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

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Discovery and history of Toxoplasma gondii

T. gondii is an obligate intracellular protozoan parasite causing the infectious disease, toxoplasmosis. It was first discovered in 1908 by two independent groups; first, in a hamster-like rodent, Ctenodactylus gundii by Charles Nicolle and Louis Manceaux, and later in a rabbit by Splendor in 1908 (Charles Nicolle & Louis Manceaux, 1908; Splendore, 1908). The name T. gondii was attributed to the isolated protozoan by Nicolle and Manceaux according to its crescent-shaped morphology; Taxon (the Greek word for arc) and plasma for form. Moreover, “gondii” derives from gundii, the organism where it was first isolated (Ferguson, 2009).

Taxonomic classification of Toxoplasma gondii

T. gondii belongs to the family of the Sarcocystidae in the class of coccidia and is the only species in the Toxoplasma genus. Coccidia are obligate, intracellular and cyst forming parasites that infect their host through the gastrointestinal tract.

Toxoplasma gondii lineages

Possibly reflecting the diversity of its natural hosts, multiple genotypes of T. gondii exist worldwide. Yet most isolated parasite strains fall within one of three clonal lineages: type I, II, or III, which are also the most studied in laboratory mice (Khan et al., 2009). These three clonal lineages are characterized by their distinct virulence in mice and their ability to form cysts (Howe and Sibley, 1995). When considering laboratory mice, type I strains kill their host prematurely due to hyper-inflammation and uncontrolled parasite dissemination, and thus fail to establish latent infections. Type I strains are characterized by their high virulence since the inoculation of a single parasite of this genotype is lethal (Boothroyd and Grigg, 2002). By contrast, type II and III strains exhibit a relatively low virulence during the acute phase of the infection and accordingly injection of around 1000 parasites is required to have a lethal effect (Saeij et al., 2005); Type II and III strains also display a slower growth rate compared with type I parasites (Fuentes et al., 2001; Grigg et al., 2001) and have a high cystogenic capacity (Boothroyd and Grigg, 2002; Saeij et al., 2005). Parasitic strains commonly used in laboratories are summarized in Table 2, yet genotyping techniques have identified the presence of isolates, which do not correspond to the three main clonal lines (Dardé, 2008; Khan et al., 2007; Robert-Gangneux and Dardé, 2012).

Toxoplasma gondii life cycle

As with many Apicomplexa, T. gondii has a dual host life cycle, first reported in 1970 (Dubey et al., 1970; Frenkel et al., 1970). The parasite alternates between the sexual reproduction phase which is limited to the intestine of felids, its only definitive hosts; and the asexual 25 replication phase, which occurs in the intermediate hosts, all warm-blooded mammals including Human (Hunter and Sibley, 2012; Montoya and Liesenfeld, 2004; Robert-Gangneux and Dardé, 2012) (Figure 3). Unlike most other apicomplexan parasites, T. gondii does not need to go through its sexual reproduction phase to be transmitted between intermediate hosts (Su et al., 2003).

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
Microtubule network
Conoid
4.3 Intracellular organelles
Micronemes
Rhoptries
Dense granules
5 Toxoplasma gondii lytic cycle:
5.1 Gliding motility and adhesion
The glideosome
Actin dynamics
5.2 Invasion
ROP and GRA proteins implication in PV formation
5.3 Cell cycle and intracellular replication
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
Rab11 regulators
Rab11 in diseases
6.5 FTS/HOOK/FHIP complex
FTS and FHIP
7 Protein trafficking in T. gondii
7.1 T. gondii endo-secretory system
The retrograde pathway in T. gondii:
7.2 Dense granule biogenesis and secretion
7.3 T. gondii Rab11
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
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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