Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication

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Gliding motility and adhesion

Gliding motility is a critical process for the parasite dissemination and invasion of the host cell. This process can be divided into several steps including the secretion of MIC adhesins and their insertion into the parasite membrane, the anchoring of these adhesins to actin filaments allowing their translocation via the actomyosin motors, and the disassembly of MICs/cellular receptor complex at the basal pole (Blader et al., 2015).

Microneme secretion and parasite adhesion

The first contact between the host cell and the parasite is mediated by adhesins belonging to the family of SAGs (Surface Antigen Glycoproteins) attached to the parasite surface. SAG proteins recognize and bind to cellular receptors of the sulfated proteoglycan type, thus allowing parasite attachment to the host cell (Carruthers and Boothroyd, 2007; He et al., 2002). Following this contact, the parasite secretes the MIC proteins, which once inserted into 56 the plasma membrane; recognize carbohydrates on the host cell surface. The first burst of microneme protein secretion is mediated by the increase of intracellular calcium levels (Lovett et al., 2002) in intracellular parasites. Indeed, a peak of intracellular Ca2+ level was observed during parasite invasion (Arrizabalaga and Boothroyd, 2004) and before egress (Moudy et al., 2001; Withers-Martinez et al., 2014). This increase in the level of intracellular calcium is induced by several stimuli (Wetzel et al., 2004). Actually, Ca2+ level increases during intracellular replication and contributes to the egress of the parasite. It involves the sensing of an increase in phosphatidic acid (PA) generated by a diacylglycerol (DAG) kinase 2 (TgDGK2) secreted into the parasitophorous vacuole (Bisio et al., 2019). Soon after parasite initial contact with the host cell, the level of extracellular potassium increases, and phosphatidylinositol-4,5-biphosphate (PI-(4,5)-P2), which is a substrate of phosphoinositide phospholipase C (PI-PLC), is formed. PI-PLC generates the secondary messengers, DAG and inositol triphosphate (IP3), which all trigger MIC protein secretion (Bullen et al., 2016). More specifically, DAG is converted into PA, which is known to be involved in various cellular processes such as signal transduction or exocytosis in mammals (Chasserot-Golaz et al., 2010). In fact, the study of Bullen et al (Bullen et al., 2016) showed a predominant role of TgDGK1 (diacylglycerol kinase-1) in MIC secretion via the presence of PA. Moreover, IP3 formation stimulates the release of calcium leading to the activation of calcium-dependent protein kinases (CDPKs) which trigger the fusion of microneme organelles with the parasite plasma membrane and thereby the secretion of MIC proteins. This membrane fusion would be initiated by the protein TgAPH (acetylated-pleckstrin-homology domain-containing protein) which can bind the PA present at the parasite membrane. Furthermore, the major roles of TgAPH and TgCDPK1 proteins in MIC secretion have been identified by knock-down (KD) and knock-out (KO) strategies respectively (Bullen et al., 2016; Lourido et al., 2010). After secretion, MIC proteins are found at the apical pole after proteolytic cleavage by ROM proteins to allow, with the glideosome, the reorientation of the parasite necessary for the invasion (Frenal and Soldati-Favre, 2013; Rugarabamu et al., 2015). Among the characterized TgROMs, TgROM4, localized at the plasma membrane, is required for the cleavage of TgMIC2 (Shen et al., 2014b). The absence of TgROM4 induces an accumulation of TgMIC2 over the entire surface of the parasite impairing the establishment of a MIC protein gradient at the apical pole necessary for the reorientation of the parasite and therefore host invasion (Shen et al., 2014b). Several phenotypical characterization experiments have been carried out on different MIC protein depleted strains to decipher their functions. The majority of the MIC examined (TgMIC1, TgMIC2, TgM2AP, TgMIC3, TgMIC4, TgMIC5, TgMIC6, and TgSUB1) are not essential for parasite survival in-vitro. However, the absence of the proteins TgMIC1, TgMIC2 or TgM2AP negatively impact parasite adhesion to host cells (Cérède et al., 2005; Gras et al., 2017; Harper et al., 2006). In vivo virulence experiments in mice carried out for the proteins TgMIC1 and TgMIC3 showed that the absence of TgMIC1 or TgMIC3 mildly affects parasite survival. However, a double KO mutant for both proteins strongly attenuates the virulence of the parasite, revealing a synergistic effect (Cérède et al., 2005).
The gliding motility mechanism is well preserved in Apicomplexa and involves a multiprotein complex called glideosome, dependent on the actomyosin motor located between the IMC and the parasite’s membrane. The actomyosin complex allows the parasite to move across a 2D substrate (Håkansson et al., 1999) and through 3D matrices (Leung et al., 2014). When parasites move over 2D substrates, they display three distinct forms of displacement: circular, helical, and twirling. Circular gliding is when parasites move across the substrate in a circular motion at average speeds of 1.5µm/s. They may also exhibit a helical motion, during which they project forward about one body length over the substratum in a biphasic flip along the longitudinal axis of the parasites. Finally, the parasites can move using a twirling motion, where they appear to spin clockwise while balancing on their basal end (Håkansson et al., 1999). During these three types of motions, the parasites shed their surface membrane, leaving behind a trail of surface antigens (SAG1) along with a variety of other proteins. However, in an extracellular 3D gel matrix, the parasites move exclusively in a spiral corkscrew-like manner (Leung et al., 2014).
The glideosome allows linking the parasite cytoskeleton via anchoring the GAPs to the IMC, and MIC adhesins to the parasite surface. This structure is composed of the protein Myosin A (TgMyoA), the main component of the actomyosin motor, of the proteins TgMLC1 (Myosin Light Chain 1), TgELC1 and TgELC2 (Essential Light Chain 1 and 2) regulating the motor activity (Herm-Götz et al., 2002), and the proteins TgGAP40, TgGAP45, and TgGAP50 (Frénal et al., 2010; Gaskins et al., 2004). The motility of the parasite results from the displacement of TgMyoA on the polymerized actin filaments. TgMLC1 allows the anchoring of TgMyoA at the 58 IMC level through its interaction with TgGAP45 which acts as a junction between the IMC and the parasite membrane (Frénal et al., 2010). In T. gondii, there are two functional protein homologs of the protein TgGAP45. At the apical pole, TgGAP70 also interacts with TgMyoA and TgGAP80 present at the basal pole recruits the myosin TgMyoC (Frénal et al., 2014) (Figure 19). Previously, the connection between TgMIC2 and the actomyosin complex was attributed to the aldolase enzyme (TgALD) due to its ability to bind both the cytoplasmic tail of adhesins (here TgMIC2) and actin. However, a recent study has shown that in the absence of TgALD, the parasite retains its capacity for motility and invasion (Shen and Sibley, 2014). The role of connector would rather be attributed to the protein TgGAC (Glideosome Associated Connector) capable of binding actin, TgMIC2, and PA (Jacot et al., 2016) (Figure 19).

TgAMA1 and TgRON complex to form the MJ

As mentioned earlier, the multistep invasion process involves the formation of a transient structure, the moving junction (MJ). The MJ results from the interaction of a RON protein complex with the microneme protein TgAMA1. More precisely, TgAMA1, which plays a central role in the invasion (Mital et al., 2005), is anchored at the parasite plasma membrane (Donahue et al., 2000; Hehl et al., 2000) and interacts with the RON proteins complex via TgRON2, inserted into the membrane of the host cell. TgAMA1 has long been considered essential for parasite survival since no inducible mutant could be obtained (Mital et al., 2005). However, the development of genetic tools in T. gondii made it possible to generate a KO strain for the TgAMA1 gene (Bargieri et al., 2013). Using this mutant, it has been demonstrated that the RON proteins always localize at the MJ, even in the absence of TgAMA1. This result would, therefore, indicate that TgAMA1 has a role in the attachment stage preceding the invasion and not a role in the formation of the MJ which must require the intervention of another protein. Subsequently, Lamarque et al (Lamarque et al., 2014) showed that in the absence of TgAMA1, the parasite can adapt by overexpressing proteins homologous to TgAMA1 and TgRON2. Thus, the parasite expresses TgAMA2 protein capable of interacting with TgRON2 to allow parasite’s invasion. The double KO of both TgAMA2 and TgAMA1 genes led to a further reduction in the invasion, which however remained partly effective. This result, therefore, suggests the existence of another alternative route to the TgAMA2/TgRON2 pair and led to the finding of another TgAMA1 homolog, TgAMA4. TgAMA4 can bind to the protein TgRON2L1, a homolog of TgRON2 expressed in the sporozoite stage, also expressed in the TgAMA1 KO. Another TgAMA/TgRON complex exists (TgAMA3/TgRON2L1), but is specific for the sporozoite stage. In summary, all these compensation mechanisms highlight the key role of TgAMA/TgRON complexes in the entry process, but also the adaptive capacity of the parasite to ensure its entry into host cells and perpetuate its lytic cycle (Figure 21).

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ROP and GRA proteins implication in PV formation

The PV is a protective structure in which the parasite develops inside the host cell while escaping the immune system. The formation of the PV results from the invagination of the host cell membrane shortly after the MJ formation (Suss-Toby et al., 1996). Once formed, the PV membrane (PVM) undergoes several modifications which allow the exclusion of host transmembrane and lipid raft proteins except for GPi anchor proteins (Charron and Sibley, 2004; Mordue et al., 1999). All these changes allow the PVM to escape the endosomal pathway and thus protect it from lysosomal degradation providing a safe environment for the parasite to proliferate (Mordue et al., 1999). The PV also consists of ROP proteins previously secreted in small vesicles called e-vacuoles in the cytoplasm of the host cell (Håkansson et al., 2001). Among the ROPs secreted, some of them have proven to be crucial for the in-vivo virulence in mice by modulating the host cell immune responses. GRAs secretion is carried out in two stages, firstly, a discharge just after the invasion in the lumen of the nascent vacuole, then a continuous secretion during the development of the parasite within the PV (Carruthers and Sibley, 1997). As mentioned before, these GRA proteins are found associated with several structures such as the PVM, the IVN (intravacuolar network) allowing the connection of the parasite to the PVM (Masatani et al., 2013), and an organelle sequestration structure “HOST” (such as GRA7) involved in the delivery of endolysosomes from the host cell to the PV (Coppens et al., 2006). Moreover, a sub-membrane network is set up thanks to GRA proteins (Lemgruber et al., 2008; Magno et al., 2005a). Thus, the PV will be able to recruit certain host cell organelles such as the ER (Magno et al., 2005b) and the mitochondria (Magno et al., 2005a; Pernas et al., 2014) and constitutes a physical barrier for the passage of host components to the parasite (Gold et al., 2015).

Table of contents :

Résumé
Abstract
Acknowledgments
Scientific Output
Table of content
Table of Figures
List of Tables
List of Abbreviations
Chapter I – Introduction
1 The Apicomplexa
2 Toxoplasma gondii
2.1 Discovery and history of Toxoplasma gondii
2.2 Taxonomic classification of Toxoplasma gondii
2.3 Toxoplasma gondii lineages
2.4 Toxoplasma gondii life cycle
2.5 Tachyzoite to bradyzoite differentiation
3 Toxoplasmosis
3.1 Modes of transmission to Humans
3.2 Pathogenesis
3.3 Diagnosis
3.4 Treatments and Vaccination
3.5 Prophylaxis
3.6 Immunity against toxoplasmosis
4 Tachyzoite architecture and ultrastructural organization:
4.1 Pellicle
4.2 Cortical cytoskeleton
4.2.1.Microtubule network
4.2.2 Conoid
4.3 Intracellular organelles
4.3.1 Micronemes
4.3.2 Rhoptries
4.3.3 Dense granules
5 Toxoplasma gondii lytic cycle:
5.1 Gliding motility and adhesion
5.1.1 The glideosome
5.1.2 Actin dynamics
5.2 Invasion
5.2.1 ROP and GRA proteins implication in PV formation
5.3 Cell cycle and intracellular replication
5.3.1 Cellular division and daughter cell formation
5.4 Egress
6 Regulation of protein trafficking:
6.1 The Anterograde/Secretory pathway
6.2 The Retrograde/Recycling pathway
6.3 Rab GTPases
6.4 Rab11
6.4.1 Rab11 regulators
6.4.2 Rab11 in diseases
6.5 FTS/HOOK/FHIP complex
6.5.1 FTS and FHIP
7 Protein trafficking in T. gondii
7.1 T. gondii endo-secretory system
7.1.1 The retrograde pathway in T. gondii:
7.2 Dense granule biogenesis and secretion
7.3 T. gondii Rab11
7.4 Objectives
Chapter II – Materials and Methods
1 Cell culture
1.1 Culture maintenance and growth of host cells and parasites:
2 Molecular Biology:
2.1 Genomic parasite DNA extraction
2.2 List of primers generated by our lab and used in our study
2.3 Cloning methods
2.4 Schemes describing the different molecular cloning strategies used in our project
2.5 Parasite transfection
2.6 Drug selection and cloning of transgenic parasites
3 Cell biology:
3.1 Immunofluorescence assays (IFA)
3.2 Plaque Assay
3.3 Parasite intracellular growth assay
3.4 Invasion assay
3.5 Attachment assay:
3.6 Motility (Trail deposition) assay
3.7 Conoid extrusion assay
3.8 Conoid extraction assay
3.9 Excreted secreted antigens assay
3.10 In vivo virulence test
3.11 Statistics
4 Microscopy
4.1 Transmission electron microscopy (TEM)
4.2 Scanning Electron microscopy (SEM)
4.3 Videomicroscopy
4.4 Automatic tracking and vesicle co-distribution using the Imaris software
4.5 Manual tracking and mathematical modeling with MATLAB
5 Biochemistry
5.1 Total protein extract and Western Blot:
5.2 Immunoprecipitation
5.3 GST pull-down
Chapter III – Results
1 Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication
1.1 TgRab11A localizes to dynamic cytoplasmic vesicles
1.2 TgRab11A-positive vesicles dynamically co-distribute with DGs
1.3 TgRab11A promotes DG exocytosis
1.4 TgRab11A regulates transmembrane protein localization at the PM
1.5 TgRab11A regulates adhesion and motility of extracellular parasites
1.6 TgRab11A-positive vesicles accumulate at the apical pole during parasite motility and host cell invasion
1.7 TgRab11A regulates polarized secretion of DG content during parasite motility and host cell invasion
2 Implication of the Toxoplasma gondii HOOK-FTS-HIP complex in microneme secretion 
2.1 The adaptor molecule TgHOOK, a novel partner of TgRab11A
2.2 TgHOOK localizes at the apical pole in T. gondii
2.3 TgHOOK contributes to parasite motility and host cell adhesion, and modestly to invasion and egress
2.4 Identification of TgHOOK associated proteins, TgFTS and TgHIP
2.5 TgFTS and TgHIP accumulate at the apical tip of intracellular replicating and extracellular parasites
2.6 TgFTS and TgHOOK interact together; and HOOK depletion leads to FTS degradation
2.7 TgFTS and TgHIP promote microneme proteins secretion
Chapter IV – Discussion and Perspectives
1 TgHOOK interacts with TgFTS and HIP to form a stable HFH complex implicated in the process of microneme secretion
2 Topology of the TgHFH complex
3 TgHOOK interacts with TgRab11A to regulate different vesicle trafficking processes
Bibliography

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