Elements that are foreign to the human body, such as bacteria, viruses and fungi, are recognised by cells of the innate immune system. Through a process termed phagocytosis, microorganisms are bound, internalised and destroyed. In this thesis, we focus upon how host cells respond to IgG-opsonised targets, studying both the initial stages of Fc-receptor (FcR) ligation and the later stages of phagocytic cup formation. We provide evidence that after clustering of the receptors, the mobility of diacylated probes such as those found in Src-family of kinases, was reduced. This immobilisation was found to be insensitive to cholesterol depletion, arguing against a role for conventional ‘lipid rafts’ in the initiation of receptor signalling. Furthermore, decreased mobility was only partially dependent upon the presence of actin which could provide a physical restriction. Importantly, inhibiting Src-family kinase activity, completely abrogated immobilisation. These results are highly suggestive of a previously unrecognised mechanism for the initation of FcR signalling.
At later stages, receptor-derived signalling leads to the formation of an actin-rich phagocytic ‘cup’. We found that even before a large particle was fully internalised, actin cleared from the base of the phagocytic cup. This clearance was necessary for the internalisation of large particles, as chemically stabilising actin prior to clearance, abrogated internalisation. Actin clearance was shown to be the indirect result of the localised disappearance of phosphatidylinositol 4,5-bisphosphate and the dephosphorylation of tyrosine-phosphorylated proteins. Strikingly, phosphatidylinositol 3-kinase activity was required for both the protein dephosphorylation and for the phosphatidylinositol 4,5-bisphosphate hydrolysis that was responsible for actin disassembly. We propose that actin disassembly is required to recycle actin to the advancing pseudopods, in order to complete phagocytosis.
For many microorganisms, internalisation through phagocytosis means certain death. Obligate intracellular bacteria, such as Salmonella enterica serovar Typhimurium however, can readily survive inside host cells. This is achieved through modulation of the host-cell signalling pathways that normally lead to microbial destruction. In S. Typhimurium, a needle-like complex, delivers small protein effectors that aid in the survival of the bacterium. We studied one such effector: SigD, that had been suggested to have phosphatidylinositol phosphatase activity. Indeed, we showed that when the cDNA for SigD was exogenously expressed in mammalian cells, phosphatidylinositol 4,5-bisphosphate was depleted and phosphatidylinositol 5-phosphate was formed. We characterised the physiological effects of this 4-phosphatase activity and furthermore, describe the use of SigD as a research tool for modulating host cell phospholipids.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/26372 |
| Date | 23 February 2011 |
| Creators | Mason, David |
| Contributors | Grinstein, Sergio |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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