The pathogenic bacterium Pseudomonas aeruginosa uses a variety of highly efficient chelating compounds (siderophores) to acquire sufficient iron for growth and virulence. These siderophores can either be endogenous or acquired from exogenous sources such as other bacteria or fungi. The transport of the endogenous siderophore pyoverdine activates a signal-transduction pathway that increases the synthesis of both the ferripyoverdine receptor protein (FpvA) and pyoverdine itself. Signal-transduction systems similar to this have three specific proteins involved: a receptor protein specific for one siderophore in the outer membrane, an anti-sigma factor in the cytoplasmic membrane and a sigma factor that activates gene expression in the cytoplasm.
The aim of the research presented in this thesis was to study the roles of the proteins in three different iron uptake and signalling pathways of P. aeruginosa. The substrates for each receptor protein were confirmed and the roles of each protein in the pathways were compared to the P. aeruginosa pyoverdine signalling pathway. The pyoverdine, desferrioxamine and ferrichrome transport pathways were studied to find whether interactions occur between them and if so, the mechanism(s) for that interaction. Furthermore, a technique for analysing gene expression of P. aeruginosa in sputum from the cystic fibrosis (CF) lung was developed. This technique was subsequently used to study the levels of iron responsive gene expression.
The receptor, sigma factor and anti-sigma factors were all found to have a role in the siderophore-induced expression of their own signalling pathway. The experimental data provide evidence of similarities in the roles of the sigma and receptor proteins within each pathway but different roles for the anti-sigma factors. In the absence of the cognate sigma factor or anti-sigma factor the expression of the desferrioxamine and ferrichrome receptors could not be upregulated. Without its cognate sigma factor fpvA could no longer be upregulated in the presence of pyoverdine. However, unlike the other systems, in the absence of the cognate anti-sigma factor, expression of fpvA was always observed. This is consistent with the anti-sigma factors being required for the activity of the cognate sigma factor in the ferrichrome and desferrioxamine signalling pathways but not the pyoverdine signalling pathway.
The siderophore signalling pathways were found to be upregulated in the presence of multiple siderophores, but generally to a lesser extent than if only one siderophore was available. This suggests that in the presence of multiple siderophores, P. aeruginosa uses all available iron chelators. The study of the role of the receptor, sigma factor and anti-sigma factor into these effects indicate sigma factor competition for RNA polymerase has a major role in the effects of multiple siderophores on pathways upregulation.
The gene expression studies of P. aeruginosa in sputum from the lungs of CF patients provided support for the hypothesis that the bacteria were growing in an environment where iron levels were sufficient for bacterial growth, but not storage of iron. The expression of the sigma factor gene pvdS that is required for pyoverdine synthesis was studied because expression of this gene is a sensitive reporter of intracellular iron levels. It was found to be downregulated in bacteria in sputum compared to laboratory grown bacteria. This result suggests the bacteria are inhabiting a more iron-replete environment within the lung. This finding advances our understanding of the CF lung environment and the impact it has on P. aeruginosa infection. This knowledge has medical implications for the development of novel therapies to combat P. aeruginosa infection.
| Identifer | oai:union.ndltd.org:ADTP/207881 |
| Date | January 2008 |
| Creators | Mettrick, Karla Adelle, n/a |
| Publisher | University of Otago. Department of Biochemistry |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Karla Adelle Mettrick |
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