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Proteomic and genetic analysis of the antibiotic-induced stress response in Pseudomonas aeruginosa

Reference	BB/C500252/1
Principal Investigator / Supervisor	Dr Martin Welch
Co-Investigators / Co-Supervisors	Professor Kathryn Lilley
Institution	University of Cambridge
Department	Biochemistry
Funding type	Research
Value (£)	279,978
Status	Completed
Type	Research Grant
Start date	01/01/2005
End date	31/12/2008
Duration	48 months

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen that accounts for some 10 per cent of all hospital-acquired infections in the industrialized world. P. aeruginosa infections are difficult to treat because the organism is resistant to many antibiotics, especially when these are applied at conventionally active therapeutic concentrations. However, even when antibiotics are applied at sub-inhibitory concentrations, proteomic analysis reveals that P. aeruginosa can sense their presence and responds by modulating the expression levels of specific proteins. In the current study we aim to investigate these observations in detail and begin to define how antibiotics might modulate gene expression. Planktonic and biofilm cultures of P. aeruginosa will be treated with low (less than MIC) concentrations of a range of structurally diverse translational inhibitors. The effects of this treatment on the soluble proteome will be analysed using 2D-DiGE (difference gel electrophoresis). Modulated proteins will be identified by LC-MS/MS and MASCOT database searching. The hydrophobic integral-membrane proteins (not normally resolved by DiGE) that are antibiotic-modulated will be identified using ICAT (isotope coded affinity tagging). Targeted mutagenesis will then be used to inactivate a selection of genes encoding the modulated proteins. Inactivation will be achieved by insertion of gfp (green fluorescent protein) and lacZ reporter gene cassettes into the coding region of each gene to generate transcriptional fusions. If any of the gene products play a role in helping the cell to survive exposure to the applied antibiotic, then the corresponding mutants should be more sensitive to these agents, and this will be tested. By monitoring expression of the gfp reporter genes we will be able to follow the expression of each tagged gene through the growth curve and in different growth conditions. The lacZ reporter gene fusions will be submitted to transposon mutagenesis and blue-whiteselection will be used to identify some of the regulators potentially involved in controlling gene expression. Proteomics (DiGE and ICAT) will be used to establish the full spectrum of gene products that are affected by the mutation of each potential regulatory gene.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	Proteomics and Cell Function (PCF) [2003-2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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