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Investigating the difference between a Pseudomonas aeruginosa planktonic cell, a colony and a biofilm.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes chronic infections in immunocompromised individuals. These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms.

The three most common ways of growing bacteria in vitro are as planktonic cultures, colonies on agar plates, and biofilms in continuous-flow systems. Biofilms are known to express genes different from those of planktonic cells, and biofilm cells are generally believed to closely resemble planktonic cells in stationary phase. However, few, if any, studies have examined global gene expression in colonies.

We used a proteomic approach to investigate the interrelationships between planktonic cells, colonies, and biofilms under comparable conditions. Our results show that protein profiles in colonies resemble those of planktonic cells. Furthermore, contrary to what has been reported previously, the protein profiles of biofilms were found to more closely resemble those of exponentially growing planktonic cells than those of planktonic cells in the stationary phase. These findings raise some intriguing questions about the true nature of biofilms.

Further to this, transcriptomic analysis is being carried out.

Investigating the factors secreted by a Pseudomonas aeruginosa biofilm.

Investigating the response and resistance mechanisms of Pseudomonas aeruginosa to Ciprofloxacin treatment.

Instances of bacterial resistance to the billion-dollar drug ciprofloxacin are still on the increase. Much transcriptomic data has been collected on the bacterial response to ciprofloxacin treatment. We have complemented this research with proteomic data using 2-Dimensional Fluorescence Difference in-Gel Electrophoresis (2D-DiGE) to investigate the response of Pseudomonas aeruginosa to the presence of ciprofloxacin. Treatment with two different sub-inhibitory concentrations results in a concentration-dependent increase in the number of proteins modulated, as identified using multivariate statistical analysis. Additionally we have characterized the proteomes of two clinically relevant spontaneous ciprofloxacin-resistance phenotypes: drug target site alteration and multi-drug efflux pump upregulation. This combined approach provides insight into the mechanisms by which P. aeruginosa responds to the drug and initializes such survival and resistance phenotypes.

Subsequent work is now focussed on the permeability of the bacterial membrane and how this is altered in multidrug efflux mutants

Investigating the response of Pseudomonas aeruginosa to Aminoglycoside treatment.

Pseudomonas aeruginosa is an opportunistic human pathogen. Serious infections are often treated with aminoglycosides, which act by inhibiting prokaryotic ribosome function. We have investigated the responses of P. aeruginosa to low levels of tobramycin and gentamicin using 2D-DIGE.

In addition to changes in translation-associated factors and central-intermediary metabolism, our results showed a global down-regulation of proteins involved in iron uptake and metabolism. Furthermore, the siderophore concentration in the culture supernatants was decreased in response to both tobramycin and gentamicin. Interestingly, the same effect was observed in the presence of the structurally unrelated antibiotics, ciprofloxacin and azithromycin, but not in the presence of ethidium bromide. Aminoglycoside antibiotics are known to cause oxidative stress in living cells. Moreover, Fe(II) catalyses the formation of oxygen radicals in the presence of hydrogen peroxide. Decreasing intracellular iron may therefore be a means of reducing oxidative stress in the cells in the presence of aminoglycosides.

Investigating the response of Pseudomonas aeruginosa to Azithromycin treatment and how it affects quorum sensing regulation.

The macrolide antibiotic, azithromycin (AZM), has been reported to improve the clinical outcome of cystic fibrosis patients with chronic Pseudomonas aeruginosa infections. However, the highest clinically-achievable concentrations of this drug are well below its minimal inhibitory concentration for this organism, raising the question of why this drug exhibits therapeutic activity. One possibility is that AZM inhibits quorum sensing by P. aeruginosa, thereby reducing virulence factor production and biofilm formation.

We have tested this hypothesis using quantitative 2D-difference gel electrophoresis and 1H-NMR metabolomic analysis to examine whether a range of clinically-relevant AZM concentrations elicited changes similar to those seen in defined quorum sensing mutants that had been cultured in identical conditions. Our data show that AZM does not have a major effect on the global quorum sensing regulon in these growth conditions, even though it does influence certain phenotypes that are known to be under quorum sensing control. Instead, we show that AZM affects a small quorum sensing sub-regulon. Furthermore, multivariate analyses of proteomic and metabolomic data show that AZM-treated samples do not cluster with any of the quorum sensing mutants and a number of quorum sensing-independent effects of AZM have been identified.

Our research has now moved into elucidating the cellular targets of azithromycin and the mechanism by which it has it's bacteriostatic effects.

Quorum sensing and metabolism in Pseudomonas aeruginosa.

Quorum sensing (QS) is a key virulence factor in P. aeruginosa. QS signalling systems control the expression of genes - including toxins - in response to population density. This is thought to allow P. aeruginosa to minimise immune responses until it has amassed sufficient population density to can overwhelm host defenses, by rapid upregulation of other virulence factors, including toxins and degradative enzymes.

This model implies a highly sigmoidal QS output function, that effectively acts like a binary switch, i.e. there is zero QS output below a threshold population density and maximal output above this threshold. However, it has become clear that QS regulatory activity varies across a range of population density. We are working to improve our understanding of how the bacteria's metabolic state changes along this continuum of QS activity.

In addition to fascinating biology, this is of medical significance: changes in bacterial metabolism as P. aeruginosa transitions from commensal to pathogen are likely to affect the efficacy of drug therapy and could guide the development of new treatment.

Investigating the production and function of Rhamnolipids by Pseudomonas aeruginosa.

Quantitative Proteomics for the profiling of Pseudomonas aeruginosa Mex Pumps.

Proteomic approaches now seek to reflect quantitative changes to a sub-proteome following a cellular perturbation, however Systems Biology often requires the stoichiometry of these changes to be reported. For multiplexed absolute protein quantification (molecules/cell) we monitor and quantify signals from peptide surrogates then interpolate their concentration from isotopically distinct internal standards of known concentration. To affordably multiplex this approach internal standards are concatenated and expressed as a protein which upon digestion liberates these peptide standards at stoichiometric unity. Currently, accurate absolute protein quantification assumes two components: firstly the peptide surrogate can accurately be distinguished and quantified, the second assumes the peptide an accurate surrogate for the protein.

An aspect of my work seeks to establish if current methodologies meet these assumptions. Also of keen interest to me is to reduce redundancy in data acquisition such that many protein species can be quantified within a short time scale. Such developments would make this approach a viable alternative to current diagnostic assays. I have designed internal standards against a family of efflux pumps encoded within P. aeruginosa genome. Appling these techniques will enable a family wide absolute expression analysis of these pumps following antibiotic or other stimuli.

Investigating the anaerobic stress response in Erwinia carotovora.

Phage Display to identify novel AHL-degrading catalytic antibodies.

"Quorum quenching" enzymes are enzymes that are capable of breaking down bacterial quorum sensing molecules. As quorum sensing is extremely important in the mechanisms of virulence and biofilm formation in Pseudomonas aeruginosa, the enzymes that can interrupt this cell-cell communication are of great interest. Using phage display, libraries of antibody fragments will be screened for affinity to quorum sensing molecules and subsequently for "quorum quenching" activity.