1 Bioscience and Technology, Department of Systems Biology, Technical University of Denmark2 Department of Systems Biology, Technical University of Denmark3 Department of Microbiology, Technical University of Denmark
Pseudomonas aeruginosa is an increasingly prevalent opportunistic pathogen which causes chronic pneumonia in cystic fibrosis patients and severe life-threatening infections in immunocompromised persons. This pathogen produces a range of malicious virulence factors such as toxins, tissue degrading enzymes and components capable of impairing the hosts’ immunity. P. aeruginosa readily assumes the biofilm lifestyle which confers efficient protection against the activity of the host defence system. In addition, P. aeruginosa exhibit an inherent tolerance to many of the antibiotics most commonly used, which emphasises the urgent need for development of novel strategies that will help us to defeat this pathogen. P. aeruginosa biofilm cells display a multicellular-like coordinated behaviour and control expression of virulence factors, elements involved in biofilm development and immunomodulating factors by means of signal molecule mediated communication, known as quorum sensing. This thesis explores a strategy which aims to counteract P. aeruginosa virulence and pathogenicity by impeding its cell-to-cell communication. A treatment regime, which focuses on targeting bacterial communication instead of growth, has the immediate advantage that it does not impose a harsh selection pressure for resistance which is the main drawback of conventional antibiotics. The first two articles included in the present thesis (article 1 and 2) describe the screening of extracts of eukaryotic organisms (microfungi and marine invertebrates) for the presence of quorum sensing antagonists. As revealed in these two articles, production of compounds capable of interfering with bacterial communication seems to be common among eukaryotes, supporting the idea that the ability interact with coordinated bacterial behaviour provides higher organisms with an evolutionary advantage. Interestingly, quorum sensing inhibitors such as patulin and penicillic acid (article 1) and the three manoalide compounds (article 2) share the 2(5H)-furanone moiety. Since these furanones are found in extracts of diverse groups of organisms, it suggests that the ability to produce them either has evolved in parallel in different organisms or that the ability to produce such compounds is a reminiscence of an ancient shared ancestor (of a range of marine invertebrates and microfungi), the trait of which has been maintained through times by selective forces. Most of the antibiotics used today originate from natural sources. The general assumption is that antibiotics have evolved in nature to counteract growth of competitors; however, this idea has recently been challenged, and it has been suggested that the ecological role of antibiotics is as communication messengers instead of defeating competitors. Article 3 included in this thesis investigates the application of low concentrations of natural as well as synthetic antibiotics as inhibitors of bacterial communication and in turn production of virulence factors. Three renowned antibiotics which are frequently used in the clinic to treat P. aeruginosa infections were found to decrease the expression and production of a range of quorum sensing regulated virulence factors when administered in subminimum inhibitory concentrations. This suggest that antibiotics may display dual activities; as bactericidal or bacteriostatic agents and as antagonists of bacterial communication. Taking this into account, it is fair to assume that we have not even fully explored and exploited the potential of the drugs we know today. As described in the final manuscript (article 4), P. aeruginosa also produces multifunctional compounds; two quorum sensing signal molecules; the Pseudomonas Quinolone Signal and N-3-oxododecanoyl L-homoserine lactone exhibit the ability to modulate activities of the immune defence in addition to functioning as quorum sensing mediators. The two signal molecules impair activities of immune cells crucial for the pro-inflammatory and antibacterial responses of the host, and this phenomenon may contribute to the successful establishment of P. aeruginosa in its host. The key to combat this bacterium may lay in the development of a novel chemotherapy which aims at impeding the coordinated behaviour of this pathogen, its production of virulence factors and its ability to alter the response of the immune defence. This key probably already exists and may be found among eukaryotic organisms which have evolved to produce quorum sensing antagonists.