Identification of compounds affecting Pseudomonas aeruginosa social behaviour and virulence

Abstract

Pseudomonas aeruginosa is the most common Gram-negative bacterium responsible for hospital-acquired infections and is a serious threat to immune compromised individuals. P. aeruginosa infections are difficult to eradicate because this pathogen is resistant to conventional antibiotic therapies. Resistance to antibiotics is mainly due to the ability of this microorganism to form biofilm and to express efflux pumps that reduce the intracellular concentration of the drug. Targeting the bacterial pathogenic potential rather than bacterial growth has the advantage of reducing the bacterial adaptability to the host environment and the severity of the infection without creating the selective pressure generally caused by conventional antibiotics. The use of virulence inhibitors could ultimately provide the host immune system with a better chance of clearing the infection. The overall rationale and aim of this project has been to pave the way for the development of innovative anti-virulence therapies specifically targeting P. aeruginosa. The objective of the study described in Chapter 3 has been the identification of compounds inhibiting P. aeruginosa quorum sensing (QS), a cell-cell communication process playing a key role in the expression of virulence factors and biofilm development. It is well known that searching for new side activities in drugs which use in humans has already been approved is an intelligent strategy for the development of novel drugs. This strategy is expected to reduce the time and cost associated with standard drug discovery processes. About thousand compounds already used as drugs in humans were screened for their anti-QS activity, using a biosensor developed in our laboratory. Seven compounds with anti-QS activity were identified; the most effective drug was the anthelmintic drug niclosamide. Microarray analysis showed that niclosamide (at 20 μM) affects the transcription of 258 genes, with a high degree of target specificity towards the QS-dependent genes. 69 genes were up-regulated and 189 genes were down-regulated, 121 of the latter group were previously included within the QS regulon. Phenotypic assays demonstrated that niclosamide suppresses surface motility, production of secreted virulence factors (elastase, pyocyanin and rhamnolipids) and reduces biofilm formation. In accordance with the strong anti-virulence activity disclosed in vitro, niclosamide prevented P. aeruginosa pathogenicity in the Galleria mellonella insect model of acute infection. To our knowledge, this is the first study in which a drug-repurposing strategy has been applied to the development of P. aeruginosa antivirulence drugs. Besides the finding that niclosamide, an FDA-approved drug has a promising anti-virulence activity against one of the most antibiotic-resistant bacterial pathogens, this work provides a proof of concept that a lateral anti-QS activity can be detected among drugs already used in humans, validating a new approach to identify virulence inhibitors that could easily move into clinical applications. Chapter four of this thesis describes a study aimed at investigating the inhibition of P. aeruginosa virulence via chemical inactivation of Resistance-Nodulation-Cell Division (RND) efflux pumps. Efflux pumps of this family are important for multiple drug-resistance in many pathogenic bacteria, including P. aeruginosa. Since no close human homologues of RND transporters have been described, efflux pumps inhibitors (EPI) are considered promising scaffolds and lead compounds for the development of drugs aimed at potentiating antibiotic activity. Since in some bacterial pathogens RND efflux pumps are also involved in virulence, EPIs have been proposed as virulence inhibitors. However, no literature data are available concerning the potential antivirulence activity of EPIs. The main aim of this study has been to provide a first proof of concept that, apart from their role in antibiotic resistance, EPIs may act as antivirulence drugs against P. aeruginosa, by using phenyl-arginine β- naphthylamide hydrochloride (PAβN) as model compound. Here, we demonstrate that PAβN 6.5 μM abrogates swarming motility, an in vitro phenotype strictly related to virulence in vivo. Moreover, microarray analysis showed that PAβN affects the transcription of about 109 genes. P. aeruginosa transcriptome is affected by PAβN in a specific way, since it negatively affects the expression of particular groups of genes, mainly related to iron and phosphate acquisition, while it increases the expression of genes involved in nitrogen metabolism. Overall, our results demonstrate that in P. aeruginosa PAβN has pleiotropic effects that go far beyond the increased susceptibility to antibiotics. In particular, PAβN decreases the transcription of virulence related genes, resulting in a reduced pathogenic potential in the G. mellonella infection model system. In conclusion, the use of EPIs in therapy is a promising challenge since these compounds, besides their effect as antibiotic adjuvants, may display anti-virulence properties.