 was still seen at the periphery of vacuoles however abnormal structures were visible and there was no delineation between parasites in the same vacuole. (<b>b</b>) Similar results were observed upon deletion of <i>gap50</i>, with a loss of peripheral MyoA staining and abnormal morphology using MLC1 and GAP45 antibodies. Scale bar 10 μm.</p

Overexpression of MyoA_tail leads to morphological disruption of the parasite’s structure.

<p>MyoA_tail-expressing parasites were treated with 1 μM Shld-1 and fixed after 24 h. <b>(a)</b> Electron microscopy of ddMyc-MyoA_tail untreated (i, iii) or treated with 1 μM Shld-1 (ii, iii) for 24 h. (<b>i</b>) Longitudinal section of a cell undergoing endodyogeny showing the conical shaped IMC (I) of the two daughters enclosing the dividing nucleus (N). (<b>ii</b>) Section showing an enlarged nucleus (N) with the cytoplasm containing partially disorganised plates of IMC (I). (<b>iii</b>) Late stage in endodyogeny showing the folding of the plasmalemma to form the pellicle of the daughters. (<b>iv</b>) Section showing a parasite with multiple nuclei (N) and the cytoplasm contain a number of partially formed daughters. C—conoid; G—Golgi body; I—IMC; R—rhoptries; M—Microneme. Scale bar 1 μm. (<b>b</b>) MyoA_tail-expressing parasites co-expressing mitochondrial marker <i>hsp60-rfp</i> were stained using anti-IMC1. Overexpression of MyoA_tail resulted in an alteration of mitochondrial morphology. (<b>c</b>) The localisation of components of the motor complex was analysed using specific antibodies (MLC1, GAP40, GAP45) or co-expression of YFP-tagged constructs (<i>yfp-gap50</i>, <i>imc1-yfp</i>, <i>yfp-myoA</i>). MyoA_tail expression, as visualised using a specific antibody against the epitope tag Myc, resulted in a severe defect in parasite morphology, however glideosome components remained at the periphery of the mother cell. Scale bar 10 μm.</p

<i>gap40</i> KOi parasites show dissociation between the plasma membrane and the IMC and show reduced ability to resist osmotic shock.

<p>(<b>a</b>) <i>gap40</i> KOi parasites were transiently transfected with <i>imc1-tomato</i>, incubated overnight to allow protein expression then excision was induced. 4 h after the addition of rapamycin, imaging was initiated and images captured every 7 mins, every second image is displayed. YFP signal was observed to appear outwith the IMC boundaries (arrows) followed by loss of the parasite integrity by 42 min (arrowhead). Scale bar 5 μm. (<b>b</b>) At 24 h post induction, parasites were mechanically lysed and treated with either HBSS, HBSS diluted with distilled H₂O (hypoosmotic) or 1 M sorbitol/HBSS (hyperosmotic) as indicated for 5 mins. The percentage of parasites that remained impermeable to PI was then scored and normalized to untreated GFP parasites. <i>P</i> values are shown between the loxP<i>gap40</i> and <i>gap40</i> KOi strains. NS—not significant. Results mean of three independent experiments ± standard deviation.</p

Conditional deletion of <i>gap40</i> and <i>gap50</i> leads to collapse of the IMC.

<p>(<b>a</b>) Growth assays of <i>gap40</i> and <i>gap50</i> KOi parasites. While control parasites showed normal growth behavior after 5 days of incubation, parasites lacking either <i>gap40</i> or <i>gap50</i> displayed no plaque formation on HFF monolayers. Scale bars represent 0.2 mm (upper panels) and 20 μm (lower panels). (<b>b</b>) The area of vacuoles at various time points post induction was quantified. In loxP<i>gap40</i> and loxP<i>gap50</i> parasites, vacuole size increased up to 48 h before reducing due to egress and reinvasion. Both <i>gap40</i> KOi and <i>gap50</i> KOi vacuoles behaved in a similar manner to the controls up to 48 h. However, no parasite egress was seen and vacuoles within host cells were maintained for at least 120 h post induction. Each point represents one vacuole (black for loxP and gray for KO) and results are representative of three independent experiments. Red line indicates mean vacuole area. (<b>c</b>) At 24 h post induction an antibody against IMC1 was used to visualise the IMC. Affected parasites lost peripheral staining and instead sheets of IMC1-positive structures were seen throughout affected vacuoles. Scale bar 10 μm (<b>d</b>) Ultrastructural appearance of WT (i, iv), <i>gap40</i> KOi (ii, v) and <i>gap50</i> KOi (iii, vi) parasites at 18 h (i, ii, iii) and 36 h (iv, v, vi) post induction. Scale bar 1 μm. (<b>i</b>) Longitudinal section through a parasite undergoing endoyogeny showing the conical shaped IMCs (I) of the two daughters partially enclosing the dividing nucleus (N). (<b>ii</b>) and (<b>iii</b>). Sections through the parasites showing the nucleus and areas of disorganised IMC (<b>iv</b>) Section through a parasitophorous vacuole showing a number of daughters forming a rosette. (<b>v</b>) and (<b>vi</b>) Sections through the parasite showing the cytoplasm containing multiple nuclei and area of apical formation consisting of the conoid and associated IMC, rhoptries and micronemes. The IMC appeared to be disorganised and does not form the conical structures associated with daughter formation. N—nucleus, I—IMC, C—conoid, R—rhoptry; M—microneme; D—dense granule G—Golgi body.</p

Disruption of <i>gapm</i> genes using CRISPR-Cas9 leads to collapse of the IMC Parasites were co-transfected with Cas9-GFP and gRNA targeted to indicated genes.

<p>(<b>a</b>) At 48 h post transfection, parasites were fixed and visualized using anti-IMC1 and anti-GAP40 antibodies. Parasites transfected with sgRNA targeting <i>gap40</i> showed collapse of the IMC and an absence of GAP40 staining, while disruption of <i>gap50</i> also resulted in structural collapse, confirming the utility of this technique. Disruption of <i>gapm</i> genes resulted in morphological abnormalities to varying degrees, suggesting that <i>gapm1a</i> and <i>gapm3</i> are essential while disruption of <i>gapm1b</i> had a more subtle effect on parasite morphology. Scale bar 5 μm. (<b>b</b>). Quantification of percentage of vacuoles positive for Cas9-GFP that showed disruption of the IMC at 48 h post transfection. While between 60–70% of GFP-positive vacuoles showed disruption to the IMC upon co-transfection with Cas9-GFP and <i>gap40</i>, <i>gapm1a</i> and <i>gapm3</i>, less than 30% of vacuoles co-transfected with <i>gapm1b</i>, <i>gapm2a</i> and <i>gapm2b</i> demonstrated severe phenotypes. Results average of three independent experiments, performed in duplicate ± standard deviation.</p