Quorum quenching quandary: resistance to antivirulence compounds

The ISME Journal (2012) 6, 493–501 & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12 www.nature.com/ismej ORIGINAL ARTICLE Quorum quenching quandary: resistance to antivirulence compounds Toshinari Maeda1,2,8, Rodolfo Garcı́a-Contreras3,4,8, Mingming Pu1,8, Lili Sheng1,5, Luis Rene Garcia6, Maria Tomás7 and Thomas K Wood1,6 1 Department of Chemical Engineering, Texas A&M University, College Station, TX, USA; 2Department of Biological Functions and Engineering, Kyushu Institute of Technology, Kitakyushu, Japan; 3Department of Biochemistry, National Institute of Cardiology, México, Mexico; 4Department of Molecular Cell Physiology, VU University, Amsterdam, The Netherlands; 5State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China; 6Department of Biology, Texas A&M University, College Station, TX, USA and 7Unidad Investigación-Microbiologı´a, CH Universitario A Coruña INIBIC, La Coruña, Spain Quorum sensing (QS) is the regulation of gene expression in response to the concentration of small signal molecules, and its inactivation has been suggested to have great potential to attenuate microbial virulence. It is assumed that unlike antimicrobials, inhibition of QS should cause less Darwinian selection pressure for bacterial resistance. Using the opportunistic pathogen Pseudomonas aeruginosa, we demonstrate here that bacterial resistance arises rapidly to the bestcharacterized compound that inhibits QS (brominated furanone C-30) due to mutations that increase the efflux of C-30. Critically, the C-30-resistant mutant mexR was more pathogenic to Caenorhabditis elegans in the presence of C-30, and the same mutation arises in bacteria responsible for chronic cystic fibrosis infections. Therefore, bacteria may evolve resistance to many new pharmaceuticals thought impervious to resistance. The ISME Journal (2012) 6, 493–501; doi:10.1038/ismej.2011.122; published online 15 September 2011 Subject Category: microbial population and community ecology Keywords: drug resistance; efflux pump; Pseudomonas aeruginosa; quorum sensing; quorum quenching Introduction Bacteria have been identified that are resistant to all known antibiotics (Defoirdt et al., 2010), and infectious diseases remain the leading cause of death (Rasko and Sperandio, 2010); hence, it is important to develop new antimicrobials. Indeed, it has been decreed that we live in a post-antibiotic era and heralded that anti-quorum sensing (QS)/antivirulence methods hold great promise for treating bacterial infections (Rasko and Sperandio, 2010). One of the most attractive features of this approach is that by interrupting cell signaling, these approaches do not impose harsh or direct selective pressure like antibiotics (Bjarnsholt et al., 2010), so there is less evolutionary pressure to develop resistance to antivirulence compounds (Bjarnsholt et al., 2010; Rasko and Sperandio, 2010). Correspondence: TK Wood, Department of Chemical Engineering, Texas A&M University, 220 Jack E. Brown Building, 3122 TAMU, College Station, TX 77843-3122, USA. E-mail: 8 These authors contributed equally to this work. Received 30 May 2011; revised 29 July 2011; accepted 29 July 2011; published online 15 September 2011 The best-characterized of the antivirulence compounds are the brominated furanones, which are secreted by the seaweed Delisea pulchra to prevent biofilms from inhibiting its photosynthesis; to date, no bacteria have been identified that are resistant to them. The natural compound (5Z)-4bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone from D. pulchra inhibits both acyl-homoserine lactone-based and autoinducer 2-based QS (Ren et al., 2001), and the synthetic furanone C-30 (Figure 1a inset) has been shown to decrease acylhomoserine lactone-based signaling as well as decrease the virulence of P. aeruginosa in a mouse pulmonary infection model (Hentzer et al., 2003). These brominated furanones interrupt QS by interacting with transcriptional regulators that propagate the QS response (Defoirdt et al., 2007), and they do not affect bacterial growth in rich medium (Gram et al., 1996; Ren et al., 2001; Hentzer et al., 2003). However, growth of pathogens in the host during infections is more likely to involve non-robust carbon sources and may involve compounds whose utilization depends on QS (Defoirdt et al., 2010). Under these conditions, there will be selection pressure to evolve resistance against the compounds Resistance to antivirulence compounds T Maeda et al 494 Figure 1 The mexR and nalC mutations render P. aeruginosa less sensitive to QQ compound C-30 during growth in adenosine minimal medium. (a) Growth (at 37 1C) of wild-type P. aeruginosa PA14 (blue squares), transposon mutant mexR (red circles) and transposon mutant nalC (green triangles). (b) Growth of spontaneous quorum quenching quandary mutants (sQQQ) 1 (red circles) and sQQQ3 (green triangles) vs wild-type PA14 (blue squares). (c) Growth of CF clinical isolate 1253 (red circles) and CF Liverpool epidemic strain 12142 (green triangles) with enhanced mexA expression vs control strain AHP (blue squares). Open symbols indicate the presence of 50 mM C-30, whereas closed symbols indicate the absence of C-30. Data represent the mean±s.d.; n ¼ 3. that block QS, and it has been theorized, but not shown, that resistance may arise (Defoirdt et al., 2010). Note that previously mutations to the QS response regulator LuxR were generated in Escherichia coli, which altered both the binding of the natural ligand as well as that of quorum quenching (QQ) compounds, but resistance was not investigated and was predicted not to occur (Koch et al., 2005). The ISME Journal To investigate whether QQ-resistant bacterial mutants may arise in the presence of antivirulence compounds, we utilized the best studied bacterium for QS, P. aeruginosa, which is an opportunistic pathogen that is responsible for many infections, including those of ventilator-associated pneumonia, urinary and peritoneal dialysis, catheter infections, bacterial keratitis, otitis externa, burns, wound infections and those of the lung (Macé et al., 2008). Wild-type P. aeruginosa PA14 was used instead of PAO1 because PA14 is more virulent than PAO1 in diverse infection models (Harrison et al., 2010) and because of the availability of the complete mutant library (Liberati et al., 2006). Our strategy was to utilize a minimal medium (so it resembles more closely clinical situations) using a growth compound whose assimilation requires QS; therefore, QS and growth were inhibited by the antivirulence compound C-30, which has become the gold standard for antivirulence compounds. Growth on adenosine by P. aeruginosa depends on the degradative enzyme, nucleoside hydrolase, which is positively controlled by LasR (Heurlier et al., 2005). LasR is the transcriptional regulator that mediates acyl-homoserine lactone-based QS in this strain via N- (...truncated)