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Toxoplasma gondii and toxoplasmosis
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the disease toxoplasmosis. The parasite was first identified by Charles Nicolle and Louis Manceaux at the Pasteur Institute in Tunis in 1908. They found the parasite in the tissues of a hamster-like organism called Ctenodactylus gundi. The same year the parasite was also identified by Splendore in Brazil, in the tissues of a rabbit. The word Toxoplasma is derived from the Greek word “toxon” which means “bow”, referring to the crescent shape of the organism (Black and Boothroyd, 2000), and “plasm” meaning form and hence together “Toxoplasma gondii”.
T. gondii life cycle
The parasite life cycle is divided into the sexual and asexual phases that occur in the feline intestine and mammalian/avian populations, respectively. The sexual phase of the parasite life cycle is hosted by the members of the Felidae family, for example domestic cats. The male and female gametes fuse to produce the zygote or oocyst within the intestinal epithelial cells. The unsporulated oocysts when shed by the cat via its faeces reach the environment. Sporogony follows, when the unsporulated oocyst matures under certain environmental conditions to produce infectious oocysts, each of which contain two sporocysts, which in turn contain four sporozoites each (Jackson M.H. and Hutchinson W. M., 2000, Dubey J.P. and Frenkel J.K., 1972). While unsporulated oocysts are resistant to adverse environmental conditions, sporulated oocysts are sensitive (Dubey J.P. et al., 1970, Ito et al 1975). Ingestion of oocysts via contaminated water or food by any warm-blooded animal including livestock and humans leads to the asexual phase of the parasite life cycle. The asexual phase is characterised by two distinct stages of growth, which correspond to the acute and chronic states of infection. During the acute phase following the rupture of the ingested oocysts, the released sporozoites develop into the endozoites or tachyzoites, which represent the rapidly growing form of the parasite. The very special feature of the tachyzoite is its ability to invade nearly all type of cells and even fish and insect cell lines (Werk R. 1985) and to grow within a protective vacuole lined by the parasitophorous vacuolar membrane (PVM). The tachyzoite goes through multiple replication cycles in a process called endodyogeny wherein two daughter parasites bud from within the mother parasite (detailed in chapter 4.3.2) (Ogino and Yoneda, 1966). This unique division process repeats itself several times in a wide range of cell types, producing the heavy parasitemia that is responsible for the various clinical manifestations of the disease. If the parasite is challenged by stress conditions such as the host immune response, the tachyzoites differentiate into latent bradyzoites encapsulated in sturdy cysts. The cysts may remain dormant for the entire life span of the intermediate host or rupture and convert again to the tachyzoite forms to cause disease conditions, upon a lowered immune response as in immune-compromised humans (Blader I.J. et al,. 2015). The cysts are mostly localized in muscular and neural tissues or the eye, but can be also detected in visceral organs such as lungs, liver and kidneys (Dubey J.P., 1993). In the horizontal transmission route, bradyzoite cysts contained in the intermediate hosts (for example, mice) can be consumed by either a definitive or an intermediate host by carnivorism. When bradyzoites are ingested by a definitive host, the asexual phase resumes as the parasite differentiates in accordance to its new host and the sexual life cycle is initiated.
Dissemination and Pathogenesis
During acute T. gondii infection, the ingested cysts or sporulated oocysts that reach the intestinal lumen, rupture to release the bradyzoites and sporozoites, respectively. The parasite then rapidly spreads from the initial site of infection, to the various near and far tissues by crossing different biological barriers such as the intestinal epithelium, the endothelial cells of the blood vessels, and organs such as the brain, eyes and placenta (Dubey J.P. et al., 1997; Harker K.S. et al., 2015). In an in vitro study, the tachyzoites have been reported to migrate across tight junctions of the intestinal epithelial cells by a paracellular route (Barragan A. et al., 2005). In addition, neutrophils harboring live non-replicating parasites were also shown to greatly enhance the spreading of parasites within the intestine and likely beyond (Coombes J.L. et al., 2013). Likewise, dendritic cells (DCs) harboring T. gondii, and particularly the Type II strain, were shown to exhibit elevated levels of migratory capacity and were proposed
to behave like ‘Trojan horses’ to facilitate parasite spreading, primarily to mesenteric lymph nods and the spleen (Lambert H. et al., 2006; Lambert H. et al., 2009). This observation and other studies added further understanding to the initial findings of lymphadenopathy in mice during acute T. gondii infection (Courret N et al., 2006; Zenner L. et al., 1998). Similarly macrophages infected with T. gondii also promote parasite dissemination to distal lymph nodes (Da Gama L.M. et al., 2004). Notably, tachyzoites stimulate macrophages and DCs to produce Interleukin 12 (IL-12), which activates Natural Killer cells (NK cells) and T cells to produce interferon gamma (IFN-γ) (Gazzinelli R.T. et al., 1994) (Figure 3B). IFN-γ together with tumor necrosis factor (TNF) trigger and activate macrophage and pro-inflammatory monocytes to mediate killing of tachyzoites (Sibley L.D. et al., 1991, Butcher and Denkers 2002) (Figure 3B.). In the bloodstream, tachyzoites were observed to preferentially infect monocytes than other leukocytes (Channon J.Y. et al., 2000; Sylveira C et al., 2011). The bloodstream flow helps the parasites carried by the monocytes to reach the vascular endothelium and further spread towards different organs, in particular the brain (Ueno N. et al 2014, Berenreiterova M et al., 2011). The parasite hosted by circulating monocytes is capable of crossing the blood brain barrier (Lachenmaeier S. et al., 2011). The local pro-inflammatory immune response, in particular IFN-γ and TNF, which induce the production of Nitric oxide (NO), triggers the rapid conversion of tachyzoites into latent bradyzoites. Usually asymptomatic in immune-competent individuals, toxoplasmosis can lead to harmful effects on behavior and physiological responses. Reactivation of the brain cysts under immune-supressive conditions can lead to severe toxoplasmic encephalitis (Luft B.J. et al., 1992).
Retinochoroiditis (Butler N.J et al., 2012) is the most common clinical manifestation of T. gondii infection in adults or in new-borns during congenital toxoplasmosis (Ajzenberg D. et al., 2002). Human DCs infected with tachyzoites were found to migrate across the retinal endothelium by molecular interactions with ICAM-1(Intercellular Adhesion Molecule-1), VCAM-1(Vascular Cell Adhesion Molecule) and ALCAM (Activated Leukocyte Cell Adhesion Molecule) (Furtado J.M. et al., 2012).
Table of contents :
Résumé
Summary
Acknowledgements
Scientific output
List of Abbreviations
Chapter I. Introduction
1. The Apicomplexa
2. Toxoplasma gondii
2.1 Classification
2.2 Life cycle
2.3 Toxoplasmosis
2.3.1 Modes of transmission
2.3.2 Dissemination and Pathogenesis
2.3.3 Diagnosis and Treatment
2.3.4 Prophylaxis
3. Parasite Architecture and ultrastructural organization
3.1 Intracellular organelles
3.1.1 The apicoplast
3.1.2 Micronemes
3.1.3 Rhoptries
3.1.4 Dense Granules
3.1.4.1 Dense granule proteins localization and function
3.1.4.2 Dense granule proteins structure and secretion
3.2 The cytoskeleton
3.2.1 The pellicle
3.2.2 The microtubule network
3.2.3 The conoid
4. The Lytic Cycle
4.1Gliding motility and adhesion
4.1.1 Microneme secretion
4.1.2 The glideosome
4.1.3 Actin dynamics
4.2 Invasion
4.2.1 Formation of the Moving Junction
4.2.2 PVM formation
4.3 Intracellular parasite replication
4.3.1 A modified cell cycle
4.3.2 Centrosome initiated division process
4.4 Egress
5. Eukaryotic protein trafficking
5.1 The anterograde trafficking pathway
5.1.1 Vesicle budding at the TGN
5.1.2 Membrane tubulation, scission and transport
5.1.3 Vesicular tethering, docking and fusion
5.2 The Adaptor Protein complexes
5.2.1 AP1
5.3 Epsins
5.4 Rabs
5.5 Rab11
5.5.1 Rab11 and motor proteins
5.5.2 Rab11 regulators
5.5.3 Rab11 in disease
5.6 Hook1
6. Protein trafficking in T.gondii
6.1 The T. gondii plant-like endosomal system
6.2 T.gondii Rab11
6.3 Microneme protein trafficking in the secretory pathway
6.4 Rhoptry protein trafficking in the secretory pathway and the role played by TgAP1
6.5 TgSORTLR a unique receptor for MIC and ROP transport
6.6 Dense granule biogenesis
6.7 Concluding Remarks
7. Objectives
Chapter II Materials and Methods
1. Cell culture
2. Molecular Biology
3. Cell Biology
4. Biochemistry
Chapter III Results
1. Dual role of the Toxoplasma gondii clathrin adaptor AP1 in the differential sorting of rhoptry and microneme proteins and in parasite division
2. Toxoplasma gondii Rab11A regulates surface membrane proteins and dense granule secretion during host invasion and parasite replication
Chapter IV Discussion and Perspectives
Bibliography
