Phytochrome-based fluorophores: sensitive tools for heme detection and in vivo imaging in bacterial pathogens

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Title: Phytochrome-based fluorophores: sensitive tools for heme detection and in vivo imaging in bacterial pathogens
Authors: Mullineaux Sanders, Caroline Elizabeth
Item Type: Thesis or dissertation
Abstract: The acquisition of iron is crucial for bacterial pathogenesis. In this thesis I describe the development and validation of a biosensor for heme in bacteria, an important host source of iron for human pathogens. I demonstrate that the co-expression of a phytochrome-based fluorophore (PBF), which utilises the heme-derived metabolite biliverdin IXα as a chromophore, with a heme oxygenase (HO) in Gram-negative bacteria results in heme-dependent near infra-red fluorescence. Using this PBF+HO reporter, I probe heme metabolism in the opportunistic human pathogen Pseudomonas aeruginosa and for the first time provide direct evidence linking the previously putative heme receptor HxuC to heme metabolism. Further, coupling of the heme biosensor to transposon mutagenesis and Fluorescence-Activated Cell Sorting was used to screen a P. aeruginosa transposon library leading to the identification of several putative novel components of heme metabolism. Finally, I demonstrate the potential of our fluorescent biosensor to be used to study heme metabolism and host-pathogen interactions in vivo using a live adult zebrafish model of infection. The PBF+HO heme biosensor allows the highthroughput, non-invasive study of bacterial heme metabolism in real-time and proves a useful tool for gaining further insight into iron-heme metabolism in bacterial pathogens.
Content Version: Open Access
Issue Date: Sep-2016
Date Awarded: Feb-2017
Supervisor: Ulijasz, Andrew
Holden, David
Sponsor/Funder: Imperial College London
Department: Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses

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