Identification of new proteins and biological processes in the apicomplexan Toxoplasma gondii

Abstract: The apicomplexan protozoan Toxoplasma gondii is a ubiquitous parasite of humans and animals. Entering its host through the digestive system, it possesses the ability to rapidly disseminate in the body and infect a wide range of tissues and cells. Thanks to various surface and secreted proteins with different receptor specificities and a pliable but resistant cytoskeleton, the tachyzoite is able to move, attach and actively penetrate all types of cells. The aim of the work described in this thesis was to investigate the existence of new proteins and processes that mediate or support the interaction between the parasite and its host. During our exploration of novel parasite ligands to ubiquitous host cell receptors, we discovered that the parasites bound and internalized heparin-like glycosaminoglycans in a specific, saturable manner. Discrete binding of the glycan occurred at the anterior third of the tachyzoite, where it was rapidly concentrated and internalized in tubulo-vesicular compartments, with no significant acidification or exocytosis observed. Incubation in continuous presence of the glycan enhanced the binding and internalization of this ligand by live tachyzoites; additionally, two tachyzoite surface polypeptides exhibited strong binding and specificity for heparin, making them candidate receptors for this ligand. Proteomics analysis of surface-associated heparin-binding proteins led to the discovery of two new internal membrane complex-associated proteins, IMC6 and IMC7. Internal membrane complex (IMC) associated proteins are components of the parasite membrane cytoskeleton and play an important role in cell membrane structure and in the physical separation of the newly formed parasites during division. Found in a membrane preparation along with the previously described IMC3 and IMC5 proteins, IMC6 is localized in the parasite pellicle and participates in the composition of the subpellicular network that underlies the IMC. Primary sequence analysis of IMC6 and IMC7 together with all the previously described IMC proteins showed that these proteins were not very homologous to each other, although they shared similar characteristics that put them together in related phylogenetic groups. Topology analysis showed that these proteins did not possess signal peptides or transmembrane domains, even though they were associated to the IMC-membrane structures in immunolocalization experiments and showed partial insolubility during detergent extraction. Additionally, IMC6 was not only found in the subpellicular network, but also labeled the parasite conoid and extended beyond the known distribution of that structure. Although Toxoplasma parasites are exposed to host trypsin proteases during invasion, they are resistant to milligram levels of this enzyme. We describe here the presence of an inhibitor of trypsin, TgTI, encoded by a gene that was found at low abundance as an expressed sequence tag (EST) in both the bradyzoite and tachyzoite stages. The inhibitory binding region of the predicted protein was found to be in the N-terminus of TgTI where aminoacid-alignment demonstrated 75% similarity to earlier described trypsin and tryptase protease inhibitors. In functional analysis, recombinant TgTI-protein inhibited the activity of trypsin approximately 10 times more efficiently than an inhibitor isolated from soybean. The presence of this type of protease inhibitor probably facilitates the survival of the parasite in the upper gastrointestinal tract andlor during attack by immune cells recruited during infection. In conclusion, we describe here the discovery of three new proteins of Toxoplasma gondii as well as a previously undescribed endocytic process that, in different ways, may help parasites during host infection, parasite reproduction and survival in extracellular and intracellular environments.