title: A comparison of host-parasite interactions between Toxoplasma gondii and Neospora caninum
Creators: name: Mohammed, Mariwan
Publisher: name: University of Liverpool Repository
Year: 2016

description: Toxoplasma gondii and Neospora caninum are closely related obligatory intracellular apicomplexan parasites that invade and multiply in almost all mammalian host cells. They cause disease in immunocompromised animals, abortion in the intermediates host and great economic losses to the farming industry. However, there are several biological differences between these parasites, including host range, zoonotic capacity, transmission, virulence and definitive host. What causes these biological differences is not well understood. To fully understand these biological differences, the host-parasite interactions of these parasites have been investigated in this study using several different approaches at the molecular level. Due to the importance of protein-protein interactions (PPIs) and multiple protein complexes (MPCs) in host-parasite interactions, blue native polyacrylamide gel electrophoresis (BN-PAGE) combined with liquid chromatography tandem mass spectrometry (LC MS/MS) was used to study T. gondii and N. caninum tachyzoites. Several interesting complexes were identified in N. caninum tachyzoites and include mitochondrial complexes, proteasome, glideosome and moving junction molecules that play an important role in the physiology and invasion of host cells. In addition, in T. gondii the microneme MIC1/6 complex was found migrated and/or co-associated with the important surface antigen glycoprotein SAG1, which is critical in the initial interaction with host surface peptidoglycan. In order to understand the direct interactions between parasite secretory proteins such as dense granule proteins (GRA2 and GRA7) and host cell proteins; a pull-down assay has been used to elucidate the binding partners of expressed recombinant GRA2 and GRA7 in both parasites within the host cell lysate. Several methods were applied to purify the recombinant GRA proteins such as affinity chromatography using nickel or cobalt, salting-out, denaturing buffer using urea and reverse phase HPLC. TgGRA2 was successfully purified by HPLC and attempts have been made to study its role in host-parasite interactions using a pull-down assay. Since these parasites secrete an array of secretory proteins, including kinases, to manipulate host cell responses; phosphopeptide enrichment, combined with LC MS/MS has been used to study the global response in the host signalling pathway through protein phosphorylation and signal transduction in response to infection with T. gondii and N. caninum. Three important differences were identified; about one-third of the phosphoproteomes of the host cell in response to infection by T. gondii and N. caninum was different. Approximately 21 % of the phospho-motifs were found differentially enriched between host cells infected with T. gondii compared to N. caninum infection and finally the pathway analysis showed that a few pathways were differentially enriched between infections with these parasites, such as glycolysis/gluconeogenesis and mTOR signalling pathway in infection with T. gondii Abstract vi but not with N. caninum. The differences in the host cell phosphoproteome indicated that these parasites interact with the host cell differently. As a means of understanding the broader host response to infection with these parasites at the systems biology level, integrated data analyses were performed on quantitative data from the transcriptome, proteome and phosphoproteome of host cells infected with the two parasites. Data analyses showed that host cells produce more proteins in response to infection with T. gondii than with N. caninum after 36 hours post infection (p.i.). In addition, data analysis showed that T. gondii inhibits apoptosis and acute inflammatory responses more when compared to N. caninum. Overall, the results presented in this thesis have provided new insights into the biological differences between T. gondii and N. caninum. Several interesting differences in host-parasite interactions at both the qualitative and quantitative levels were identified. These interactions are related to the virulence and transmission strategy of the parasites and so are potentially associated with the biological differences between these parasites.