Gallium-mediated siderophore quenching as an evolutionarily robust antibacterial treatment

18 o r i gi na l research article Evolution, Medicine, and Public Health [2014] pp. 18–29 doi:10.1093/emph/eou003 Adin Ross-Gillespie*1,y, Michael Weigert1,2,y, Sam P. Brown3 and Rolf Kümmerli1,2 1 Institute of Plant Biology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland; 2Swiss Federal Institute of Aquatic Science and Technology (Eawag), Environmental Microbiology, Überlandstrasse 133, 8600 Dübendorf, Switzerland; 3Institute of Evolutionary Biology and Centre for Immunity, Infection and Evolution, University of Edinburgh, West Mains Road, Ashworth Laboratories, Edinburgh EH9 3JT, UK *Correspondence address. Institute of Plant Biology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland. Tel: þ41 44 635 2905; Fax: þ41 44 634 8204; E-mail: y These authors contributed equally to this work. Received 19 December 2013; revised version accepted 24 January 2014 ABSTRACT Background and objectives: Conventional antibiotics select strongly for resistance and are consequently losing efficacy worldwide. Extracellular quenching of shared virulence factors could represent a more promising strategy because (i) it reduces the available routes to resistance (as extracellular action precludes any mutations blocking a drug’s entry into cells or hastening its exit) and (ii) it weakens selection for resistance, as fitness benefits to emergent mutants are diluted across all cells in a cooperative collective. Here, we tested this hypothesis empirically. Methodology: We used gallium to quench the iron-scavenging siderophores secreted and shared among pathogenic Pseudomonas aeruginosa bacteria, and quantitatively monitored its effects on growth in vitro. We assayed virulence in acute infections of caterpillar hosts (Galleria mellonella), and tracked resistance emergence over time using experimental evolution. Results: Gallium strongly inhibited bacterial growth in vitro, primarily via its siderophore quenching activity. Moreover, bacterial siderophore production peaked at intermediate gallium concentrations, indicating additional metabolic costs in this range. In vivo, gallium attenuated virulence and growth—even more so than in infections with siderophore-deficient strains. Crucially, while resistance soon evolved against conventional antibiotic treatments, gallium treatments retained their efficacy over time. Conclusions: Extracellular quenching of bacterial public goods could offer an effective and evolutionarily robust control strategy. K E Y W O R D S : antivirulence therapy; public good quenching; resistance; experimental evolution; Pseudomonas ß The Author(s) 2014. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Gallium-mediated siderophore quenching as an evolutionarily robust antibacterial treatment Quenching public goods as a robust antibacterial treatment Ross-Gillespie et al. | 19 INTRODUCTION frequently involve intracellular action, against which many potential resistance-conferring adaptations could arise (e.g. modified membrane properties to block a drug’s entry into a cell, or upregulated efflux pumps to hasten its exit [15]). Second, QS regulates not only PGs but also certain essential private goods [16], giving QQ resistants substantial personal benefits over susceptibles—and therefore a means to spread. For maximal evolutionary robustness, we need therapies where resistance mutations are unlikely to arise in the first place (e.g. extracellular action restricts potential routes to resistance) and are also unlikely to spread, because fitness differences between resistant and susceptible pathogens are minimized. The latter should be the case when collective traits are targeted, because fitness consequences are shared across many individuals. Of course, the extent and evenness of this sharing will depend on the relatedness and spatial structure of the pathogen population and the diffusive properties of the environment, and these factors would also need to be considered during therapy design [3]. In this study, we investigate—in a test case—the hypothesis that extracellular PG quenching is an effective and evolutionarily robust strategy for pathogen control. The PG trait we target is siderophores, important exoproducts whose regulation is not linked to any exclusively private goods. Siderophores are diffusible molecules with a high affinity for ferric iron (Fe3þ) and are secreted by most bacteria to scavenge this important but generally bio-unavailable form of iron from their environment or, in the case of pathogens, from their host’s own iron-chelating compounds [17]. Once loaded with Fe3þ, siderophores are taken up by producer cells—or other nearby individuals equipped with appropriate receptors— stripped of their iron, and secreted once again into the environment [18]. Although their primary function may be to scavenge iron, siderophores also bind, with varying success, several other metals [19, 20]. Among these, gallium is the closest mimic of iron. Ga3þ and Fe3þ ions have very similar ionic radii and binding propensities but, crucially, while Fe3þ reduces readily, Ga3þ does not [19]. Ga3þ therefore cannot replace iron as a co-factor in redox-dependent enzymes. We investigated the iron-mimicking effects of gallium on pyoverdine, the primary siderophore of Pseudomonas aeruginosa [21], a widespread opportunistic pathogen with a broad host range and, in humans, the cause of Like all organisms, pathogens acquire genetic mutations, and, in time, even ‘pure’ cultures will inevitably come to harbor mutant lineages. Such genetic variability can make some pathogen variants less sensitive to therapeutic interventions than others, and under strong or sustained therapy, these resistant variants will have a selective advantage and will come to predominate over more susceptible variants. Consequently, the therapy will lose efficacy [1, 2]. To avoid this situation, we can try to prevent resistant variants from arising and/or from spreading [3]. To prevent resistance arising, we could attempt to reduce mutation supply, through limiting effective population size or by employing interventions with specialized modes of action where relatively few ‘routes to resistance’ are possible. To prevent spread, meanwhile, we must aim to minimize fitness differences across individual pathogens. Killing every individual, the conventional antibiotic strategy, could certainly quash fitness evenly, but this is difficult in practice and whenever incomplete gives resistant pathogens a strong relative fitness advantage. ‘Antivirulence’ treatments, meanwhile, ostensibly disarm but do not harm pathogens, suc (...truncated)