International Journal of Antimicrobial Agents 40 (2012) 246–251 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Contents lists available at SciVerse ScienceDirect provided by Elsevier - Publisher Connector International Journal of Antimicrobial Agents j ournal homepage: http://www.elsevier.com/locate/ijantimicag Raloxifene attenuates Pseudomonas aeruginosa pyocyanin production and virulence a,1,2 a,1 a a Shannan J. Ho Sui , Raymond Lo , Aalton R. Fernandes , Mackenzie D.G. Caulfield , c b,d b a a,∗ Joshua A. Lerman , Lei Xie , Philip E. Bourne , David L. Baillie , Fiona S.L. Brinkman a Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada b Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California–San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA c Bioinformatics and Systems Biology Program, University of California–San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA d Department of Computer Science, Hunter College, The City University of New York, New York, NY 10065, USA a r t i c l e i n f o a b s t r a c t Article history: There has been growing interest in disrupting bacterial virulence mechanisms as a form of infectious Received 29 January 2012 disease control through the use of ‘anti-infective’ drugs. Pseudomonas aeruginosa is an opportunistic Accepted 4 May 2012 pathogen noted for its intrinsic antibiotic resistance that causes serious infections requiring new thera- peutic options. In this study, an analysis of the P. aeruginosa PAO1 deduced proteome was performed to Keywords: identify pathogen-associated proteins. A computational screening approach was then used to discover Anti-virulence drug repurposing opportunities, i.e. identifying approved drugs that bind and potentially disrupt the Pseudomonas aeruginosa pathogen-associated protein targets. The selective oestrogen receptor modulator raloxifene, a drug cur- Pyocyanin Raloxifene rently used in the prevention of osteoporosis and/or invasive breast cancer in post-menopausal women, was predicted from this screen to bind P. aeruginosa PhzB2. PhzB2 is involved in production of the blue pigment pyocyanin produced via the phenazine biosynthesis pathway. Pyocyanin is toxic to eukaryotic cells and has been shown to play a role in infection in a mouse model, making it an attractive target for anti-infective drug discovery. Raloxifene was found to strongly attenuate P. aeruginosa virulence in a Caenorhabditis elegans model of infection. Treatment of P. aeruginosa wild-type strains PAO1 and PA14 with raloxifene resulted in a dose-dependent reduction in pyocyanin production in vitro; pyocyanin production and virulence were also reduced for a phzB2 insertion mutant. These results suggest that raloxifene may be suitable for further development as a therapeutic for P. aeruginosa infection and that such already approved drugs may be computationally screened and potentially repurposed as novel anti-infective/anti-virulence agents. © 2012 Elsevier B.V. and the International Society of Chemotherapy. Open access under CC BY-NC-ND license. 1. Introduction antibiotics owing to its impermeable outer membrane, efflux capa- bilities, tendency to colonise surfaces in a biofilm form and ability Pseudomonas aeruginosa is an opportunistic, Gram-negative to acquire and maintain antibiotic plasmids [1]. Novel approaches bacterial pathogen that causes infections in immunocompromised for the treatment of P. aeruginosa infection are urgently needed. hosts, burn victims, individuals in intensive care and patients with The concept of targeting virulence using anti-infective/anti- cystic fibrosis (CF). The lungs of nearly all CF patients are chron- virulence drugs that ‘disarm’ pathogenic bacteria rather than kill ically colonised by P. aeruginosa, which significantly reduces life them has garnered increasing attention in recent years. It is expectancy and is the leading cause of morbidity and mortality believed that such an approach places less selective pressure on for CF patients. The effectiveness of P. aeruginosa as a pathogen bacteria to evolve new strategies for survival, thereby being more can be attributed to its arsenal of virulence mechanisms and its specific to pathogenic bacteria and subject to less selection for large metabolic capacity, including the ability to intrinsically resist drug resistance [2]. To this end, in a previous analysis we iden- tified pathogen-associated proteins having homologues only in pathogenic bacteria and not in non-pathogens [3]. Such proteins are more likely to have virulence-related functions. ∗ Corresponding author. Tel.: +1 778 782 5646; fax: +1 778 782 5583. The list of pathogen-associated proteins identified included E-mail address: [email protected] (F.S.L. Brinkman). 1 components of the phenazine biosynthesis pathway. Strains of P. These two authors contributed equally to this work. 2 aeruginosa produce and secrete a variety of redox-active phenazine Present address: Department of Biostatistics, Harvard School of Public Health, 655 Huntington Ave., Boston, MA 02115, USA. compounds, the most well studied being pyocyanin. Pyocyanin 0924-8579 © 2012 Elsevier B.V. and the International Society of Chemotherapy. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.ijantimicag.2012.05.009 S.J. Ho Sui et al. / International Journal of Antimicrobial Agents 40 (2012) 246–251 247 is responsible for the blue–green colour characteristic of Pseu- elegans pathogenesis model. When P. aeruginosa strain PA14 is domonas spp. and is considered both a virulence factor and a grown on low-salt media, it accumulates in the C. elegans intestine, quorum sensing (QS) signalling molecule for P. aeruginosa [4]. killing the worms relatively slowly over the course of 2–3 days. QS-regulated virulence factors, and pyocyanin in particular, are This infection-like process referred to as ‘slow killing’ differs from active in lung infections associated with CF and produce numer- ‘fast killing’ on high-osmolarity media, which involves the secre- ous effects that are relevant to CF (reviewed in [4,5]). Pyocyanin tion of diffusible toxins and takes place over the course of several modulates redox cycling and generates reactive oxygen species hours [13]. Many P. aeruginosa virulence-related genes required for capable of causing significant oxidative stress, which in turn affects mammalian infection have been shown to play a role in C. elegans calcium homeostasis [4,5]. It inhibits cellular respiration, depletes killing, including proteins that regulate the transcription and export intracellular cAMP and ATP levels, and thus affects chloride ion of virulence factors via the type III secretion machinery as well as channels that are controlled by ATP-driven conformational changes proteins involved in QS [14]. Novel genes not previously known to [5]. Pyocyanin inhibits prostacyclin release and can inactivate be involved in virulence have also been identified in this manner human V-ATPases (involved in receptor-mediated endocytosis), [15]. ␣ 1-protease inhibitor (which modulates serine protease activity, Here we describe our application of drug repurposing method- including neutrophil elastase) and nitric oxide (which influences ology to identify potential anti-infective drug targets and drug blood flow, blood pressure and immune functions) [5]. Pyocyanin leads for P. aeruginosa. The computational screen identified poten- also alters the host immune response in several ways to aid tial interactions between PhzB2 from the phenazine biosynthesis evasion of the immune system and establish chronic infection. pathway and the selective oestrogen receptor modulator ralox- Evidence suggests that pyocyanin could prevent the development ifene. We present evidence that raloxifene attenuates P. aeruginosa of an effective T-cell response against P. aeruginosa and prevents virulence and promotes C. elegans survival. A pyocyanin quantifi- the activation of monocytes and macrophages (through inhibi- cation assay demonstrated that pigment production is reduced in tion of cytokine production) [5]. High concentrations of pyocyanin the presence of raloxifene. These results suggest that raloxifene in the sputum of CF patients suggest that this compound plays could be a novel source of anti-infective agents for the treatment a role in pulmonary tissue damage observed with chronic lung of P. aeruginosa infection and, furthermore, that drug repurpos- infections, and early (in the growth curve) overexpression of QS- ing is a promising, efficient approach for novel antimicrobial drug regulated virulence factors such as LasA, elastase and pyocyanin discovery. is common among populations of a highly successful CF epidemic strain [6]. Additional evidence demonstrates that pyocyanin signif- 2. Materials and methods icantly contributes to lung destruction during chronic P. aeruginosa infection of bronchiectasis airways in a mouse model and sup- 2.1. Database of protein–drug complexed structures ports the hypothesis that pyocyanin contributes to the accelerated decline in lung function of CF and other bronchiectasis patients Drug repurposing uses existing approved drugs for new indi- once they are infected with P. aeruginosa [7]. Pyocyanin biosyn- cations by targeting a new site or sites of interest on a protein thesis is therefore an attractive target for anti-infective drug involved in a different disease. A database of all protein–drug com- intervention. plexed structures was generated by