Manipulation of the host cell response to infection with Toxoplasma gondii

The principal aim of the work presented in this thesis was to investigate the host-pathogen interaction in Toxoplasma gondii infected cells and to elucidate which proteins and pathways were involved in the host response to infection. Toxoplasma gondii is of clinical importance for both human and veterinary disease. There are currently no drugs available to treat the chronic stage of disease. The transcriptional response of host cells to Toxoplasma infection has previously been investigated using microarrays. Proteins are the functional molecules in the cell and the protein expression response is likely to differ from the transcriptional response due to gene expression control and post translational modifications. The advantage of a global proteomic expression profiling study of Toxoplasma gondii infected cells is that, in principle, all possible host responses could be identified without pre-supposing a specific mechanism. Advanced proteomic methods were developed and used to investigate the host protein response to infection with Toxoplasma gondii. A conventional gel-to-gel approach was used in concert with difference gel electrophoresis (DIGE) to investigate the protein expression in infected cells. This part of the work concluded that the patterns of protein expression in infected cells showed clear differences from that of the non-infected cell. This resulted in the unambiguous identification of one hundred and fifty seven host proteins which changed in expression during infection with Toxoplasma gondii. Many proteins not previously implicated in response to infection were identified. The results from the proteomic studies were compared with the transcriptional study in order to determine the gene to protein correlation in Toxoplasma gondii infected cells. A weakly positive correlation between gene and protein expression was determined. To investigate whether the host response is uniquely tailored to an individual pathogen or whether cells have a common response to infection, the protein response to a second intracellular parasite, Leishmania major, was investigated. Far fewer changes were seen in the L.major infected cell proteome, proving that the host response to infection is unique to the pathogen. Phosphorylation is one the most common post translational modifications therefore the host cell phosphoproteome was investigated alongside the steady state proteome of infected cells using three techniques and proteins specifically phosphorylated due to the presence of the parasite were identified. Functional analysis of the modulated proteins revealed extensive modulation of many pathways and functions within the infected host cell including glycolytic, mitotic and structural proteins. Over one third of all the modulated proteins identified were mitochondrial. Overall the findings detailed in this thesis indicate that the host cell has a highly specific response to Toxoplasma gondii infection and that many host cell functions are modulated due to the presence of the parasite and reveal the intimate molecular relationship between host and parasite