NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. ----- Toxoplasma gondii and N. caninum are closely related and similar in many aspects. They are closely related phylogenetically, have similar structure and nearly identical morphology, comparable antigenic characteristics, and they both infect a wide range of vertebrate hosts. However, they are biologically distinct as separate species as they employ different definitive hosts (dogs for N. caninum, cats for T. gondii), and they differ in many other features such as surface carbohydrate composition, antigenicity, and some ultrastructural features.
The aim of this thesis is to investigate the hidden causes of these similarities and differences between these two parasites focusing on some basic cell biology aspects. The thesis begins with an examination of the surface protein composition of the two parasites. Using methods described previously for the purification of pellicle and plasmalemma fractions from T. gondii, I evaluated the same methodology for the preparation of pellicles and plasmalemma for N. caninum. The approach used involved subcellular fractionation and sucrose gradient centrifugation to prepare fractions containing pellicles. Plasmalemma was prepared by extraction of this fraction with a high salt glycerol treatment. Fractions containing membrane structures were identified by electron microscopy, and SDS-PAGE and Western blotting subsequently studied the proteins and antigens present in them. Electron microscopy of the pellicle fractions of N. caninum demonstrated a preservation of the triple-membrane structure which is identical to that found in T. gondii. SDS-PAGE of the pellicle fractions revealed it contained several major proteins. Analyses revealed that the plasmalemma of N. caninum contained two abundant proteins in addition to other much lower abundance antigens detectable by monoclonal antibodies. These studies therefore report for the first time, a detailed molecular characterisation of the pellicle and plasmalemma of N. caninum.
The second part of the dissertation compares attachment and invasion of T. gondii and N. caninum to a cat and a dog fibroblast cell line and two epithelial cell lines (a cat kidney and Vero) using a fluorescence antibody methodology. In addition, trypsin treatment of tachyzoites was used to determine whether protein molecules were essential to the process of invasion. The results show that both T. gondii and N. caninum invaded all four cell lines, and that pre-treatment of T. gondii tachyzoites with trypsin caused an increase in the ability of the parasite to invade these host cells. Furthermore T. gondii, in comparison to N. caninum, invaded all four-cell lines at greater levels. The results here support the conclusion that both T. gondii and N. caninum have the ability to invade a variety of cell types including both dog and cat cells, and questions the utility of Vero cells as an appropriate host cell for in vitro studies on the biology of these taxa.
The thesis continues exploring the contribution of the host cell membrane to the attachment and invasion process and parasitophorous vacuole (PV) formation. In order to determine whether host cell membrane proteins are internalised into the PV membrane or onto the parasite surface during the host-parasite interaction process, the host cell surface proteins were labelled with fluorescent probes DTAF or, biotin NHS before their interaction with tachyzoites. The fate of the labelled host cell proteins were followed and analysed by immunofluorescence, confocal laser scanning microscopy, Western blotting and immunoelectron microscopy. Following attachment and invasion of the parasites into Vero cells, intense labelling of the parasite surface was observed, demonstrating transfer of proteins from the Vero cell surface to the parasite surface. Subsequent labelling of the membrane of the parasitophorous vacuole containing parasites was also observed. In contrast, in dog and cat fibroblast cells fluorescence was absent in the parasitophorous vacuole with no apparent transfer occurring to the tachyzoites. These results show for the first time that T. gondii and N. caninum employ different mechanisms while invading different host cells. The results demonstrate for the first time that during host cell invasion to N. caninum host cell surface proteins are internalised into the PV membrane.
Finally autofluorescence of bradyzoites and tissue cysts of the two species were studied by fluorescence microscopy during their differentiation from tachyzoites to bradyzoites in vitro under alkaline conditions. Stage conversion into bradyzoites and cysts was confirmed by immunofluorescence microscopy and Western blot analysis using SAG1- and BAG 1-specific antibody respectively. From day 4 p.i, pale blue auto fluorescence of the bradyzoites and cysts was observed with UV light, coinciding with the onset of cyst development. This autofluorescence under UV light of bradyzoites and cysts increased in intensity from day 8 to day 10 p.i. In contrast, to the autofluorescence shown by bradyzoites and cysts, tachyzoites and PV containing tachyzoites never showed autofluorescence at any time examined. The autofluorescence of the cystic stages was of sufficient intensity and duration to allow the detection of cysts and bradyzoites of T. gondii and N. caninum.
In summary, the findings presented in this thesis support the view that although T. gondii and N. caninum are closely related and share many common features (autofluorescence under UV light, attachment and invasion of host cells involving surface membrane proteins, PV formation), T. gondii and N. caninum are different in the mechanisms they use for these processes. This evidence provides support for the argument that the evolution of N. caninum and T. gondii as species from a common ancestor has involved fine adjustments to the cell biology of these organisms.