Abstract
The heterotrimeric G-proteins are critically important intracellular signalling molecules that regulate fundamental processes in cellular homeostasis. A unique bacterial toxin, Pasteurella multocida toxin (PMT) modifies 3 of the 4 families of G-proteins (Gq, Gi, and G12) by deamidation which leads to a plethora of downstream signalling. PMT induces drastic morphological changes such as loss of adherence to the matrix, foci and stress fibre formation in Swiss 3T3 and Vero cells. PMT is mitogenic for many cell types but not all, and the work reported in this thesis aims to compare two cell lines that respond differently to PMT.Swiss 3T3 and Vero cells were treated with different concentrations of PMT to determine the effects on cell proliferation, cytoskeletal reorganisation and cell death. PMT induced prominent cytoskeletal changes, mitogenic and anti-cell death responses in Swiss 3T3 cells while delayed cytoskeletal changes with no evidence of mitogenicity and cell death were observed in Vero cells. PMT modified G-proteins at different times in Swiss 3T3 and Vero cells. The PMT-induced anti-cell death response in Swiss 3T3 cells was dose-dependent while the delayed cytoskeletal response in Vero cells was linked to the late PMT-mediated G12 activation. The amino acid sequence of Gα12 differed in the two cell types – the G12 subunit in Vero cells is missing N-terminal cysteine residue, which may have contributed to the differences. Gq/11 signalling is active and sustained in Swiss 3T3 cells, but not in Vero cells. Gβγ may have inhibited adenylyl cyclase activity so it is unknown whether Gi signalling is active and sustained over the 3-day period as the forskolin-stimulated cAMP level decreased in both serum-starved untreated and PMT-treated cell lines. In summary, I have identified changes in both the primary effect of PMT on the two cell types and also on downstream signalling that is likely to reflect the differential cell response.
| Date of Award | 2016 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Alistair Lax (Supervisor) & Agamemnon Grigoriadis (Supervisor) |