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Type: Thesis
Title: Biogenesis of Shigella flexneri IcsA protein.
Author: Grabowicz, Marcin
Issue Date: 2010
School/Discipline: School of Molecular and Biomedical Science
Abstract: The IcsA autotransporter is a vital virulence factor for Shigella flexneri, a humanspecific causative agent of bacillary dysentery that accounts for over a million global deaths annually. Ingested Shigellae invade and spread throughout the colonic epithelium. IcsA confers motility to intracellular bacteria by engaging host actin regulatory proteins to polymerise filaments of actin in a processes termed actin-based motility. This IcsA-dependent motility potentiates the intercellular spreading. IcsA is displayed at one pole of the bacterium, thereby providing a functional focus for actin polymerisation that generated propulsive force. This work investigated the biogenesis of IcsA, seeking to identify factors that direct the cytoplasmic deliver of the nascent protein towards the pole. A refined polar targeting region, IcsA₅₃₂₋₅₇₀ has been identified. Additionally, insertion mutant, i532 and i563, within the recognised targeting sequence IcsA₅₀₆₋₆₂₀ that have been identified that are defective, though not entirely deficient, in polar targeting. GFP+ fusions to these mutated targeting sequences revealed rapid motion of fluorescent foci throughout the cytoplasm, a process that likely precedes polar targeting in the wild-type. The delivery of the polarly targeted IcsA₅₀₆₋₆₂₀ region was shown to occur contemporaneously with segregating origins of chromosomal replication (oriC) and likely shares a common cell cycle cue. The diffusive properties of exported IcsA in outer-membrane have also been investigated, addressing whether the protein diffuses in the outer membrane or is masked by LPS. To directly observe the behaviour of IcsA soon after it appears at the cell surface, a strategy exploiting metabolic biotinylation was developed and used to rapidly and specifically label nascent IcsA in the outer membrane. In further investigation of the IcsA-LPS interplay, the profile of polar LPS was shown to be uniform in comparison to the lateral cell body in S. flexneri. Reciprocal co-purification presented biochemical evidence that confirmed IcsAIcsA interactions in the outer-membrane, supporting functional oligomerisation of IcsA in the outer-membrane.
Advisor: Morona, Renato
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2010
Keywords: Shigella; flexneri; IcsA; autotransporter; polarity
Provenance: Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.
Appears in Collections:Research Theses

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